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Excerpted from: Rediscovering the American Republic, vol. 1: 1492–1877
From Pre-Columbian to British North America, 1492–1763
Following Columbus's Caribbean landing in 1492 (see p. 32), numerous European nations competed for territory in North America, dragging African slaves and Native Americans into the fray. The land that one day would become the United States served as the grand chess board in this geopolitical struggle that lasted some two and half centuries—longer than the United States has since then existed. The near simultaneously establishment of Jamestown by England (1607), Santa Fe by Spain (1608), and Quebec by France (1609) offers a small glimpse of the diverse settlements competing for primacy in the New World (for other examples, see pp. 43–46).
England's own colonies were themselves a mixed bag (see p. 49). Puritans came to New England to establish a state-church that would shame the Church of England into reform (see p. 60). William Penn, however, drew a different lesson from the religious controversies experienced in England: he came to America not to perfect the state-church, but to abandon it, forming Pennsylvania as a haven for religious dissenters (see p. 103). Farther south, along the Chesapeake and in the Carolinas, slave-based agriculture, more than religion, defined the emerging social order (see p. 89). What all of these English settlers had in common, however, was a shared heritage dating back to the Magna Carta (1215) and renewed by the Glorious Revolution (1688): the right to limit the monarchy, which came to mean also the right to a locally elected representative government (see pp. 113–14). This would become the colonists' legacy to the founding fathers of the United States, and the founders' legacy to the world. But first the monarchies of France and Spain had to be dealt a decisive blow.
After a long series of imperial wars—which hardly gave advantage to any of the participants—a decisive British victory at last was achieved in the Seven Years' War, culminating in 1763 with the Treaty of Paris (see pp. 147–48). France was virtually evicted from the New World, with Spain possessing the southwestern half of North America and Britain possessing everything between the eastern seaboard and the Mississippi River. Britain's American colonists had fought alongside imperial soldiers, sharing a patriotic unity that never had been stronger—nor would it ever be again. The British Empire had reached its zenith in North America, and soon would find itself defeated by a veteran of the Seven Years' War: George Washington.
The Creation of the American Republic, 1763–1789
Saddled with debt from the Seven Years' War, and now with more territory to protect than ever before, Britain sought a new way to raise revenue: tax the colonists. Parliament had taxed them before, but primarily for regulating trans-Atlantic commerce, not for funding the national debt. Moreover, earlier taxes had often been evaded by the colonists and forgiven by the mother country. The Stamp Act of 1765, however, marked a new approach: taxing for revenue and enforcing the collection (see p. 187). As heirs of the Glorious Revolution, America's colonists thought themselves entitled to a local representative government. They looked increasingly at their own assemblies, not distant Parliament, as their legitimate law-making body. Parliament, meanwhile, wavered from one minister to the next, but the long-term trend was obviously in the direction of tighter control over the colonies (see pp. 183–189). When King George III refused to hear the colonists' appeals, the Americans united in July 1776 to declare their independence (see p. 206). A war of ideas gave way to a contest of arms.
The Second Continental Congress designated General George Washington as commander of the patriot forces. Although he lost more battles than he won, he knew better than most generals throughout history how to execute a tactical retreat (see, for example, p. 213). Most of all, Washington's integrity won the hearts of his soldiers. Thomas Paine, whose treatise Common Sense had inspired the colonists to seek independence, wrote an encouraging masterpiece that boosted troop morale on the crucial eve of Washington's attack against Trenton (see pp. 216–19). In 1777, American soldiers secured a victory at Saratoga that attracted an alliance with France (see pp. 223–25). Bolstered by French money, men, and materiel, the patriots forced a British surrender at Yorktown in 1781 (pp. 235–37). The world's greatest empire at the time lost her greatest colony as the American Republic was born.
Two things were now certain for the thirteen original states. First, they had become independent of Great Britain. Second, they somehow remained interdependent upon one another. Throughout most of the Revolutionary War, the Continental Congress served as an emergency government. Although Congress had adopted a formal political arrangement, the Articles of Confederation in 1777 (see p. 248), the states did not unanimously agree to it until 1781, by which time the war was nearly finished. In the years that followed, the Confederation successfully established a federal lands policy through the Northwest Land Ordinance and related legislation, but failed to hold the founders' confidence as a sustainable arrangement for uniting the states. Responding to the deficiency, the Philadelphia Convention of 1787 drafted a new frame of government, the U.S. Constitution (see p. 266). Although this new plan had some advantages over the Articles of Confederation, it also risked delegating too much power to the federal government. To protect the liberties of the people and the authority of their state governments, amendments were promptly proposed in 1789, ten of which were ratified collectively as the Bill of Rights (see p. 295). Meanwhile, the presidential electors unanimously selected George Washington as the nation's first leader under the new constitution.
The Power of Political Parties, 1789–1836
During the 1790s, America's first political party system formed. Federalists, led by Alexander Hamilton and John Adams, sought to establish the federal government's authority to unite the states into a national economy through a centralized bank and the funding of a permanent national debt that could alleviate the states of their own debts (see p. 314). Although George Washington, as the nation's first president, initially appeared to steer clear of political factions, his sympathies placed him squarely in the Federalist camp. Meanwhile, the Republicans, led by Thomas Jefferson and James Madison, emphasized the tradition of local representative government that had underwritten the American Revolution (for example, see p. 354). Whereas Jefferson's party feared the federal government was becoming too powerful, Hamilton's party feared that unless the federal government and national economy were stabilized, the United States would be vulnerable to foreign invasion.
As Federalists and Republicans vied for power, America conducted its greatest experiment: testing the political theory embodied in the U.S. Constitution against lived experience. Two questions were crucial. First, what is the relationship between the federal government and the various state governments? Second, how are the three branches of the federal government related to each other? As every American middle school student knows, the answer was spelled out on paper in terms of the "checks and balances" of the Constitution, but it remained to be seen how Americans would apply those principles in practice. Republicans favored a strict construction of the Constitution, reserving to the people and their states whatever powers were not explicitly delegated to the federal government in the Constitution. Federalists preferred a looser interpretation, regarding the "necessary and proper" clause of Art. I, Sec. 8 (see p. 270) as permission, for example, for Congress to establish a national bank (see p. 314). And while the framers may have sincerely expected that the three distinct branches of government would keep each other in check, the lived reality was that by 1798 all three branches were populated by the same party—the Federalists—which enacted, enforced, and upheld legislation limiting the free speech of Republicans (see p. 352). Was this a necessary sacrifice of liberty in order to preserve order, or was this an imposition of one party's sense of order designed to crush the other party's liberty? The people's answer came in 1800, when Republicans won sweeping electoral victories for both the presidency and Congress. The Supreme Court, however, soon elevated itself above partisan squabbles, affirming its own authority to nullify any law that violated the supreme law of the land—the U.S. Constitution (see pp. 369–70).
Although the Federalist Party fizzled away when America proved victorious in the Republican-supported War of 1812, many of the Federalists' ideals shaped the thinking of a new breed of Republicans, the National Republicans, led by John Quincy Adams and Henry Clay. Then the appearance of a "corrupt bargain" between these two men in 1824 led to the rise of a new party, the Democrats (see p. 421). Organized by Martin van Buren and led by Andrew Jackson, the Democrats appealed to the people while also perfecting the tactics of political power to solidify a national base (see pp. 422–24). In response to Jackson's startling success, the Whig Party formed, ostensibly committed to the anti-Jacksonian policies of a protective tariff, federally funded roads, and the national bank (see pp. 433, 467–72). But the issues of the day also could be twisted inside out as personal vendetta crossed party lines: Jackson's own vice president, John C. Calhoun, on the one hand led his home state of South Carolina in protesting the tariff, but soon after joined the tariff-sponsoring Whig Party to spite Jackson, who had dared to enforce the tariff as president. It was a tense time, and the future of the nation may have been determined by whose wife was excluded from the latest gala (see pp. 434–45). Meanwhile, one subject had become taboo: slavery—the mere mention of it made national politics impossible, so both the Democrats and the Whigs learned to ignore it.
Liberty, Slavery, and American Destiny, 1836–1860
Just as the British Empire had broken apart after it became too large to manage in the wake of the Seven Years' War, so also the United States nearly busted loose at the seams when Texas was annexed in 1845 and the present-day states of New Mexico, Arizona, California, Nevada, Utah, and Colorado were acquired from Mexico in 1848. As southern slaveholders migrated westward, the topic of slavery could no longer be ignored. Southerners thought it only natural to extend slavery into the newly acquired lands, while northerners who had learned to tolerate slavery in the South loathed the idea of its expanding westward. What some history books call the "Compromise of 1850" was in fact a series of uncompromising measures that, by benefiting each side slightly, managed to hold the nation in a delicate balance for a few more years (see p. 534).
