Sterol-binding proteins in late endosomes : Regulation of endosome motility and lipid metabolism

Despite its bad reputation in the mass media, cholesterol is an indispensable constituent of cellular membranes and vertebrate life. It is, however, also potentially lethal as it may accumulate in the arterial intima causing atherosclerosis or elsewhere in the body due to inherited conditions. Studying cholesterol in cells, and research on how the cell biology of cholesterol affects on system level is essential for a better understanding of the disease states associated with cholesterol and for the development of new therapies for these conditions. On its way to the cell, exogenous cholesterol traverses through endosomes, transport vesicles involved in internalizing material to cells, and needs to be transported out of this compartment. This endosomal pool of cholesterol is important for understanding both the common disorders of metabolism and the more rare hereditary disorders of cholesterol metabolism. The study of cholesterol in cells has been hampered by the lack of bright fluorescent sterol analogs that would resemble cholesterol enough to be used in cellular studies. In the first study of my thesis, we present a new sterol analog, Boron-Dipyrromethene (BODIPY)-cholesterol for visualizing sterols in living cells and organism. This fluorescent cholesterol derivative is shown to behave similarly to cholesterol both by atomic scale computer simulations and biochemical experiments. We characterize its localization inside different types of living cells and show that it can be used to study sterol trafficking in living organisms. Two sterol binding proteins associated with the endosomal membrane; the Niemann-Pick type C disease protein 1 (NPC1) and the Oxysterol Binding Protein Related Protein 1 (ORP1) are the subjects of the rest of this study. Sensing cholesterol on endosomes, transporting lipids away from this compartment and the effects these lipids play on cellular metabolism are considered. In the second study we characterize how the NPC1 protein affects lipid metabolism. We show that this cholesterol binding protein affects synthesis of triglycerides and that genetic polymorphisms or a genetic defect in the NPC1 gene affect triglyceride on the whole body level. These effects take place via regulation of carbon fluxes to different lipid classes in cells. In the third part we characterize the effects of another endosomal sterol binding protein, ORP1L on the function and motility of endosomes. Specifically we elucidate how a mutation in the ability of ORP1L to bind sterols affects its behavior in cells, and how a change in ORP1L levels in cells affects the localization, degradative capacity and motility of endosomes. In addition we show that ORP1L manipulations affect cholesterol balance also in macrophages, a cell type important for the development of atherosclerosis.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

The Proto-Lucianic Problem in 1 Samuel

The Lucianic text of the Septuagint of the Historical Books witnessed primarily by the manuscript group L (19, 82, 93, 108, and 127) consists of at least two strata: the recensional elements, which date back to about 300 C.E., and the substratum under these recensional elements, the proto-Lucianic text. Some distinctive readings in L seem to be supported by witnesses that antedate the supposed time of the recension. These witnesses include the biblical quotations of Josephus, Hippolytus, Irenaeus, Tertullian, and Cyprian, and the Old Latin translation of the Septuagint. It has also been posited that some Lucianic readings might go back to Hebrew readings that are not found in the Masoretic text but appear in the Qumran biblical texts. This phenomenon constitutes the proto-Lucianic problem. In chapter 1 the proto-Lucianic problem and its research history are introduced. Josephus references to 1 Samuel are analyzed in chapter 2. His agreements with L are few and are mostly only apparent or, at best, coincidental. In chapters 3 6 the quotations by four early Church Fathers are analyzed. Hippolytus Septuagint text is extremely hard to establish since his quotations from 1 Samuel have only been preserved in Armenian and Georgian translations. Most of the suggested agreements between Hippolytus and L are only apparent or coincidental. Irenaeus is the most trustworthy textual witness of the four early Church Fathers. His quotations from 1 Samuel agree with L several times against codex Vaticanus (B) and all or most of the other witnesses in preserving the original text. Tertullian and Cyprian agree with L in attesting some Hebraizing approximations that do not seem to be of Hexaplaric origin. The question is more likely of early Hebraizing readings of the same tradition as the kaige recension. In chapter 7 it is noted that Origen, although a pre-Lucianic Father, does not qualify as a proto-Lucianic witness. General observations about the Old Latin witnesses as well as an analysis of the manuscript La115 are given in chapter 8. In chapter 9 the theory of the proto-Lucianic recension is discussed. In order to demonstrate the existence of the proto-Lucianic recension one should find instances of indisputable agreement between the Qumran biblical manuscripts and L in readings that are secondary in Greek. No such case can be found in the Qumran material in 1 Samuel. In the text-historical conclusions (chapter 10) it is noted that of all the suggested proto-Lucianic agreements in 1 Samuel (about 75 plus 70 in La115) more than half are only apparent or, at best, coincidental. Of the indisputable agreements, however, 26 are agreements in the original reading. In about 20 instances the agreement is in a secondary reading. These agreements are early variants; mostly minor changes that happen all the time in the course of transmission. Four of the agreements, however, are in a pre-Hexaplaric Hebraizing approximation that has found its way independently into the pre-Lucianic witnesses and the Lucianic recension. The study aims at demonstrating the value of the Lucianic text as a textual witness: under the recensional layer(s) there is an ancient text that preserves very old, even original readings which have not been preserved in B and most of the other witnesses. The study also confirms the value of the early Church Fathers as textual witnesses.

