Close-to-native ultrastructural preservation by high pressure freezing

The objective of modern transmission electron microscopy (TEM) in life science is to observe biological structures in a state as close as possible to the living organism. TEM samples have to be thin and to be examined in vacuum; therefore only solid samples can be investigated. The most common and popular way to prepare samples for TEM is to subject them to chemical fixation, staining, dehydration, and embedding in a resin (all of these steps introduce considerable artifacts) before investigation. An alternative is to immobilize samples by cooling. High pressure freezing is so far the only approach to vitrify (water solidification without ice crystal formation) bulk biological samples of about 200 micrometer thick. This method leads to an improved ultrastructural preservation. After high pressure freezing, samples have to be subjected to follow-up procedure, such as freeze-substitution and embedding. The samples can also be sectioned into frozen hydrated sections and analyzed in a cryo-TEM. Also for immunocytochemistry, high pressure freezing is a good and practicable way.

Shaping a Bright Future for Montana Tech: Developing State, National and Global Leaders in Resource Sectors

Presentation by Leigh W. Freeman. Rachel Carson's book Silent Spring published in 1962 marked the birth of concepts leading to the development of mineral resources with a balance of economic, environmental and social imperatives. Montana plays a special role in this story. It is 'the last best place.' As such it can serve as a bell-weather state for Carson's revelations. Consider: Butte as a poster child for legacy problems resulting from resource development under solely economic imperatives; and Montana as a first-adapter in 1971 of state environmental laws based on imperatives heralded with Silent Spring. What better place is there to educate and develop leaders and incubate the future of resource development in global sustainability?

Discrimination in Metropolitan Housing Markets: Phase 3 - Native Americans

This paper documents the results of a pilot paired testing program to examine the treatment of Native Americans by real estate agents in rental housing markets in three states and owner-occupied housing markets in one state. The study finds that the level of discrimination experienced by Native Americans in rental markets exceed those experienced by Hispanics, Blacks, and Asian-Americans.

50 Years Ago We Marched: What began on the grounds of Lincoln University, ended on the steps of the Alabama State Capitol