Senator Stephen Douglas of Illinois thought he had a solution: let the local residents decide the slavery question, and Congress should stay out of it. His doctrine of "popular sovereignty" held the promise of echoing America's founding principle of local representative government, but theory does not always equate to practice. In Kansas, two rival governments each claimed to be locally representative, one favoring slavery and the other excluding it. The result was bloodshed, or as Abraham Lincoln called it, "squatter's sovereignty" (p. 566).
Courtesy of the U.S. Supreme Court, squatter's sovereignty was soon "squatted out of existence," as Lincoln colorfully characterized the Dred Scott ruling of 1857 (p. 566, in reference to p. 563). Local governments no longer could exclude slavery, which is to say the nation was on a path toward full nationalization of the slavery regime. Meanwhile, the Fugitive Slave Act of 1850 required that northern opponents of slavery must nonetheless actively participate in the restoration of escaped slaves to their owners. The slavery debate had become an all-or-nothing contest for the allegiance of the American people.
In 1860, northern voters turned to Abraham Lincoln, a presidential candidate promising to preserve the Union while also hoping in time to end slavery. He was moderate compared to the abolitionists, who wanted slavery to end immediately and sometimes were willing to resort to violent methods to achieve this goal. At the other extreme, southerners clung to their peculiar institution and rejected Lincoln's leadership. Although he won a clear majority of the electoral votes, he did not receive any electoral support from the South. A secession convention in South Carolina took this as reason enough to vote their state out of the Union.
The Civil War and Reconstruction, 1860–1877
The nation that had been held together since 1788 by tenuous constitutional compromises concerning slavery now suffered the ultimate test. Ten other slave-holding states, following South Carolina's example, left the Union; the secessionists promptly formed the Confederate States of America. Five years of Civil War (1861–1865) pitted brother against brother in a contest that would determine not only the fate of four million slaves, but also the future form of the American Republic—if there was even to be a republic anymore. Lincoln switched from one general to another until finally discovering in Ulysses S. Grant a commander who could deliver the field into the president's hands (pp. 642–49). Meanwhile, the significance of the war had shifted in 1863 with the Emancipation Proclamation (pp. 632–33): no longer did President Lincoln seek merely to restore the Union, but also to free the slaves. Before his assassination in 1865, he had nearly accomplished both objectives.
Unfinished work fell next to President Andrew Johnson and the U.S. Congress. With Confederate General Robert E. Lee's surrender to General Grant, what should become of the Confederate leaders? Under what terms should the former Confederate states be readmitted into the Union? And what about the status of the recently freed slaves? Can they vote? Can they hold office? How will they be provided with land or education? Acting frequently against Johnson's veto, Congress answered these questions by establishing policies known collectively as Reconstruction. Southern states could be readmitted only after abolishing slavery and guaranteeing equal rights of citizenship to African Americans (see pp. 674–75). Three new amendments to the U.S. Constitution were ratified, going so far as to guarantee people of all races an equal right to vote (see pp. 676–77). A new model of ordered liberty thereby was established, with blacks participating alongside whites in the process of self-government; indeed, several blacks were elected to public office—local, state, and national (p. 679). Would Reconstruction last? The answer is found in the Conclusion (p. 681), but the quest that brought the nation there can only be discovered by beginning with America's colonial roots.
Continue with Next: A Brief Summary of American History, Part II: 1877–Present


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Gelechia trachydyta is een vlinder uit de familie van de tastermotten (Gelechiidae). De wetenschappelijke naam van de soort is voor het eerst geldig gepubliceerd in 1920 door Meyrick.

trachydyta

redpajama_set_name: RedPajamaWikipedia


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Peacemakers will tell churches about Matthew 25 work around the world
<June>
Presbyterian Peacemaking Program for 2019 has assembled 15 International Peacemakers
We see headlines every day from nations around the world telling us about crisis and conflict — and stories of people living in and overcoming extraordinary circumstances.
This fall, your church or presbytery can hear directly from Palestinians working for peace and justice in Israel-Palestine, a church worker in the Democratic Republic of Congo helping people who have HIV/AIDS, a Korean minister working for reunification of the Korean peninsula, a minister of Latvian and Russian-German descent based in Moscow, a social worker in South Sudan who lived through her own trauma and is now helping people in her nation live through theirs, and many more.
Those are snapshots of some of the 15 International Peacemakers who will be traveling to Presbyterian churches and related organizations throughout the United States from mid-September to early October as part of the 2019 International Peacemakers, an annual program presented by the Presbyterian Peacemaking Program. This is the 35th year of the Peacemakers' visits, which have brought more than 300 people from 60 countries to the United States to share their unique experiences in global peace and justice work.
While it has been around for more than three decades, the program is also ideally aligned with the Presbyterian Church (U.S.A.)'s new Matthew 25 invitation to congregations to build congregational vitality, dismantle structural racism and eradicate systemic poverty.
"Collectively, they are witnessing to the Matthew 25 church," said the Rev. Carl Horton, coordinator of the Presbyterian Peacemaking Program. "We've got someone working to provide clean water to the thirsty. We've got those providing food to the hungry. We've got someone welcoming the migrant 'stranger' in Europe. We've got people visiting those imprisoned in societal structures of racism and oppression.
"Taken as a whole, this is a great group of people to help us learn more about what it means to be a Matthew 25 church, each one doing a different aspect of that."
With 15 peacemakers, this year's class is a third larger than the 2018 group of 10. Horton said the Peacemaking Program had a lot of great suggestions from mission co-workers and others who gave input.
"We look for people who have their sleeves rolled up and are on the ground doing the work," Horton said. "They are engaged in peace and justice work. These are people working with marginalized populations, working with people at risk, working in contexts of speaking truth to power and challenging authorities — doing frontline justice work."
Horton points out that a number of Peacemakers are people whom participants in the Peacemaking Program's Travel Study Seminars and bi-annual Mosaic of Peace Conference in Israel and Palestine have encountered or will encounter in their home areas.
Peacemakers are available to visit around the country from Sept. 13 to Oct. 7. Visits are coordinated with mid councils, synods and presbyteries and can include visits to churches, seminaries, colleges, universities and similar institutions. Visits generally last three to five days, but they can be extended as needed.
"The power of the program is the personal relationship that is forged between the Peacemaker and the people who hear from them," Horton said. "It's a first-hand account of the peace and justice work that's being done around the world. It builds not only personal relationships, but in some cases sister churches have emerged out of these, presbytery partnerships have emerged — a lot of times our mid councils and congregations that hear from a Peacemaker want to show support."
Horton notes that the visits are not fundraising tours, though a desire to support peacemakers in some tangible way often emerges.
"It is educational, it's informative, it's intended to teach and widen people's understanding. It's not to collect money," Horton said. "But the reality is when our members hear what people are doing around the world — this year they're going to hear examples of being a Matthew 25 church — they will be moved and want to partner with them, and all these Peacemakers are partners of the Presbyterian Church.
"So, there are ways — spiritually, tangibly and materially — they can support these ministries."
Rich Copley, Communication Strategist, Presbyterian Mission Agency
Today's Focus: 2019 Presbyterian Peacemaking Program
Janna Wofford, APCU
Ann Wolf, BOP
God of love, thank you for modeling true compassion and peace. Fill us with your love so that we perceive and understand others the way you do. Use us to make an impact on our communities and churches, and to achieve a world of perfect peace. We pray all of this in the name of Jesus Christ. Amen.
Morning Psalms 51; 148
First Reading 1 Samuel 3:1-21
Second Reading Acts 2:37-47
Gospel Reading Luke 21:5-19
Evening Psalms 142; 65

redpajama_set_name: RedPajamaCommonCrawl


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ALI welcomes new Director: Dave Poulton
The Alberta Land Institute's objective is to connect research and policy for better land management. With a new addition this summer, the Institute has enhanced its ability to carry out that mission.
On June 1, 2016, Dave Poulton joined ALI in the new role of Director. An experienced consultant to organizations, businesses, and governments, Dave possesses a wealth of experience in promoting cross-sectoral collaboration. He is also an experienced researcher, with specific expertise in the areas of conservation offsets, market-based conservation, parks and protected areas, and land-use planning.
In the role of Director, Dave will help guide the development of ALI's ongoing strategy, while taking a lead role in outreach, and helping to further raise the Institute's profile across Alberta. He will inform and support ALI's research agenda, and help ensure that work completed by Institute researchers is shared with decision makers and stakeholders throughout the province.
Dave is no stranger to the Alberta Land Institute; he is one of only two speakers to deliver presentations at both the 2014 and 2016 editions of ALI's bi-annual Land Use Conference (the other was Peter Boxall, Principal Investigator on ALI's Alberta's Living Laboratories wetlands project). He has also built strong, long-standing relationships with many of ALI's researchers, staff, advisory board members, and partners.