Dynamic changes in mitogen-activated protein kinase (MAPK) activities in the corpus luteum of the bonnet monkey (Macaca radiata) during development, induced luteolysis, and simulated early pregnancy: A role for p38 MAPK in the regulation of luteal function

Changes in MAPK activities were examined in the corpus luteum (CL) during luteolysis and pregnancy, employing GnRH antagonist (Cetrorelix)-induced luteolysis, stages of CL, and hCG treatment to mimic early pregnancy as model systems in the bonnet monkey. We hypothesized that MAPKs could serve to phosphorylate critical phosphoproteins to regulate luteal function. Analysis of several indices for structural (caspase-3 activity and DNA fragmentation) and functional (progesterone and steroidogenic acute regulatory protein expression) changes in the CL revealed that the decreased luteal function observed during Cetrorelix treatment and late luteal phase was associated with increased caspase-3 activity and DNA fragmentation. As expected, human chorionic gonadotropin treatment dramatically increased luteal function, but the indices for structural changes were only partially attenuated. All three MAPKs appeared to be constitutively active in the mid-luteal-phase CL, and activities of ERK-1/2 and p38-MAPK (p38), but not Jun N-terminal kinase (JNK)-1/2, decreased significantly (P < 0.05) within 12 - 24 h after Cetrorelix treatment. During the late luteal phase, in contrast to decreased ERK-1/2 and p38 activities, JNK-1/2 activities increased significantly (P < 0.05). Although human chorionic gonadotropin treatment increased ERK-1/2 and p38 activities, it decreased JNK-1/2 activities. The activation status of p38 was correlated with the phosphorylation status of an upstream activator, MAPK kinase-3/6 and the expression of MAPK activated protein kinase-3, a downstream target. Intraluteal administration of p38 kinase inhibitor (SB203580), but not MAPK kinase-1/2 inhibitor (PD98059), decreased the luteal function. Together, these data suggest an important role for p38 in the regulation of CL function in primates.