LINCOLN UNIVERSITY - On March 25, 1965, a bus loaded with Lincoln University students and staff arrived in Montgomery, Ala. to join the Selma march for racial and voting equality. Although the Civil Rights Act of 1964 was in force, African-Americans continued to feel the effects of segregation. The 1960s was a decade of social unrest and change. In the Deep South, specifically Alabama, racial segregation was a cultural norm resistant to change. Governor George Wallace never concealed his personal viewpoints and political stance of the white majority, declaring “Segregation now, segregation tomorrow, segregation forever.” The march was aimed at obtaining African-Americans their constitutionally protected right to vote. However, Alabama’s deep-rooted culture of racial bias began to be challenged by a shift in American attitudes towards equality. Both black and whites wanted to end discrimination by using passive resistance, a movement utilized by Dr. Martin Luther King Jr. That passive resistance was often met with violence, sometimes at the hands of law enforcement and local citizens. The Selma to Montgomery march was a result of a protest for voting equality. The Student Nonviolent Coordinating Committee (SNCC) and the Southern Christian Leadership Counsel (SCLC) among other students marched along the streets to bring awareness to the voter registration campaign, which was organized to end discrimination in voting based on race. Violent acts of police officers and others were some of the everyday challenges protesters were facing. Forty-one participants from Lincoln University arrived in Montgomery to take part in the 1965 march for equality. Students from Lincoln University’s Journalism 383 class spent part of their 2015 spring semester researching the historical event. Here are their stories: Peter Kellogg “We’ve been watching the television, reading about it in the newspapers,” said Peter Kellogg during a February 2015 telephone interview. “Everyone knew the civil rights movement was going on, and it was important that we give him (Robert Newton) some assistance … and Newton said we needed to get involve and do something,” Kellogg, a lecturer in the 1960s at Lincoln University, discussed how the bus trip originated. “That’s why the bus happened,” Kellogg said. “Because of what he (Newton) did - that’s why Lincoln students went and participated.” “People were excited and the people along the sidewalk were supportive,” Kellogg said. However, the mood flipped from excited to scared and feeling intimidated. “It seems though every office building there was a guy in a blue uniform with binoculars standing in the crowd with troops and police. And if looks could kill me, we could have all been dead.” He says the hatred and intimidation was intense. Kellogg, being white, was an immediate target among many white people. He didn’t realize how dangerous the event in Alabama was until he and the others in the bus heard about the death of Viola Liuzzo. The married mother of five from Detroit was shot and killed by members of the Ku Klux Klan while shuttling activists to the Montgomery airport. “We found out about her death on the ride back,” Kellogg recalled. “Because it was a loss of life, and it shows the violence … we could have been exposed to that danger!” After returning to LU, Kellogg’s outlook on life took a dramatic turn. Kellogg noted King’s belief that a person should be willing to die for important causes. “The idea is that life is about something larger and more important than your own immediate gratification, and career success or personal achievements,” Kellogg said. “The civil rights movement … it made me, it made my life more significant because it was about something important.” The civil rights movement influenced Kellogg to change his career path and to become a black history lecturer. Until this day, he has no regrets and believes that his choices made him as a better individual. The bus ride to Alabama, he says, began with the actions of just one student. Robert Newton Robert Newton was the initiator, recruiter and leader of the Lincoln University movement to join Dr. Martin Luther King’s march in Selma. “In the 60s much of the civil rights activists came out of college,” said Newton during a recent phone interview. Many of the events that involved segregation compelled college students to fight for equality. “We had selected boycotts of merchants, when blacks were not allowed to try on clothes,” Newton said. “You could buy clothes at department stores, but no blacks could work at the department stores as sales people. If you bought clothes there you couldn’t try them on, you had to buy them first and take them home and try them on.” Newton said the students risked their lives to be a part of history and influence change. He not only recognized the historic event of his fellow Lincolnites, but also recognized other college students and historical black colleges and universities who played a vital role in history. “You had the S.N.C.C organization, in terms of voting rights and other things, including a lot of participation and working off the bureau,” Newton said. Other schools and places such as UNT, Greenville and Howard University and other historically black schools had groups that came out as leaders. Newton believes that much has changed from 50 years ago. “I think we’ve certainly come a long way from what I’ve seen from the standpoint of growing up outside of Birmingham, Alabama,” Newton said. He believes that college campuses today are more organized in their approach to social causes. “The campus appears to be some more integrated amongst students in terms of organizations and friendships.” Barbara Flint Dr. Barbara Flint grew up in the southern part of Arkansas and came to Lincoln University in 1961. She describes her experience at Lincoln as “being at Lincoln when the world was changing.