In addition to his appointment with ALI, Dave will continue to serve as the Executive Director of the Alberta Association for Conservation Offsets and the Principal of Poulton Environmental Strategies. He is a member of the board of the Environmental Law Centre, was Executive Director of the Southern Alberta Chapter of the Canadian Parks and Wilderness Society from 2000-2008, and served as Conservation Director of that organization from 1999-2000.
Dave holds a Master of Laws degree in Natural Resources, Energy and Environment program at the University of Calgary Faculty of Law, as well as a B.A. and M.A. in political science from that institution. His LL.B. was completed at Dalhousie University in Halifax.


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What is Belladonna and its Health Benefits
Belladonna derives its name from the Italian word 'Bella' meaning beautiful and 'donna' meaning lady. Belladonna was introduced for the first time in the London Pharmacopoeia in 1809. Historically, it was used as witch-craft in the middle-ages. Its juice was used as a cosmetic.
Belladonna coming under tropane alkaloids consists of dried leaves and aerial parts of Atropa belladonna Linn.(European belladonna) or Atropa acuminata Royle ex Lindley(Indian belladonna) or mixture of both the species collected during the flowering stage of the plants. These species belongs to family Solanaceae. It contains not less than 0.3% of tropane alkaloids, calculated as 'hyoscyamine'. Its popular names include Folia belladonna, Deadly night shade leaves, Poison Black cherry or simply belladonna herb.
It's Plant is a herb about 1.5 meters in height. Root is fleshy, tapering and bears numerous rootlets. Stem is straight, branched, glabrous and hairy.
Leaves are green or brownish-green colored; arranged alternately and are seen in pairs, of which one leaf is larger than the other. Flowers are purple to yellowish-brown whereas fruits are green to brown.
Odour is slight and characteristic.
Taste is bitter and acrid.
Chemical constitution
Major part of belladonna herb possess alkaloids namely hyoscyamine, atropine, apoatropine and scarcely hyoscine. These alkaloids only are responsible for medicinal property. Among the non-medicinal constituents are volatile bases, pyridine, pyrrolidine and a diamine. It also contains a fluorescent coumarin derivative, scopoletin or beta-methyl aesculetin.
Health Benefits of Belladonna
Belladonna leaves and root are used entirely for medicinal utility and henceforth belladonna phrase will mean belladonna leaves or root only now onwards. The berries and fruits of belladonna plant are highly toxic.
As an antispasmodic agent:
Belladonna relaxes the smooth muscles of the respiratory tract and hence gives relief to patients suffering from asthma, bronchitis and whooping cough.
It also has a depressant action on the intestinal tract so slows down the peristaltic movement produced during usage of strong purgatives. This also benefits in reducing the spasms arousing during the intestinal gripping.
Ophthalmologists use homatropine, a replacement of atropine for dilating the pupil of eye for diagnostic property.
Atropine present in belladonna is used to suppress vomiting and hence used as an anti-emetic.
Atropine obtained from belladonna is combined with opium and compounded as powder or tincture or suppository form to treat diarrhoea and other forms of visceral pain.
Hydrobromide salt form of hyoscyamine was formerly used in pain-killer called 'twilight-sleep' during child-birth.
As a sedative or local anaesthetic effect:
Because of its sedative action on the central nervous system, Belladonna is famously used for treating excitability, in shocks including those of aerial bombardments, motion sickness specifically of air and sea. Its sedative action is so strong that it is used as a 'truth serum' for getting information from war prisoners.
The local anaesthetic effect and the anodyne effect of belladonna are employed in liniment and plaster of belladonna to treat the peripheral pains, skin and joint pain concerned with rheumatism.
As an anticholinergic / parasympatholytic agent:
Belladonna blocks the secretions like those of salivary glands, gastrointestinal system as well as sweat glands. So it is used for treating hyperacidity, ulcer or any mucosal inflammation of the gastro-enteric lining.
It is also used to check the profuse perspiration of patients suffering from tuberculosis.
Miscellaneous health benefits and usage of Belladonna:
Belladonna is used as an antidote to treat poisoning by physostigmine and organophosphorous esters that are used in preparing insecticides. It is also used as an antidote in opium and chloral hydrate poisoning.
Apart from the entire herb use, belladonna roots are specifically used in treatment for Parkinsonism because the roots contain belladonine, which is not present in leaves.
Leaf and root are used for medicinal purpose. Internal preparations of belladonna as sedative or secretion checker involve leaves whereas preparations of roots are used for external purpose.
For centuries, belladonna is traditionally used for conditions like headache, inflammation, peptic ulcer, menstrual symptoms and motion sickness.
It is also used as a recreational drug because of its hallucinogenic and delirium effects but this sort of use brings dangerous risk of fatal damage including memory disruption.
Other Probable health uses of Belladonna
Insufficient evidence is also reported for use of belladonna in treating hay-fever, hemorrhoids, colds, spasm reliever in stomach & bile-ducts and nerve problems. It is yet to study the effectiveness of belladonna in arthritis-like pain and colic-like pain.
It is still undergoing research in its use for treating irritable bowel syndrome and acute enterocolitis as an adjuvant therapy.
Side effects of Belladonna
Oral consumption of belladonna is found unsafe because of toxic chemicals. It causes dry mouth, inability in urination and sweating, spasms and increasing the rate of cardiac pumping. Belladonna causes enlarged pupil, blurred vision, fever and skin becomes red and dry. Its severity is noted in causing mental problems, hallucinations, convulsion and coma.
Precautions while using Belladonna
Pregnant and breast feeding women must avoid taking belladonna. It is reported to reduce the production of milk and also passes into breast milk causing toxic effects.
If you are suffering from congestive heart failure then you must avoid using belladonna as it worsens the heart rate by increasing it all the more.
Belladonna makes constipation worse, increases body temperature, worsens oesophageal reflux, increases the severity of ulcers as well as ulcerative colitis, and makes your urination frequent.
If you are suffering from glaucoma, do not consume this drug as it worsens narrow angle glaucoma and hence can cause increased chances of blindness.
As belladonna slows down the intestinal emptying rate, it causes the retention of bacteria and viruses as a result of which, person becomes susceptible to infections.
Do not use belladonna if you have Down syndrome or hiatal hernia.
Drug interaction : Belladonna if used in combination with anticholinergics or antihistaminics or antidepressants cause drying of skin, dizziness, low blood pressure, tachycardia and other serious side-effects. So you must avoid using them together.
This drug finds its usage in numerous fields. Previously it was used in making poisonous arrows or as a poison to win over the enemies during wars or as a cosmetic. And now it has also made itself known in the medicinal purposes. But because belladonna is extremely poisonous, it must be used with caution.


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Best of Inland Empire
Disneyland stops selling all Magic Key annual…
Lawsuit vs. Disneyland
Disneyland stops selling all Magic Key annual passes a day after resuming sales
Disneyland vistors walk through Tomorrowland at Disneyland earlier this year. The park announced Thursday, Nov. 17 that it is pausing sales of its $1,099 Believe Key the day after resuming annual pass sales after a six-month hiatus. (Photo by Jeff Gritchen, Orange County Register/SCNG)
By Jeffrey Miller | jsmiller@scng.com | Orange County Register
PUBLISHED: November 17, 2022 at 12:54 p.m. | UPDATED: November 18, 2022 at 10:04 a.m.
The Disneyland Resort stopped selling annual passes on Thursday, Nov. 17, just a day after it resumed selling the passes following a six-month hiatus.
On Wednesday, Disneyland announced that people who didn't currently have annual passes could buy the Inspire Key for $1,599. the Believe Key for $1,099 or the Imagine Key (available only to Southern California residents) for $449. Over the course of a few hours Thursday, the company paused sales of the Believe, Imagine and Inspire keys in quick succession.
Guests who already have Magic Key passes may renew them if their pass is within 30 days of expiring, but passes are no longer available to new customers.
"While we know this will be disappointing news to Disneyland fans, it's important that we help protect the experience for Magic Key holders and the value those passes provide," Disneyland officials said in a statement.
The Magic Key program was launched in August 2021 as a replacement for Disneyland's annual pass system, which began in 1984 but was eliminated in January 2021, while the parks were shuttered during the 412-day closure mandated by the COVID-19 pandemic. Annual passholders, who in pre-pandemic times could visit on any day their passes were valid, were cited as a reason why the parks were frequently crowded.
Disneyland and Disney California Adventure reopened in April 2021 with capacity limits in place. Although state mandates to limit attendance were lifted in June of that year, Disney's Anaheim parks have continued to require reservations as a way to regulate the number of people in the parks.
Two months after rolling out Magic Key, Disneyland paused sales of the top-priced Dream Key, which has since been replaced by the Inspire Key. Sales of the Believe Key were stopped in November 2021 and sales of the two remaining pass tiers — Enchant and Imagine — ceased in the spring of this year.
When Disneyland resumed sales of Magic Key passes this week, it initially offered only the Inspire, Believe and Imagine keys to new customers. The Enchant Key could be renewed by existing passholders up to 30 days before their current passes expired.