Hot gaseous coronae of early-type galaxies and their radio luminosity function

Recent X-ray observations have revealed that early-type galaxies (which usually produce extended double radio sources) generally have hot gaseous haloes extending up to approx102kpc1,2. Moreover, much of the cosmic X-ray background radiation is probably due to a hotter, but extremely tenuous, intergalactic medium (IGM)3. We have presented4–7 an analytical model for the propagation of relativistic beams from galactic nuclei, in which the beams' crossing of the pressure-matched interface between the IGM and the gaseous halo, plays an important role. The hotspots at the ends of the beams fade quickly when their advance becomes subsonic with respect to the IGM. This model has successfully predicted (for typical double radio sources) the observed8 current mean linear-size (approx2Dsime350 kpc)4,5, the observed8–11 decrease in linear-size with cosmological redshift4–6 and the slope of the linear-size versus radio luminosity10,12–14 relation6. We have also been able to predict the redshift-dependence of observed numbers and radio luminosities of giant radio galaxies7,15. Here, we extend this model to include the propagation of somewhat weaker beams. We show that the observed flattening of the local radio luminosity function (LRLF)16–20 for radio luminosity Papproximately 1024 W Hz-1 at 1 GHz can be explained without invoking ad hoc a corresponding break in the beam power function Phi(Lb), because the heads of the beams with Lb < 1025 W Hz-1 are decelerated to sonic velocity within the halo itself, which leads to a rapid decay of radio luminosity and a reduced contribution of these intrinsically weaker sources to the observed LRLF.

Pre-eclampsia : The role of soluble VEGF receptor-1 and related anti-angiogenic factors beyond

Pre-eclampsia is a pregnancy complication that affects about 5% of all pregnancies. It is known to be associated with alterations in angiogenesis -related factors, such as vascular endothelial growth factor (VEGF). An excess of antiangiogenic substances, especially the soluble receptor-1 of VEGF (sVEGFR-1), has been observed in maternal circulation after the onset of the disease, probably reflecting their increased placental production. Smoking reduces circulating concentrations of sVEGFR-1 in non-pregnant women, and in pregnant women it reduces the risk of pre-eclampsia. Soluble VEGFR-1 acts as a natural antagonist of VEGF and placental growth factor (PlGF) in human circulation, holding a promise for potential therapeutic use. In fact, it has been used as a model to generate a fusion protein, VEGF Trap , which has been found effective in anti-angiogenic treatment of certain tumors and ocular diseases. In the present study, we evaluated the potential use of maternal serum sVEGFR-1, Angiopoietin-2 (Ang-2) and endostatin, three central anti-angiogenic markers, in early prediction of subsequent pre-eclampsia. We also studied whether smoking affects circulating sVEGFR-1 concentrations in pregnant women or their first trimester placental secretion and expression in vitro. Last, in order to allow future discussion on the potential therapy based on sVEGFR-1, we determined the biological half-life of endogenous sVEGFR-1 in human circulation, and measured the concomitant changes in free VEGF concentrations. Blood or placental samples were collected from a total of 268 pregnant women between the years 2001 2007 in Helsinki University Central Hospital for the purposes above. The biomarkers were measured using commercially available enzyme-linked immunosorbent assays (ELISA). For the analyses of sVEGFR-1, Ang-2 and endostatin, a total of 3 240 pregnant women in the Helsinki area were admitted to blood sample collection during two routine ultrasoundscreening visits at 13.7 ± 0.5 (mean ± SD) and 19.2 ± 0.6 weeks of gestation. Of them, 49 women later developing pre-eclampsia were included in the study. Their disease was further classified as mild in 29 and severe in 20 patients. Isolated early-onset intrauterine growth retardation (IUGR) was diagnosed in 16 women with otherwise normal medical histories and uncomplicated pregnancies. Fifty-nine women remaining normotensive, non-proteinuric and finally giving birth to normal-weight infants were picked to serve as the control population of the study. Maternal serum concentrations of Ang-2, endostatin and sVEGFR-1, were increased already at 16 20 weeks of pregnancy, about 13 weeks before the clinical manifestation of preeclampsia. In addition, these biomarkers could be used to identify women at risk with a moderate precision. However, larger patient series are needed to determine whether these markers could be applied for clinical use to predict preeclampsia. Intrauterine growth retardation (IUGR), especially if noted at early stages of pregnancy and not secondary to any other pregnancy complication, has been suggested to be a form of preeclampsia compromising only the placental sufficiency and the fetus, but not affecting the maternal endothelium. In fact, IUGR and preeclampsia have been proposed to share a common vascular etiology in which factors regulating early placental angiogenesis are likely to play a central role. Thus, these factors have been suggested to be involved in the pathogenesis of IUGR. However, circulating sVEGFR-1, Ang-2 and endostatin concentrations were unaffected by subsequent IUGR at early second trimester. Furthermore, smoking was not associated with alterations in maternal circulating sVEGFR-1 or its placental production. The elimination of endogenous sVEGFR-1 after pregnancy was calculated from serial samples of eight pregnant women undergoing elective Caesarean section. As typical for proteins in human compartments, the elimination of sVEGFR-1 was biphasic, containing a rapid halflife of 3.4 h and a slow one of 29 h. The decline in sVEGFR-1 concentrations after mid-trimester legal termination of pregnancy was accompanied with a simultaneous increase in the serum levels of free VEGF so that within a few days after pregnancy VEGF dominated in the maternal circulation. Our study provides novel information on the kinetics of endogenous sVEGFR-1, which serves as a potential tool in the development of new strategies against diseases associated with angiogenic imbalance and alterations in VEGF signaling.