“ She was an active member of Lincoln’s History Club, which focused on current events and issues and influenced her decision to join the Selma march. “The first idea was to raise some money and then we started talking about ‘why can’t we go?’ I very much wanted to be a living witness in history.” Reflecting on the march and journey to Montgomery, Flint describes it as being filled with tension. “We were very conscious of the fact that once we got on the road past Tennessee we didn’t know what was going to happen,” said Flint during a February 2015 phone interview. “Many of the students had not been beyond Missouri, so they didn’t have that sense of what happens in the South. Having lived there you knew the balance as well as what is likely to happen and what is not likely to happen. As my father use to say, ‘you have to know how to stay on that line of balance.’” Upon arriving in Alabama she remembers the feeling of excitement and relief from everyone on the bus. “We were tired and very happy to be there and we were trying to figure out where we were going to join and get into the march,” Flint said. “There were so many people coming in and then we were also trying to stay together; that was one of the things that really stuck out for me, not just for us but the people who were coming in. You didn’t want to lose sight of the people you came with.” Flint says she was keenly aware of her surroundings. For her, it was more than just marching forward. “I can still hear those helicopters now,” Flint recalled. “Every time the helicopters would come over the sound would make people jump and look up - I think that demonstrated the extent of the tenseness that was there at the time because the helicopters kept coming over every few minutes.” She said that the marchers sang “we are not afraid,” but that fear remained with every step. “Just having been there and being a witness and marching you realize that I’m one of those drops that’s going to make up this flood and with this flood things will move,” said Flint. As a student at Lincoln in 1965, Flint says the Selma experience undoubtedly changed her life. “You can’t expect to do exactly what you came to Lincoln to do,” Flint says. “That march - along with all the other marchers and the action that was taking place - directly changed the paths that I and many other people at Lincoln would take.” She says current students and new generations need to reflect on their personal role in society. “Decide what needs to be done and ask yourself ‘how can I best contribute to it?’” Flint said. She notes technology and social media can be used to reach audiences in ways unavailable to her generation in 1965. “So you don’t always have to wait for someone else to step out there and say ‘let’s march,’ you can express your vision and your views and you have the means to do so (so) others can follow you. Jaci Newsom Jaci Newsom came to Lincoln in 1965 from Atlanta. She came to Lincoln to major in sociology and being in Jefferson City was largely different from what she had grown up with. “To be able to come into a restaurant, sit down and be served a nice meal was eye-opening to me,” said Newsom during a recent interview. She eventually became accustomed to the relaxed attitude of Missouri and was shocked by the situation she encountered on an out-of-town trip. “I took a bus trip from Atlanta to Pensacola and I encountered the worse racism that I have ever seen. I was at bus stop, I went in to be served and they would not serve me. There was a policeman sitting there at the table and he told me that privately owned places could select not to serve you.” Newsom describes her experience of marching in Montgomery as being one with a purpose. “We felt as though we achieved something - we felt a sense of unity,” Newsom said. “We were very excited (because) we were going to hear from Martin Luther King. To actually be in the presence of him and the other civil rights workers there was just such enthusiasm and excitement yet there was also some apprehension of what we might encounter.” Many of the marchers showed their inspiration and determination while pressing forward towards the grounds of the Alabama Capitol building. Newsom recalled that the marchers were singing the lyrics “ain’t gonna let nobody turn me around” and “we shall overcome.” “ I started seeing people just like me,” Newsom said. “I don’t recall any of the scowling, the hitting, the things I would see on TV later. I just saw a sea of humanity marching towards the Capitol. I don’t remember what Martin Luther King said but it was always the same message: keep the faith; we’re going to get where we’re going and let us remember what our purpose is.” Newsom offers advice on what individuals can do to make their society a more productive and peaceful place. “We have come a long way and we have ways to change things that we did not have before,” Newsom said. “You need to work in positive ways to change.” Referencing the recent unrest in Ferguson, Mo., she believes that people become destructive as a way to show and vent anger. Her generation, she says, was raised to react in lawful ways – and believe in hope. “We have faith to do things in a way that was lawful and it makes me sad what people do when they feel without hope, and there is hope,” Newsom says. “Non-violence does work - we need to include everyone to make this world a better place.” Newsom graduated from Lincoln in 1969 and describes her experience at Lincoln as, “I grew up and did more growing at Lincoln than I think I did for the rest of my life.”