"Renewals for current Magic Key holders within their renewal window (up to 30 days before pass expiration) will continue for all available pass types at this time," Disneyland officials said.
Update: An earlier version of this story said only sales of the Believe Key had been stopped.
Jeffrey Miller | Assistant Managing Editor
Jeff Miller started at The Orange County Register in 1989 as the Costa Mesa cities reporter and later covered Huntington Beach and courts, in addition to a stint as a general-assignment reporter. He has been an editor since 1997, overseeing beats such as breaking news, courts, immigration, education, pop music, theater, visual arts, dance, TV and movies.
jsmiller@scng.com
Follow Jeffrey Miller @JeffMiller33333
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TNT's exclusive presentation of 2016 NBA Western Conference Finals Game 6 between the Oklahoma City Thunder and Golden State Warriors on Saturday, May 28, averaged 10.8 million total viewers and a 6.2 U.S. HH rating, based on Nielsen Fast Nationals, to deliver the network's most-viewed and highest-rated Conference Finals Game 6 of all time. Last night's game telecast is the most-viewed and highest-rated NBA Playoff game across any network to date this year, and it is the highest-rated and second most-viewed NBA telecast of the season across all of television.
The Warriors/Thunder telecast – peaking with an average of 14.6 million total viewers and an 8.3 U.S. HH rating from 11:30-11:45 p.m. ET – won the night across all of television (cable and broadcast).
Additionally, TNT's Inside the NBA garnered a 4.1 HH rating – its highest overnight rating of the season – to rank as television's second-best program of the night, according to overnight delivery. Throughout the NBA Conference Finals, Inside the NBA continues to out-deliver all other late night talk show programming (cable and broadcast) in total viewers and all key adult and male demos.
Turner's TV Everywhere platforms delivered all-time records for the Warriors/Thunder Game 6 including 817,000 uniques, 1.6 million video starts and 18 million minutes of video consumption.
Last night's Thunder/Warriors telecast is up 33% in total viewers and 27% in U.S. HH rating when compared with 2014, the last time TNT televised the Western Conference Finals (Oklahoma City vs. San Antonio). This year's coverage of WCF Game 6 also generated double-digit audience growth across all key demos.
Through six games, the WCF on TNT is averaging nine million viewers and a 5.4 U.S. HH rating, up 33% and 29%, respectively, when compared with the network's WCF coverage in 2014.
Content published via NBA on TNT's Facebook and Twitter accounts have generated two billion social media million impressions for this year's NBA Playoffs to date, up 43% over last year. Turner's accounts have netted more than 154 million video views, up 143% vs. 2015.
TNT's exclusive coverage of the 2016 Western Conference Finals will culminate with a decisive Game 7 between the Thunder and Warriors on Monday, May 30, at 9 p.m. The network's coverage will begin with NBA Tip-Off presented by Autotrader at 8 p.m. with host Ernie Johnson and analysts Charles Barkley, Kenny Smith and Shaquille O'Neal.
Source: Nielsen Media Research based on Live + Same Day data stream, Fast Nationals for 05/28/16 NBA on TNT 2016 NBA Western Conference Finals compared to historical NBA Conference Finals on TNT. DMA ratings and Inside the NBA data program metered market rating provided by Arianna, compared with cable ad supported programs for 05/28/16. Online and Mobile TVE Live Stream Starts, Uniques, Minutes by Quarter Hour (rounded based on actual game time) from Omniture SiteCatalyst; Facebook Insights, Twitter Analytics, Crowdtangle, Spredfast Intelligence.


Does the paleo diet exclude even dark chocolate consumption?
Does the paleo diet exclude any kinds of chocolate consumption?
Good day, everybody, my name is Silva, and I am 24. I hope that you will help me to find some answers to my question. See, I am doing diet research at the moment, and it is about the paleo diet, it's pros and cons and some cheats during it. My central question is: does this diet allow to eat dark chocolate?
Will it be an enormous problem or not? How much chocolate can I eat per week? At what time of the day?
I heard that it is the best to eat it in the morning till 9 o'clock. Is it true?
What caloric content has the dark chocolate? Does the paleo diet exclude any kinds of chocolate consumption? What amount is not harmful to the body? What sweet but dietetic products can I eat while paleo dieting? Can I make non-caloric chocolate cakes at home? What easy dietetic receipts can you suggest for me?


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Justia Patents US Patent for Synthetic polypeptides as inhibitors of HIV-1 Patent (Patent # 5,444,044)
Synthetic polypeptides as inhibitors of HIV-1
Mar 26, 1992 - New York Blood Center
A synthetic HIV-1-based polypeptide as well as methods for topically inhibiting HIV-1 infectivity or replication. The polypeptide of the present invention has an amino acid sequence substantially corresponding to a specified region of the HIV-1.sub.IIIB virus. The polypeptide of the present invention may be administered in effective amounts for topically inhibiting HIV-1 infectivity or replication. The polypeptide is useful for inhibiting the replication of the HIV-1 virus as well as HIV-1-mediated cytopathogenesis and cell fusion at levels which are within acceptable ranges of cytotoxicity.
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The present invention generally relates to antiviral agents useful for inhibiting the infectivity of human acquired immunodeficiency virus. More particularly, the present invention concerns synthetic polypeptides capable of inhibiting the infectivity of HIV-1.
Acquired Immunodeficiency Syndrome (AIDS), caused by the HIV-1 virus, has been reported in increasing numbers throughout the world. The disease is currently recognized by medical experts as approaching epidemic proportions and there is presently no cure or vaccine available to control its spread. Furthermore, new strains are developing due to the high mutation rate of the virus. Consequently, there is a tremendous need for methods of treating HIV-infected individuals and for the prevention of HIV-1 infections.
Until recently, research efforts directed toward developing anti-HIV agents has focused on several approaches. These approaches include: (1) the development of polyclonal or monoclonal antibodies to HIV antigens; (2) chemical antiviral agents; and (3) a soluble CD4 molecule corresponding to the HIV receptor on CD4.sup.+ target cells.
For example, previous research efforts have yielded both monoclonal and polyclonal antibodies to various HIV proteins. In particular, envelope glycoproteins of the HIV-1 virus, such as gp160, have been found to elicit antibodies which neutralize the infectivity of HIV-1. Consequently, gp160 and various fragments thereof have been tested extensively as candidates for a potential vaccine against HIV-1 infections. Unfortunately, it has been found that some of the immunodominant regions present on gp160 elicit the production of harmful antibodies which may trigger undesirable immunosuppression or even enhance HIV-1 infections.
For example, it is well-known that gp160 is a glycosylated polypeptide which has a molecular weight of 160,000 daltons and is processed to yield envelope glycoproteins gp120 and gp41. Although gp120 and gp41 both elicit immune responses which result in antibodies to HIV-1, these responses are generally immuno-specific to individual sub-types of the virus or may cause undesirable immunosuppressive effects. In particular, certain selected regions from gp120 and gp41 can suppress lymphocyte blastogenic responses and inhibit the activity of natural killer T-cells. Furthermore, some of the immunodominant regions of gp120 and gp41 do not contribute to protective immunity and may behave as immunological decoys.
An additional problem is encountered in developing a vaccine which is effective against HIV-1 infections because the virus exhibits frequent sequence variations during replication. More specifically, individual HIV-1 virus isolates demonstrate sequence variations, particularly in critical immunodominant regions of the virus sequence. Consequently, most virus-neutralizing antibodies are immuno-specific to individual clinical sub-types of the virus and the antibodies elicited by a vaccination against one clinical sub-type would likely be ineffective against another strain of the virus.
Other research efforts have been directed toward developing chemical agents which are effective against HIV-1 infections. For example, 3'azido-3'deoxythymidine (AZT) and other dideoxynucleoside analogs have been used to treat HIV-1 infections, however, these drugs are used in dosages which are often toxic to the patient. Furthermore, the use of chemical agents frequently induces the formation of viral mutants that are drug-resistant. Viral replication of drug-resistant mutants is not effectively inhibited by these drugs.
Additional research efforts have been directed toward the development of synthetic molecules containing receptor sites that mimic the receptor sites on target cells which the HIV-1 virions use to attach themselves to the cell membrane. For example, the CD4 molecule was expected to become the most hopeful anti-HIV agent for both therapy and prophylaxis of HIV-1 infections since it effectively inhibited viral replication in vitro. Unfortunately, it was later discovered that the molecule was not capable of inhibiting the infectivity of many clinical sub-types or strains of the virus.
It is therefore an object of the present invention to provide an antiviral substance which is capable of effectively inhibiting the replication of HIV-1, HIV-1-mediated cytopathogenesis and cell fusion at a concentration which falls within acceptable limits of cytotoxicity.
It is a further object of the present invention to provide an antiviral substance which is not specific to only one sub-type of HIV-1, but is effective against a variety of sub-types or strains of the virus.