Chemical approaches to penicillin allergy—V. Nature of reaction between rabbit antigen (CRP-penicillin conjugate) and rabbit antipenicillin antibody

The availability of an electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) and rabbit antipenicillin antibody afforded an idealin vitro system to calculate the thermodynamic parameters of the binding of14C benzyl penicillin CRP conjugate (antigen) to the purified rabbit antipenicillin antibody. The thermodynamic parameters of this antigen-antibody reaction has been studied by radio-active assay method by using millipore filter. Equilibrium constant (K) of this reaction has been found to be 2·853×109M−2 and corresponding free energy (ΔG) at 4°C and 37°C has been calculated to be −12·02 and −13·5 kcal/mole, enthalpy (ΔH) and entropy (ΔS) has been found to be 361 kcal/mole and +30 eu/mole respectively. Competitive binding studies of CRP-analogue conjugates with the divalent rabbit antibody has been carried out in the presence of14C-penicilloyl CRP. It was found that 7-deoxy penicillin-CRP complex and 6-amino penicilloyl CRP conjugate binds to the antibody with energies stronger than that with the14C-penicilloyl CRP. All the other analogue conjugates are much weaker in interfering with the binding of the penicilloyl CRP with the antibody. The conjugate of methicillin,o-nitro benzyl penicillin and ticarcillin with CRP do not materially interfere in the process.

Early biochemical events during adventitious root initiation in the hypocotyl of Phaseolus vulgaris

Indole butyric acid (IBA) initiates roots in the hypocotyl tissue of Phaseolus vulgaris (French bean). The response is dependent on the concentration of IBA and the duration of exposure to the hormone. IBA enhances the rate of total protein synthesis in ca 30 min after exposure of the hypocotyl segments to the hormone. There is no detectable change in total or poly(A)-containing RNA synthesis in this period although significant increases are seen 2 hr after hormone pre-treatment. The early IBA-mediated increase in protein synthesis (30 min) is not sensitive to Actinomycin D but the antibiotic blocks the increase manifested 2 hr after hormone pre-treatment. Inhibition of early protein synthesis by cycloheximide depresses and delays root initiation. Cytosol prepared from IBA-treated hypocotyl tissue stimulates protein synthesis in vitro to a greater extent than that of the control.

TLR 9 Activation in Dendritic Cells Enhances Salmonella Killing and Antigen Presentation via Involvement of the Reactive Oxygen Species

Synthetic CpG containing oligodeoxynucleotide Toll like receptor-9 agonist (CpG DNA) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. It was observed that Salmonella Typhimurium growth can be inhibited by the CpG DNA treatment in the murine dendritic cells. This inhibitory effect was mediated by an increased reactive oxygen species production. In addition, it was noted that CpG DNA treatment of dendritic cells during Salmonella infection leads to an increased antigen presentation. Further this increased antigen presentation was dependent on the enhanced reactive oxygen species production elicited by Toll like receptor-9 activation. With the help of an exogenous antigen it was shown that Salmonella antigen could also be cross-presented in a better way by CpG induction. These data collectively indicate that CpG DNA enhance the ability of murine dendritic cells to contain the growth of virulent Salmonella through reactive oxygen species dependent killing.