2.6 Judge William F. Frank, Chief Justice, Missouri State Supreme Court

In February, 1937, the Court, in an opinion by Chief Justice William F. Frank, issued their verdict that supported the Missouri statute that allowed for out-of-state tuition for Missouri blacks to continue their education if the desired program was not offered at Lincoln University Once again, the legal team of Lloyd Gaines was not undaunted.

2.5 Missouri State Supreme Court Building, Jefferson City, Missouri

On December 12, 1936, Charles Hamilton Houston was in Jefferson City, Missouri arguing Gaines’ appeal in front of the Missouri Supreme Court. Houston added some teeth to his argument by adding that neither “the slender hope” that Gaines may someday attend a new law program at Lincoln nor the provision of tuition scholarships to attend an out-of-state law school met the US Constitution’s requirement of equal treatment regardless of race.

1.4 Lloyd C. Stark, 39th Governor of State of Missouri, 1937-1941

After weeks of debate Taylor’s proposal, House Bill No. 195 was passed and signed by Governor Lloyd Stark.

3.5 The Missouri State Supreme Court Conferred a Honorary Law License for Lloyd Gaines.

On September 28th, 2006, the Missouri State Supreme Court conferred a posthumous law license for Mr. Gaines.

Working to Form a More Perfect Union: Honoring Women in Public Service in the State of Missouri

https://bluetigercommons.lincolnu.edu/pli/1010/thumbnail.jpg

Working to Form a More Perfect Union: Honoring Women in Public Service in the State of Missouri

https://bluetigercommons.lincolnu.edu/pli/1009/thumbnail.jpg

In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen

T cells recognizing poorly displayed self determinants escape tolerance mechanisms and persist in the adult repertoire. The process by which these T cells are primed is not clear, but once activated, they can cause autoimmunity. Here, we show that dendritic cells treated with interleukin 6 (IL-6) process and present determinants from a model native antigen in a qualitatively altered hierarchy, activating T cells in vitro and in vivo against determinants that were previously cryptic because of poor display. IL-6 does not induce conventional maturation of dendritic cells but alters the pH of peripheral, early endosomal compartments and renders the cells more susceptible to killing by chloroquine. Acidification of endosomes by ouabain mimics the effect of IL-6 and allows processing of the same cryptic determinant. These results suggest that cytokines such as IL-6 could initiate and help to propagate an autoimmune disease process by differentiating dendritic cells into a state distinct from that induced by normal maturation.

Influenza hemagglutinin is spring-loaded by a metastable native conformation

Enveloped viruses enter cells by protein-mediated membrane fusion. For influenza virus, membrane fusion is regulated by the conformational state of the hemagglutinin (HA) protein, which switches from a native (nonfusogenic) structure to a fusion-active (fusogenic) conformation when exposed to the acidic environment of the cellular endosome. Here we demonstrate that destabilization of HA at neutral pH, with either heat or the denaturant urea, triggers a conformational change that is biochemically indistinguishable from the change triggered by low pH. In each case, the conformational change is coincident with induction of membrane-fusion activity, providing strong evidence that the fusogenic structure is formed. These results indicate that the native structure of HA is trapped in a metastable state and that the fusogenic conformation is released by destabilization of native structure. This strategy may be shared by other enveloped viruses, including those that enter the cell at neutral pH, and could have implications for understanding the membrane-fusion step of HIV infection.

Transition-state structure as a unifying basis in protein-folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

A specific transition state for S-peptide combining with folded S-protein and then refolding

We measured the folding and unfolding kinetics of mutants for a simple protein folding reaction to characterize the structure of the transition state. Fluorescently labeled S-peptide analogues combine with S-protein to form ribonuclease S analogues: initially, S-peptide is disordered whereas S-protein is folded. The fluorescent probe provides a convenient spectroscopic probe for the reaction. The association rate constant, kon, and the dissociation rate constant, koff, were both determined for two sets of mutants. The dissociation rate constant is measured by adding an excess of unlabeled S-peptide analogue to a labeled complex (RNaseS*). This strategy allows kon and koff to be measured under identical conditions so that microscopic reversibility applies and the transition state is the same for unfolding and refolding. The first set of mutants tests the role of the α-helix in the transition state. Solvent-exposed residues Ala-6 and Gln-11 in the α-helix of native RNaseS were replaced by the helix destabilizing residues glycine or proline. A plot of log kon vs. log Kd for this series of mutants is linear over a very wide range, with a slope of −0.3, indicating that almost all of the molecules fold via a transition state involving the helix. A second set of mutants tests the role of side chains in the transition state. Three side chains were investigated: Phe-8, His-12, and Met-13, which are known to be important for binding S-peptide to S-protein and which also contribute strongly to the stability of RNaseS*. Only the side chain of Phe-8 contributes significantly, however, to the stability of the transition state. The results provide a remarkably clear description of a folding transition state.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.