It is still a further object of the present invention to provide an antiviral substance which is effective against AZT-resistant variants of HIV-1.
It is also an object of the present invention to provide a method of treating HIV-1 infections in a subject without inducing undesirable immunosuppressive effects.
The present invention provides a synthetic HIV-1-based polypeptide which comprises an amino acid sequence substantially corresponding to the amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB virus from the amino acid residue 600 to amino acid residue 862, and most preferably from amino acid residue 637 to amino acid residue 666.
Additionally, the present invention also provides a method for treating a test subject infected with HIV-1 virus. The method involves administering to the subject an effective amount of the synthetic HIV-1-based polypeptide of the present invention in order to inhibit replication of the virus.
The polypeptide of the present invention includes an amino acid sequence which substantially corresponds to a portion of the amino acid sequence of the HIV-1 virus containing a domain that is critical to the ability of the virus to infect cells. This domain appears to be common to most clinical sub-types of HIV-1. Consequently, the present invention provides an antiviral substance specific to HIV-1 infections which is effective against variants and sub-types of the virus, especially those variants which have become resistant to chemical antivirals such as AZT.
Unlike the chemical antivirals currently available for treating HIV-1 infections, the present invention provides a method of effectively treating HIV-1 infected individuals while avoiding some of the undesirable physiological side effects associated with treatments using AZT or other similar substances. Additionally, the present invention provides both a method of treating HIV-1 infected individuals as well as a new antiviral substance against HIV-1 which does not induce undesirable side effects.
Moreover, low cytotoxicity and high antiviral activity are also important factors to be considered when evaluating an antiviral substance. For example, there are many chemical antiviral agents capable of inhibiting viral replication of HIV-1, but most of them are unacceptable due to high levels of cytotoxicity. The polypeptide of the present invention is quite advantageous as it falls within acceptable ranges of cytotoxicity while effectively inhibiting HIV-1 infection.
For a better understanding of the present invention, together with other and further objects, reference is made to the following description as well as the accompanying sequence listing and drawings, the scope of which will be pointed out in the appended claims.
FIG. 1 illustrates the inhibition of HIV-1.sub.IIIB replication in MT-2 cells by synthetic peptides from gp120/gp41 of HIV-1.sub.IIIB.
FIG. 2 illustrates the inhibition of HIV-1.sub.IIIB -mediated cytopathogenesis in MT-2 cells by synthetic peptides from gp120/gp41 of HIV-1.sub.IIIB.
FIG. 3 illustrates cytotoxicity mediated by the various synthetic peptides.
FIG. 4 illustrates the inhibition of HIV-1.sub.IIIB replication in U937 cells by synthetic peptides from HIV-1.sub.IIIB gp120/gp41.
FIG. 5 illustrates the dose-dependent inhibition of HIV-1.sub.IIIB infection of MT-2 cells by synthetic peptide (637-666).
FIG. 6 illustrates the inhibition of various HIV-1 isolates by synthetic peptide (637-666).
FIG. 7 illustrates that antiserum to synthetic peptide (637-666) blocks its antiviral activity.
FIG. 8 illustrates a comparison of monomers and dimers of synthetic peptide (637-666) as inhibitors of HIV-1 infection.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention provides for a synthetic polypeptide which effectively inhibits infectivity and viral replication of HIV-1. As previously mentioned, the polypeptide of the present invention has an amino acid sequence substantially corresponding to the amino acid sequence of the envelope glycoprotein of the HIV-1.sub.IIIB virus from amino acid residue 600 to amino acid residue 862, and more preferably from amino acid residue 620 to 680. In the most preferred embodiment, the polypeptide is present as a dimer and has an amino acid sequence substantially corresponding to the amino acid sequence of the envelope glycoprotein of the HIV-1.sub.IIIB virus from amino acid residue 637 to 666. Additionally, the synthetic polypeptide of the present invention preferably includes a Gly-Gly-Cys amino acid triplet coupled at its C-terminus, thereby facilitating dimerization or alternatively affording a means for coupling the polypeptide to other entities.
It is known in the art of protein chemistry that an identical copy of a particular polypeptide can be readily produced where every amino acid in the copy appears in exactly the same sequential position as it does in the original. In short, the copy has an amino acid sequence which corresponds exactly to the amino acid sequence of the original polypeptide from which it was copied. Alternatively, certain amino acids in the copy can be substituted with natural or synthetic analogs without altering any of the desired biological characteristics of the resulting polypeptide, provided that the regions containing biologically important residues are conserved. For example, certain synthetic amino acid analogs can be substituted at desired positions within an amino acid sequence to impart resistance to proteolytic cleavage while maintaining the desired immunological character of the resulting polypeptide. Therefore, an identical copy of a particular polypeptide can receive certain substitutions at non-conserved regions and still retain the same biological character as the original polypeptide from which it was copied.
Generally, the synthetic peptide of the present invention can be substituted with natural or synthetic analogs as long as the regions responsible for the biological character of the peptide remain conserved. These substitutions may be induced to impart a desired a character to the resulting polypeptide or, alternatively, may occur as natural variations in the sequence as observed in identified sub-types of HIV-1. For example, each clinical isolate of the HIV-1 virus has its own variant sequence and it is known in the art that the relative numerical alignment of the amino acid sequences for each isolate will be slightly different.
Reconciliation of these differences during attempts to fashion polypeptides of the present invention after regions of interest in a particular isolate of HIV-1 can be readily accomplished using techniques known in the art. Accordingly, for purposes of the present application, the polypeptide of the present invention can be said to "substantially correspond with" the original polypeptide or isolate from which it was copied despite natural variations in sequence and/or numerical alignment or substitutions as discussed above.
In constructing the polypeptide of the present invention, a numerical frame of reference or standardized reading frame corresponding to the amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB should be consulted in order to establish the region of interest, amino acid residues 637-666. Several publications containing standardized amino acid sequence data for the HIV-1 virus are currently available. See, for example, Ratner, et al., "Complete nucleotide sequence of the AIDS virus," Nature, Volume 313, 277, (January, 1985). See also, "Human Retroviruses and AIDS 1991, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences," (Myers et al. Eds.), Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex., USA.
Upon establishing the proper reading frame, the amino acid region of interest can be reproduced using a variety of procedures available in the art. For example, a variety of automated synthesis units are currently available including the NPS 4,000 semi-automated multichannel peptide synthesizer and the Biosearch Model 9,600 automatic peptide synthesizer. Alternatively, certain recombinant DNA techniques or proteolytic cleavage techniques may be incorporated for constructing the polypeptide of the present invention. For example, a carefully orchestrated proteolysis of gp160 or gp41 using proteolytic enzymes or chemical cleaving agents would be an acceptable method for producing a polypeptide of the present invention. Accordingly, the term "synthetic polypeptide", as it appears throughout the present Application, is taken to mean any non-naturally-occurring polypeptide constructed by one of the methods mentioned above or by other suitable methods known in the art.
The Sequence Listing portion of the present Application provides examples of several synthetic polypeptides having amino acid sequences substantially corresponding to the amino acid sequences of the envelope glycoprotein of various clinical isolates of HIV-1 virus from amino acid residue 637 to 666 (see SEQ ID NOS: 1-10). As previously mentioned, the polypeptide of the present invention may preferably contain a Gly-Gly-Cys amino acid triplet coupled to its C-terminus in order to facilitate dimerization or attachment to another entity. The Sequence Listing portion of the present Application also contains each of the amino acid sequences set forth in SEQ. ID NOS: 1-10 further comprising a Gly-Gly-Cys triplet coupled to its C-terminus (see SEQ ID NOS: 11-20). These amino acid sequences constitute various synthetic polypeptides constructed in accordance with the present invention.
In addition to the monomeric forms of the synthetic polypeptides set forth in the Sequence Listing portion of the present Application, the present invention also includes dimeric, trimeric and other polymeric forms of these peptides. In particular, the best mode of the present invention is the dimeric form of the polypeptide, wherein dimerization is achieved via the Gly-Gly-Cys triplet positioned at the C-terminus of the peptide, as illustrated in SEQ ID NOS. 11-20. Dimerization or polymerization of the polypeptide of the present invention can be induced using any number of acceptable methods known in the art.
For example, a variety of covalent chemical or physical linkages can be employed to produce multimers of the polypeptide of the present invention. In particular, multimers of the polypeptide can be generated via attachment to multimeric peptide carriers known in the art or other approaches, such as multiple antigen peptide systems (MAP) or ISCOM. Furthermore, in addition to inducing dimerization via the Gly-Gly-Cys triplet coupled to the C-terminus of the polypeptides illustrated in SEQ ID NOS. 11-20, additional cysteine residues found outside of the 637-666 region may be utilized to induce dimerization where the polypeptide to be dimerized has amino acid residues extending beyond residue 666.