Dietary aspects of cow's milk allergy in young children

Cow s milk allergy (CMA) affects about 2-6% of infants and young children. Environmental factors during early life are suggested to play a role in the development of allergic diseases. One of these factors is likely to be maternal diet during pregnancy and lactation. The association between maternal diet and development of CMA in offspring is not well known, but diet could contain factors that facilitate development of tolerance. After an established food allergy, another issue is gaining tolerance towards an antigen that causes symptoms. The strictness of the elimination depends on the individual level of tolerance. This study aimed at validating a questionnaire used to inquire about food allergies in children, at researching associations between maternal diet during pregnancy and lactation and subsequent development of cow s milk allergy in the offspring, and at evaluating the degree of adherence to a therapeutic elimination diet of children with CMA and factors associated with the adherence and age of recovery. These research questions were addressed in a prospective birth cohort born between 1997 and 2004 at the Tampere and Oulu University Hospitals. Altogether 6753 children of the Diabetes Prediction and Prevention (DIPP) Nutrition cohort were investigated. Questionnaires regarding allergic diseases are often used in studies without validation. High-quality valid tools are therefore needed. Two validation studies were conducted here: one by comparing parentally reported food allergies with information gathered from patient records of 1122 children, and the other one by comparing parentally reported CMA with information in the reimbursement records of special infant formulae in the registers of the Social Insurance Institution for 6753 children. Both of these studies showed that the questionnaire works well and is a valid tool for measuring food allergies in children. In the first validation study, Cohen s kappa values were within 0.71-0.88 for CMA, 0.74-0.82 for cereal allergy, and 0.66-0.86 for any reported food allergy. In the second validation study, the kappa value was 0.79, sensitivity 0.958, and specificity 0.965 for reported and diagnosed CMA. To investigate the associations between maternal diet during pregnancy and lactation and CMA in offspring, 6288 children were studied. Maternal diet during pregnancy (8th month) and lactation (3rd month) was assessed by a validated, 181-item semi-quantitative food frequency questionnaire (FFQ), and as an endpoint register-based information on diagnosed CMA was obtained from the Social Insurance Institution and complemented with parental reports of CMA in their children. The associations between maternal food consumption and CMA in offspring were analyzed by logistic regression comparing the highest and lowest quarters with two middle quarters of consumption and adjusted for several potential confounding factors. High maternal intake of milk products (OR 0.56, 95% CI 0.37-0.86 p = 0.002) was associated with a lower risk of CMA in offspring. When stratified according to maternal allergic rhinitis or asthma, a protective association of high use of milk products with CMA was seen in children of allergy-free mothers (OR 0.30, 95% CI 0.13 - 0.69, p < 0.001), but not in children of allergic mothers. Moreover, low maternal consumption of fish during pregnancy was associated with a higher risk of CMA in children of mothers with allergic rhinitis or asthma (OR 1.47, 95% CI 0.96 - 2.27 for the lowest quarter, p = 0.043). In children of nonallergic mothers, this association was not seen. Maternal diet during lactation was not associated with CMA in offspring, apart from an inverse association between citrus and kiwi fruit consumption and CMA. These results imply that maternal diet during pregnancy may contain factors protective against CMA in offspring, more so than maternal diet during lactation. These results need to be confirmed in other studies before giving recommendations to the public. To evaluate the degree of adherence to a therapeutic elimination diet in children with diagnosed CMA, food records of 267 children were studied. Subsequent food records were examined to assess the age at reintroduction of milk products to the child s diet. Nine of ten families adhered to the elimination diet of the child with extreme accuracy. Older and monosensitized children had more often small amounts of cow s milk protein in their diet (p < 0.001 for both). Adherence to the diet was not related to any other sociodemographic factor studied or to the age at reintroduction of milk products to the diet. Low intakes of vitamin D, calcium, and riboflavin are of concern in children following a cow s milk-free diet. In summary, we found that the questionnaires used in the DIPP study are valid in investigating CMA in young children; that there are associations between maternal diet during pregnancy and lactation and the development of CMA in offspring; and that the therapeutic elimination diet in children with diagnosed CMA is rigorously adhered to.

