Análise das principais dificuldades dos alunos de uma turma do 2.º ano do 1º CEB na construção e interpretação de gráficos

Relatório Final apresentado para a obtenção de grau de mestre em Educação pré-escolar e ensino do 1º ciclo do ensino básico

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.”

Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: A novel mechanism of growth hormone receptor regulation

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 ± 2.9% and 23.0 ± 2.7% of control (mean ± SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (P ≤ 0.0005), whereas GHR translocation was completely abolished by 75–90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

Streaming potential measurements in αβγ-rat epithelial Na+ channel in planar lipid bilayers

Streaming potentials across cloned epithelial Na+ channels (ENaC) incorporated into planar lipid bilayers were measured. We found that the establishment of an osmotic pressure gradient (Δπ) across a channel-containing membrane mimicked the activation effects of a hydrostatic pressure differential (ΔP) on αβγ-rENaC, although with a quantitative difference in the magnitude of the driving forces. Moreover, the imposition of a Δπ negates channel activation by ΔP when the Δπ was directed against ΔP. A streaming potential of 2.0 ± 0.7 mV was measured across αβγ-rat ENaC (rENaC)-containing bilayers at 100 mM symmetrical [Na+] in the presence of a 2 Osmol/kg sucrose gradient. Assuming single file movement of ions and water within the conduction pathway, we conclude that between two and three water molecules are translocated together with a single Na+ ion. A minimal effective pore diameter of 3 Å that could accommodate two water molecules even in single file is in contrast with the 2-Å diameter predicted from the selectivity properties of αβγ-rENaC. The fact that activation of αβγ-rENaC by ΔP can be reproduced by the imposition of Δπ suggests that water movement through the channel is also an important determinant of channel activity.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332 and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

Ex vivo evaluation of a Taylor-Couette flow, immobilized heparinase I device for clinical application

Efficient and safe heparin anticoagulation has remained a problem for continuous renal replacement therapies and intermittent hemodialysis for patients with acute renal failure. To make heparin therapy safer for the patient with acute renal failure at high risk of bleeding, we have proposed regional heparinization of the circuit via an immobilized heparinase I filter. This study tested a device based on Taylor-Couette flow and simultaneous separation/reaction for efficacy and safety of heparin removal in a sheep model. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. The device, referred to as a vortex flow plasmapheretic reactor, consisted of two concentric cylinders, a priming volume of 45 ml, a microporous membrane for plasma separation, and an outer compartment where the immobilized heparinase I was fluidized separately from the blood cells. Manual white cell and platelet counts, hematocrit, total protein, and fibrinogen assays were performed. Heparin levels were indirectly measured via whole-blood recalcification times (WBRTs). The vortex flow plasmapheretic reactor maintained significantly higher heparin levels in the extracorporeal circuit than in the sheep (device inlet WBRTs were 1.5 times the device outlet WBRTs) with no hemolysis. The reactor treatment did not effect any physiologically significant changes in complete blood cell counts, platelets, and protein levels for up to 2 hr of operation. Furthermore, gross necropsy and histopathology did not show any significant abnormalities in the kidney, liver, heart, brain, and spleen.

Cholecystokinin-8 protects central cholinergic neurons against fimbria–fornix lesion through the up-regulation of nerve growth factor synthesis

In this study, we demonstrate that cholecystokinin-8 (CCK-8) induces an increase in both nerve growth factor (NGF) protein and NGF mRNA in mouse cortex and hippocampus when i.p. injected at physiological doses. By using fimbria–fornix-lesioned mice, we have also demonstrated that repeated CCK-8 i.p. injections result in recovery of lesion-induced NGF deficit in septum and restore the baseline NGF levels in hippocampus and cortex. Parallel to the effects on NGF, CCK-8 increases choline acetyltransferase (Chat) activity in forebrain when injected in unlesioned mice and counteract the septo-hippocampal Chat alterations in fimbria–fornix-lesioned mice. To assess the NGF involvement in the mechanism by which CCK-8 induces brain Chat, NGF antibody was administrated intracerebrally to saline- and CCK-8-injected mice. We observe that pretreatment with NGF antibody causes a marked reduction of NGF and Chat activity in septum and hippocampus of both saline- and CCK-8-injected mice. This evidence indicates that the CCK-8 effects on cholinergic cells are mediated through the synthesis and release of NGF. Taken together, our results suggest that peripheral administration of CCK-8 may represent a potential experimental model for investigating the effects of endogenous NGF up-regulation on diseases associated with altered brain cholinergic functions.

Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel

The epothilones are naturally occurring, cytotoxic macrolides that function through a paclitaxel (Taxol)-like mechanism. Although structurally dissimilar, both classes of molecules lead to the arrest of cell division and eventual cell death by stabilizing cellular microtubule assemblies. The epothilones differ in their ability to retain activity against multidrug-resistant (MDR) cell lines and tumors where paclitaxel fails. In the current account, we focus on the relationship between epothilone and paclitaxel in the context of tumors with multiple drug resistance. The epothilone analogue Z-12,13-desoxyepothilone B (dEpoB) is >35,000-fold more potent than paclitaxel in inhibiting cell growth in the MDR DC-3F/ADX cell line. Various formulations, routes, and schedules of i.v. administration of dEpoB have been tested in nude mice. Slow infusion with a Cremophor-ethanol vehicle proved to be the most beneficial in increasing efficacy and decreasing toxicity. Although dEpoB performed similarly to paclitaxel in sensitive tumors xenografts (MX-1 human mammary and HT-29 colon tumor), its effects were clearly superior against MDR tumors. When dEpoB was administered to nude mice bearing our MDR human lymphoblastic T cell leukemia (CCRF-CEM/paclitaxel), dEpoB demonstrated a full curative effect. For human mammary adenocarcinoma MCF-7/Adr cells refractory to paclitaxel, dEpoB reduced the established tumors, markedly suppressed tumor growth, and surpassed other commonly used chemotherapy drugs such as adriamycin, vinblastine, and etoposide in beneficial effects.

Rapid Up-Regulation of HKT1, a High-Affinity Potassium Transporter Gene, in Roots of Barley and Wheat following Withdrawal of Potassium1

High-affinity K+ uptake in plant roots is rapidly up-regulated when K+ is withheld and down-regulated when K+ is resupplied. These processes make important contributions to plant K+ homeostasis. A cDNA coding for a high-affinity K+ transporter, HKT1, was earlier cloned from wheat (Triticum aestivum L.) roots and functionally characterized. We demonstrate here that in both barley (Hordeum vulgare L.) and wheat roots, a rapid and large up-regulation of HKT1 mRNA levels resulted when K+ was withdrawn from growth media. This effect was specific for K+; withholding N caused a modest reduction of HKT1 mRNA levels. Up-regulation of HKT1 transcript levels in barley roots occurred within 4 h of removing K+, which corresponds to the documented increase of high-affinity K+ uptake in roots following removal of K+. Increased expression of HKT1 mRNA was evident before a decline in total root K+ concentration could be detected. Resupply of 1 mm K+ was sufficient to strongly reduce HKT1 transcript levels. In wheat root cortical cells, both membrane depolarizations in response to 100 μm K+, Cs+, and Rb+, and high-affinity K+ uptake were enhanced by K+ deprivation. Thus, in both plant systems the observed physiological changes associated with manipulating external K+ supply were correlated with levels of HKT1 mRNA expression. Implications of these findings for K+ sensing and regulation of the HKT1 mRNA levels in plant roots are discussed.

Vaccination with syngeneic, lymphoma-derived immunoglobulin idiotype combined with granulocyte/macrophage colony-stimulating factor primes mice for a protective T-cell response.

The idiotype of the Ig expressed by a B-cell malignancy (Id) can serve as a unique tumor-specific antigen and as a model for cancer vaccine development. In murine models of Id vaccination, formulation of syngeneic Id with carrier proteins or adjuvants induces an anti-idiotypic antibody response. However, inducing a potent cell-mediated response to this weak antigen instead would be highly desirable. In the 38C13 lymphoma model, we observed that low doses of free granulocyte/macrophage colony-stimulating factor (GM-CSF) 10,000 units i.p. or locally s.c. daily for 4 days significantly enhanced protective antitumor immunity induced by s.c. Id-keyhole limpet hemocyanin (KLH) immunization. This effect was critically dependent upon effector CD4+ and CD8+ T cells and was not associated with any increased anti-idiotypic antibody production. Lymphocytes from spleens and draining lymph nodes of mice primed with Id-KLH plus GM-CSF, but not with Id-KLH alone, demonstrated significant proliferation to Id in vitro without any biased production of interferon gamma or interleukin 4 protein or mRNA. As a further demonstration of potency, 50% of mice immunized with Id-KLH plus GM-CSF on the same day as challenge with a large s.c. tumor inoculum remained tumor-free at day 80, compared with 17% for Id-KLH alone, when immunization was combined with cyclophosphamide. Taken together, these results demonstrate that GM-CSF can significantly enhance the immunogenicity of a defined self-antigen and that this effect is mediated exclusively by activating the T-cell arm of the immune response.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.