In particular, dimerization or polymerization may be mediated through additional -Cys- groups incorporated in or attached to the polypeptide. Additionally, the polypeptides described herein may be coupled to protein or high molecular weight (macromolecular) polymeric carriers (or substrates) through several types of functional groups on the polypeptides. For example, thiol (--SH) groups, aromatic rings (e.g., Tyr or His), amino or carboxyl groups, etc. would be acceptable linking moieties. Methods designed to avoid the use of carriers by polymerizing synthetic polypeptides to give peptide polymers are also known in the art. Another particular approach, known as the multiple antigen peptide system (MAP), utilizes a small peptidyl core matrix with covalently attached, radially branching, multiple synthetic peptides. See, for example, Tam, J.P. (1988) "Synthetic Peptide Vaccine Design: Synthesis and Properties of a High-Density Multiple Antigenic Peptide System" Proc. Natl. Acad. Sci. U.S.A., 85, 5409-5413.
As illustrated in the Examples below, the polypeptide of the present invention effectively inhibits infections of various cell lines, such as the monocytic cell line U937 and CD4.sup.+ T-cell lines MT-2 and H9, by several HIV-1 isolates, including the HIV-1 isolates MN, RF, SF2, IIIB and AZT-resistant isolates. In addition to effectively inhibiting HIV-1 viral replication, the polypeptide of the present invention provides for excellent inhibition of HIV-1-mediated cytopathogenesis and cell fusion, as compared with other synthetic polypeptides fashioned from other regions of the HIV-1 virus amino acid profile. Furthermore, the polypeptide of the present invention performs effective inhibitory function at acceptable levels of cytotoxicity.
The following Examples serve to further illustrate the present invention but are not meant in any way to limit or restrict the effective scope of invention.
In accordance with the present invention, 29 peptides covering almost the entire sequence of HIV-1.sub.IIIB gp160 and 21 peptides corresponding to the full-length V3 hypervariable loops of distinct HIV-1 isolates were synthesized. The syntheses were carried out on either an NPS 4000 semi-automated multichannel peptide synthesizer or a Biosearch Model 9600 automatic peptide synthesizer. The purity of the product was confirmed by HPLC and by amino acid analysis. The sequence of each peptide was determined using an Applied Biosystems Protein Sequence Model 477A with on-line HPLC analysis of PTH amino acids using an Applied Biosystems Model 120A PTH analyzer.
For virus inhibition assays, H9, MT-2 and U937 cells were used as target cells. Five HIV-1 isolates (IIIB, MN, RF, SF2, and an AZT-resistant mutant) were cultivated in MT-2 cells, collected, and aliquoted. The titer of infectivity of the virus stocks were 10.sup.4 -10.sup.6, 50% tissue culture infective doses(TCID.sub.50).
HIV-1 infectivity was determined by two distinct methods; an ELISA measuring HIV-1 core protein p24 production and a colorimetric assay for HIV-mediated cytopathogenesis. Briefly, 1.times.10.sup.4 MT-2 cells in 96 well plates were infected with dilutions of HIV-1.sub.IIIB in 200 .mu.l RPMI 1640 medium with 10% FBS. After 24 hrs., half of the culture medium was changed. On the fourth day after incubation at 37.degree. C., 100 .mu.l of culture supernatant was collected from each well and an equal volume of fresh medium was added to the wells. The collected supernatants were mixed with equal volumes of 5% Triton X-100 and assayed for p24 using a kit from Coulter Immunology (Hialeah, Fla.). On the sixth day post infection, an indicator XTT tetrazolium dye (1 mg/ml; 50 .mu.l/well; PolySciences, Inc., Warrington, Pa.) was added to the cells. After a 4 hr. period, intracellular formazan was determined colorimetrically at 450 nm. The percentage of cytopathogenesis was calculated using the following formula: [(OD.sub.450 in negative control-OD.sub.450 in experiment)/(OD.sub.450 in negative control-OD.sub.450 in positive control)].times.100. The negative control corresponded to cells mixed with culture medium, instead of HIV, while the positive control represented cells mixed with 100 TCID.sub.50 of HIV-1.sub.IIIB which lysed 100% of the MT-2 cells.
The inhibitory effect of the peptide of the present invention on the fusion of HIV-1 infected cells with uninfected cells was also determined. In brief, 1.25.times.10.sup.4 HIV-1.sub.IIIB infected H9 cells were mixed with the peptide and an equal number of uninfected H9 cells. After incubation at 37.degree. C. for 16 hrs., the syncytium-forming cells in a titer plate well were counted under a microscope and the inhibition of cell fusion by the peptide was calculated.
Twenty-nine peptides from HIV-1.sub.IIIB gp160 (gp120/gp41) and 21 peptides from V3 hypervariable loops of distinct HIV-1 isolates were screened for their inhibitory activity on HIV-1 replication and HIV-1-mediated cytopathogenesis in MT-2 cells. As shown in FIG. 1, among the 29 peptides from gp160 screened, four peptides (amino acid sequence 61-90, 102-126, 280-306, and 637-666), at a final concentration of 12.5 .mu.g/ml, significantly inhibited HIV-1 replication, as indicated by a decrease of gag protein p24 synthesis. However, none of the peptides from V3 loops of 21 distinct HIV-1 isolates inhibited HIV-1 replication.
Additionally, studies were performed to determine whether or not any of the peptides could inhibit the cytopathogenesis mediated by HIV-1. The results are shown in FIG. 2. Only the peptide (637-666) markedly inhibited cytopathogenesis, while the other three peptides which inhibited p24 production did not exhibit this inhibitory activity.
These results demonstrate that the peptide (637-666) from gp160 of HIV-1.sub.IIIB effectively inhibited infection by the homologous HIV-1 isolate (IIIB).
Subsequently, cytotoxicity studies were performed using MT-2 cells at a final concentration of 12.5 .mu.g/ml. It was found that three peptides (61-90, 102-126, and 280-306) had significant cytotoxicity, while the peptide (637-666) did not exhibit any cytotoxicity (see FIG. 3). When the concentration of the peptide (637-666) was increased to 11,145 .mu.M, significant cytotoxicity remained undetectable. These results suggest that the inhibition of p24 production by the three peptides (61-90, 102-126, and 280-306) was actually due to the cytotoxicity of these peptides for MT-2 cells, while only the peptide (637-666) could really inhibit both p24 production and cytopathogenesis.
The same techniques were also used to screen peptides with the monocytic cell line, U937. The results are shown in FIG. 4 Among the 29 peptides from gp160, only three peptides (102-126, 280-306 and 637-666) significantly inhibited p24 production. The peptide (637-666) had the highest inhibitory activity, suggesting that this peptide could inhibit the infection of both CD4.sup.+ T lymphocytes and of monocytic cells.
Subsequently, the peptide (637-666) was tested for its inhibitory activities on the HIV-1 replication, HIV-1-mediated cytopathogenesis and cell fusion at serial dilutions. As shown in FIG. 5, the peptide inhibited p24 production, cytopathogenesis and cell fusion in a dose-dependent manner. The ED.sub.50 for inhibition of HIV-1.sub.IIIB replication, as indicated by the decrease of gag protein p24 production, was 101.0 nM; for inhibition of cytopathogenesis mediated by HIV-1.sub.IIIB, 142.0 nM; and, for inhibition of cell fusion, 160.0 nM.
Further studies were conducted to determine whether or not the peptide of the present invention would also inhibit the replication of other HIV-1 isolates, such as MN, RF and SF2 isolates, as well as an AZT-resistant mutant. Results are shown in FIG. 6. The peptide (637-666) effectively inhibited infections by all the five HIV-1 isolates, although the ED.sub.50 for inhibition of both replication and cytopathogenesis mediated by the distinct HIV-1 isolates were different. These results suggest that the anti-HIV activity of the peptide (637-666) is not sub-type (isolate) limited. Furthermore, the peptide is also effective against AZT-resistant HIV-1 isolates.
Additionally, experiments were performed to determine whether or not the anti-HIV activity of the peptide (637-666) could be blocked by antibodies. Rabbit antiserum directed against this peptide was prepared. Serial dilutions of rabbit anti-(637-666) antiserum were incubated with the peptide (637-666) at a final concentration of 1 .mu.g/ml at 37.degree. C. for 30 minutes. The mixture was tested for antiviral activity. As shown in FIG. 7, in the absence of the antiserum, the peptide (637-666) significantly inhibited p24 production. However, after incubation with the anti-peptide (637-666) antiserum, the inhibitory activity of the peptide (637-666) was significantly blocked, while the antiserum alone did not affect p24 production. This result indicates that the inhibitory activity was indeed mediated by the peptide (637-666).
The aforementioned peptide (637-666) of the present invention was synthesized with a Gly-Gly-Cys amino acid triplet coupled at the C-terminus of the peptide.
Mass spectrometric data demonstrated that this peptide was completely dimerized. The dimerized form of the peptide will spontaneously form via the thiol group in the cysteine if allowed to incubate accordingly. The synthetic peptide was cleaved from resin stored from the original peptide synthesis and was shown to be in monomeric form using mass spectrometry. To determine the anti-HIV activity of the monomeric peptide (637-666), as compared with the anti-HIV activity of the dimeric peptide (637-666), the two peptide batches were assayed in a comparative experiment.
The results of the comparative assay are shown in FIG. 8. The dimerized form of the peptide is much more effective for inhibiting HIV-1 replication, HIV-1-mediated cytopathogenesis and cell fusion than the monomeric version of the same peptide. This result suggests that the peptide (637-666) will dimerize or polymerize by the addition of an extra amino acid residue, such as cysteine, at the C-terminus or the N-terminus of the peptide. Furthermore, these results also indicate that the dimerized or polymerized form of the peptide (637-666) may more effectively interfere with the process of HIV-1 infection than the monomeric version.
As illustrated in the foregoing Examples, the polypeptide of the present invention inhibited HIV-1.sub.IIIB replication, HIV-1-mediated cytopathogenesis and cell fusion. Moreover, it inhibited the replication of HIV-1 in a dose-dependent manner. Its 50% effective dose (ED.sub.50) for inhibition of HIV-1 replication was 101.0 nM. Its 50% effective dose for inhibition of cytopathogenesis mediated by HIV-1 was 142.0 nM. Its ED.sub.50 for inhibition of cell fusion mediated by HIV-1.sub.IIIB infected H9 cells was 160.0 nM. Its effective concentration is lower than that of most antiviral agents tested for inhibition of HIV-1 infection, such as aurintricarboxylic acid (ATA), and hemin. This agent had very low cytotoxicity. The 50% cytotoxic dose (CD.sub.50), determined by a colorimetric assay, is larger than 11,145 nM. Therefore, its selective index (S.I.=CD.sub.50 /ED.sub.50) is more than 110.0 for inhibition of HIV-1.sub.IIIB replication, 78.0 for inhibition of HIV-1.sub.IIIB -mediated cytopathogenesis and 70.0 for inhibition of HIV-1.sub.IIIB -mediated cell fusion.
Additionally, the peptide of the present invention inhibited the replication of not only the homologous HIV-1 isolate (IIIB), but also heterologous HIV-1 isolates tested (MN, RF, and SF2). It also effectively inhibited infection by an AZT-resistant mutant which could not be effectively neutralized by AZT. The viral inhibitory activity of the peptide was blocked by rabbit antiserum directed against this synthetic peptide, suggesting that the virus-inhibitory activity is specifically mediated by the peptide (637-666) and not by other factors which may have contaminated the peptide preparation during peptide synthesis. The peptide of the present invention can dimerize, thereby enhancing its activity against HIV-1 infections.
Consequently, the polypeptide of the present invention is an ideal candidate for an antiviral agent against HIV-1, since it has a higher antiviral activity against many HIV-1 isolates than most other anti-HIV agents. The toxicity of this peptide for lymphocytes and monocytic cells is very low, suggesting that any adverse effects on human subjects may be also lower than that of currently used anti-HIV drugs, such as AZT.
AS a result, the polypeptide of the present invention provides for a method of treating HIV-1 infected subjects in addition to inhibiting viral replication in vitro. In particular, the method includes administering the polypeptide of the present invention in an amount effective to inhibit viral replication, and preferably in an amount effective to inhibit HIV-1-mediated cytopathogenesis and cell fusion. Most preferably, the amount of polypeptide administered to the subject should be sufficient to maintain an effective blood serum concentration ranging from about 0,005 mg/ml-0.01 mg/ml.
Suitable methods of administration would include any pharmacologically acceptable approaches known in the art for delivering a therapeutic or prophylactic agent, such as oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal, parenteral (including intramuscular, sub-cutaneous and intravenous), as well as approaches utilizing inhalation. Additionally, the polypeptide can be incorporated into an antiviral substance containing other active ingredients, such as AZT.
While there have been described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that changes and modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modification as fall within the true scope of the invention.
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 20
(2) INFORMATION FOR SEQ ID NO: 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Amino Acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
GluTrpAspArgGluIleAs nAsnTyrThrSerLeuIleHis
SerLeuIleGluGluSerGlnAsnGlnGlnGluLysAsnGluGlnGlu
GluTrpGluLysLeuValAspAsnTyrThrHisIleIle TyrThrLeu
LeuAspAspAlaGlnThrGlnGlnGlyIleAsnGlnLysAsp
GluTrpAspArgGluIleSerAsnTyrThrAsnThrIlePheArgLeu
ValGluAsnGlyGlnIleGlnGlnAspLysAsnGluArgGlu
GluTrpAspArgGluIleGluAsnTyrThrGlyValIleTrpAsnLeu
MetGluLysThrGlnSerGlnGlnAsnLysAsnGluLeuGlu
GluTrpAspArgGluIleGlnAsnTyrThrGlnLysIleHisGlyLeu
IleGluGlnProGlnAsnGlnGlnGlnLysAsnGluGlnGlu
GluTrpAspArgGluIleLysAsnTyrThrAspLeuIleHisAspLeu
IleGluArgSerGlnAsnG lnGlnLysLysAsnGluGlnGlu
GluTrpAspArgGluIleArgAsnTyrThrGluLeuIleHisGluLeu
IleGluHisSerGlnAsnGlnGlnArgLysAsnGluGln Glu
GluTrpAspArgGluI leHisAsnTyrThrLysLeuIleHisGlnLeu
IleGluSerSerGlnAsnGlnGlnHisLysAsnGluGlnGlu
GluTrpAspArgGluIleThrAsnTyrThrArgLeu IleHisLysLeu
IleGluThrSerGlnAsnGlnGlnSerLysAsnGluGlnGlu
(2) INFORMATION FOR SEQ ID NO: 10 :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 :
GluTrpAspArgGluIleAsnAsnTyrThrThrLeuIleHisHisLeu
IleGluGluSerGlnAsnGlnGlnThrLysAsnGluGlnGlu
GluTrpAspArgGluIleAsnAsnTyrThrSerLeuIleHisSerLeu
IleGluGluSerGlnAsnGlnGlnGluLysAsnGluGlnGluGlyGly
GluTrpGluLysLeuValAspAsnTyrThrHisIleIleTyrThrLeu
LeuAspAspAlaGlnThrGlnGlnGlyIleAsnGlnLysAspGlyGly
( i) SEQUENCE CHARACTERISTICS:
ValGluAsnGlyGlnIleGlnGlnAspLysAsnGluArgGluGlyGly
MetGluLysThrGlnSerGlnGlnAsnLysAsnGluLeuGluGlyGly
IleGluGlnProGlnAsnGlnGlnGlnLysAsnGluGlnGluGlyGly
IleGluArgSerGlnAsnGlnGlnLysLysAsnGluGlnGluGlyGly
IleGluHisSerGlnAsnGlnGlnArgLysAsnGluGlnGluGlyGly
GluTrpAspArgGluIleHisAsnTyrThrLysLeuIleHisGlnLeu
IleGluSerSerGlnAsnGlnGlnHisLysAsnGluGlnGluGlyGly
(A ) LENGTH: 33 Amino Acids
GluTrpAspArgGluIleThrAsnTyrThrArgLeuIleHisLysLeu
IleGluThrSerGlnAsnGlnGlnSerLysAsnGluGlnGluGlyGly
IleGluGluSerGlnAsnGlnGlnThrLysAsnGluGlnGluGlyGly
1. A prophylactic method of topically inhibiting HIV-1 infectivity or replication, comprising:
topically administering to a subject an effective amount of a prophylactic antiviral substance, wherein said prophylactic antiviral substance includes:
an HIV-1-based polypeptide having an amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB or a natural variant thereof from amino acid 600 to amino acid 862, or a portion thereof containing the sequence from amino acid residue 637 to amino acid residue 666.
2. The prophylactic method of claim 1, wherein said polypeptide is attached to a carrier molecule or linking moiety capable of forming dimers of said polypeptide.
3. The prophylactic method of claim 2, wherein said polypeptide is included in dimeric form.
4. The prophylactic method of claim 1, wherein said polypeptide further comprises a Gly-Gly-Cys amino acid triplet coupled at the C-terminus of the polypeptide.
5. The prophylactic method of claim 4, wherein said polypeptide is included in a dimeric form.
6. The prophylactic method of claim 1, wherein said polypeptide has an amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB is or a natural variant thereof from amino acid residue 620 to amino acid residue 680, or a portion thereof containing the sequence from amino acid residue 637 to amino acid residue 666.
7. The prophylactic method of claim 1, wherein said polypeptide has an amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB or a natural variant thereof from amino acid residue 637 to amino acid residue 666.
8. The prophylactic method of claim 1, wherein said polypeptide is selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
9. The prophylactic method of claim 1, wherein said prophylactic antiviral substance further comprises a pharmacologically acceptable carrier.
10. The prophylactic method of claim 1, wherein said prophylactic antiviral substance further comprises another pharmacologically acceptable active ingredient.
11. An antiviral substance, comprising an HIV-1-based polypeptide having an amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB or a natural variant thereof from amino acid 600 to amino acid 862, or a portion thereof containing amino acid residue 637 to amino acid residue 666.
12. The antiviral substance of claim 11, wherein said polypeptide is attached to a carrier molecule or linking moiety capable of forming dimers of said polypeptide.
13. The antiviral substance of claim 12, wherein said polypeptide is included in dimeric form.
14. The antiviral substance of claim 11, wherein said polypeptide further comprises a Gly-Gly-Cys amino acid triplet coupled at the C-terminus of the polypeptide.
15. The antiviral substance of claim 14, wherein said polypeptide is included in a dimeric form.
16. The antiviral substance of claim 11, wherein said polypeptide has an amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB or a natural variant thereof from amino acid residue 620 to amino acid residue 680, or a portion thereof containing amino acid residue 637 to amino acid residue 666.
17. The antiviral substance of claim 11, wherein said polypeptide has an amino acid sequence of the envelope glycoprotein of HIV-1.sub.IIIB is or a natural variant thereof from amino acid residue 637 to amino acid residue 666.
18. The antiviral substance of claim 11, wherein said polypeptide is selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
19. The antiviral substance of claim 11, wherein said prophylactic antiviral substance further comprises a pharmacologically acceptable carrier.
20. The antiviral substance of claim 11, wherein said prophylactic antiviral substance further comprises another pharmacologically acceptable active ingredient.
0330359A2 August 1989 EPX
0335134A2 October 1989 EPX
PCT/US87/01294 December 1987 WOX
Ratner, et al., "Complete Nucleotide Sequence of the AIDS Virus", Nature, vol. 313, pp. 277-284, (Jan. 24, 1985). Neurath, et al., "B Cell Epitope Mapping of Human Immunodeficiency Virus Envelope Glycoproteins with Long (19- to 36-) Synthetic Peptides", Journal of General Virology, 71, pp. 85-95 (1990). Neurath et al., Virology, vol. 188, pp. 1-13, 1992.
Assignee: New York Blood Center (New York, NY)
Inventors: Shibo Jiang (New York, NY), Kang Lin (New York, NY), A. Robert Neurath (New York, NY)
Primary Examiner: Jill Warden
Assistant Examiner: A. M. Davenport
Law Firm: Hoffmann & Baron
Current U.S. Class: 514/12; Peptides Of 3 To 100 Amino Acid Residues (530/300); 25 Or More Amino Acid Residues In Defined Sequence (530/324); Proteins, I.e., More Than 100 Amino Acid Residues (530/350)
International Classification: A61K 3800; C07K 500; C07K 700; C07K 1700;


text:
Starbucks makes special delivery to ensure the future of coffee
By Raina Lang
Tags: CAFE practices coffee coffee trees farmers Guatemala One Tree for Every Bag Starbucks Sustainable Coffee Challenge
Editor's note: September 29 marks National Coffee Day in the U.S. Throughout September, Human Nature is publishing a series of reports on the Sustainable Coffee Challenge, a coalition working to make coffee the world's first sustainable agricultural product. This post is the second in the series.
This story follows Conservation International's (CI) director of sustainable coffee markets, Raina Lang, to Guatemala, with Mattea Fleischner, manager on Starbucks' global social impact team. They were in the country to see how coffee trees are grown and delivered to farmers as part of the "One Tree for Every Bag" commitment, which has raised enough funds to plant more than 30 million new coffee trees. The commitment is part of a nearly 20-year partnership between CI and Starbucks.
As we approached the Huehuetenango nursery, crossing a one-lane bridge suspended over the Valparaiso River, I realized just how complex coffee tree deliveries could be. This year, the nursery is supplying half a million seedlings to farmers in the region as part of Starbucks commitment. As a partner in this effort, CI works with Starbucks and ECOM, the administrator of the nurseries, to ensure that healthy, high-quality coffee leaf-rust-resistant trees are distributed and that farmers understand and respect key environmental and social safeguards associated with the program.
I was in Guatemala to observe the deliveries of coffee trees to C.A.F.E. Practice farmers — those who comply with a set of social, environmental and economic best practices defined as requirements to enter the Starbucks supply chain. I also visited a few farms to see where and how trees were being planted. Tracking how nurseries deliver rust-resistant coffee plants to farmers — and monitoring the quality of the trees they're delivering — is one critical step in monitoring designed to ensure healthy, sustainable coffee farms and thriving farmers.
Coffee farmers rely on productive and resilient trees to maintain their place as growers in a competitive market — and to sustain their livelihoods. Due to threats such as aging trees, climate change and significant pest and disease outbreaks in recent decades, farmers in many places are in desperate need of support. According to a 2015 study, there is a need to replant an estimated 22,000 square kilometers (13,600 square miles) globally, which translates to roughly 7 billion-10 billion coffee trees. To address this need — and build on the success of the One Tree for Every Bag program — Starbucks has committed to quadruple its commitment by providing 100 million healthy coffee trees to farmers by 2025.
This particular nursery in La Libertad — one of 12 nurseries across Guatemala, El Salvador and Mexico servicing the program — has the capacity to distribute 10,000 trees a day. But there's a challenge: The bridge connecting trees to farmers can only be used by lightweight vehicles. When the river swells during rainy season, larger trucks that could transport greater quantities of trees can't make it to the nursery, resulting in a dance of pick-up trucks entering and exiting the nursery.
When we made it to the nursery at 7 a.m., there were already two trucks waiting to be loaded. Nursery workers move the trees to the truck bed using a plastic crate, fitting roughly 700 to 1,000 coffee trees into the truck bed. The whole loading process took around 45 minutes per truck. To ensure that the trees are accounted for and tracked, there is an intricate process in place to document and record the quantity of trees, license plate, driver and date in a central registrar. Using this method, Starbucks and their local suppliers can account for the nearly 21 million coffee trees that have been distributed to C.A.F.E. Practices farmers since 2016.
That afternoon — just prior to a tropical downpour common in the tropics during rainy season — we visited a farm that had received seedlings from the program. Gustavo Alfaro is a fourth-generation farmer whose property was hit by coffee leaf rust several years back, just when he was taking it over from his father. Since taking ownership, he has made a concerted approach to increase shade cover in and around the coffee area. The trees and native vegetation in the zone regulate the climate across the farm, he explained, which can help mitigate future rust outbreaks. As we chatted, each newly delivered seedling was carried carefully to the area using a wooden backpack, then planted under a canopy of shade.
As we stood under the conacaste trees watching the seedlings being planted, we could hear the distinct calls of a tinamu chico, a flightless bird that roams the coffee fields in this region. In the face of climate change, those healthy, disease-resistant seedlings help Gustavo further build resilience on his farm.
But what if we could do more to help farmers like Gustavo adapt to a changing climate? Dozens of organizations in the Sustainable Coffee Challenge — including Starbucks — have joined forces to accelerate the responsible renovation and rehabilitation of coffee farms, committing to provide 1 billion healthy and productive trees worldwide. Together, the group is working to increase collective investment to ensure a healthy future for coffee and to make it possible for every coffee farmer to make renovation and rehabilition a regular part of doing business.
Learn more about the Challenge and the commitments of partner organizations »
Raina Lang is CI's director of sustainable coffee markets.
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Justia Company Company Announcements New Justia Web Site Enables Consumers to Easily Track Auto Recalls
New Justia Web Site Enables Consumers to Easily Track Auto Recalls
Auto Recalls Center Provides RSS Feeds for Recalls of Every Auto, Make, Model and Year
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acquired in the apical five-chamber, right parasternal long axis, or suprasternal views to obtain the highest velocities across the valve. The maximum velocity obtained is highly dependent on accurate transducer positioning, and will be underestimated when Doppler beam angle deviates from that of blood flowing through the stenotic valve (Fig. 8). A nonimaging probe can be used if inadequate envelopes are found with the duplex imaging transducer.
In most cases, the modified Bernoulli equation is an accurate and simple measure of the peak instantaneous transaortic pressure gradient.
Peak transaortic gradient measured by Doppler should be differentiated from the peak-to-peak gradient obtained at cardiac catheterization. Hemodynamically, the peak-to-peak gradient measures the difference between the peak left ventricular pressure and the peak aortic pressure—which are not measured simultaneously. Therefore, peak-to-peak gradients are not physiological, and no Doppler measurement exactly corresponds to this measurement in the cathetherization laboratory. However, mean gradients across the aortic valve are similar when measured by Doppler and by cardiac catheterization.
Fig. 7. M-mode through aortic valve showing characteristic thickening of valve leaflets in aortic stenosis (compare Chapter 3, Fig. 14A).