Noninvasive biomarkers for early smoking related lung disease

Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airway inflammation and largely irreversible airflow limitation. One major risk factor for COPD is cigarette smoking. Since the inflammatory process starts many years prior to the onset of clinical symptoms and still continues after smoking cessation, there is an urgent need to find simple non-invasive biomarkers that can be used in the early diagnosis of COPD and which could help in predicting the disease progression. The first aim of the present study was to evaluate the involvement of different oxidative/nitrosative stress markers, matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) in smokers and in COPD. Elevated numbers of inducible nitric oxide synthase (iNOS), nitrotyrosine, myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) positive cells and increased levels of 8-isoprostane and lactoferrin were found in sputum of non-symptomatic smokers compared to non-smokers, and especially in subjects with stable mild to moderate COPD, and they correlated with the severity of airway obstruction. This suggests that an increased oxidant burden exists already in the airways of smokers with normal lung function values. However, none of these markers could differentiate healthy smokers from symptomatic smokers with normal lung function values i.e. those individuals who are at risk of developing COPD. In contrast what is known about asthma exhaled nitric oxide (FENO) was lower in smokers than in non-smokers, the reduced FENO value was significantly associated with neutrophilic inflammation and the elevated oxidant burden (positive cells for iNOS, nitrotyrosine and MPO). The levels of sputum MMP-8 and plasma MMP-12 appeared to differentiate subjects who have a risk for COPD development but these finding require further investigations. The levels of all studied MMPs correlated with the numbers of neutrophils, and MMP-8 and MMP-9 with markers of neutrophil activation (MPO, lactoferrin) suggesting that especially neutrophil derived oxidants may stimulate the tissue destructive MMPs already in lungs of smokers who are not yet experiencing any airflow limitation. When investigating the role of neutrophil proteases (neutrophil elastase, MMP-8, MMP-9) during COPD exacerbation and its recovery period, we found that levels of all these proteases were increased in sputum of patients with COPD exacerbation as compared to stable COPD and controls, and decreased during the one-month recovery period, giving evidence for a role of these enzymes in COPD exacerbations. In the last study, the effects of subject`s age and smoking habits were evaluated on the plasma levels of surfactant protein A (SP-A), SP-D, MMP-9 and TIMP-1. Long-term smoking increased the levels of all of these proteins. SP-A most clearly correlated with age, pack years and lung function decline (FEV1/FVC), and based on the receiver operating characteristic curve analysis, SP-A was the best marker for discriminating subjects with COPD from controls. In conclusion, these findings support the hypothesis that especially neutrophil derived oxidants may activate MMPs and induce an active remodeling process already in the lungs of smokers with normal lung function values. The marked increase of sputum levels of neutrophil proteases in smokers, stable COPD and/or during its exacerbations suggest that these enzymes play a role in the development and progression of COPD. Based on the comparison of various biomarkers, SP-A can be proposed to serve as sensitive biomarker in COPD development.

Coated pits,endosome CURLing and intracellular receptor--ligand trafficking

In mediating endocytosis of extracellular macromolecules; the major mechanism in which cells ingest nutrients, degrade hormones and maintain the protein and lipid compositions of their organelle membrane, the cell surface receptors encounter 'coated pits', migrate continuously from one organelle to another, deliver the 'cargo' and often recycle back to the cell surface. This article is an attempt to give an account of the recent advances in our understanding of the molecular events involved in the 'round trip itinerary' of cell surface receptors.

Affine Hall-Littlewood Functions for A(1)((1)) And Some Constant Term Identities of Cherednik-Macdonald-Mehta Type

We study t-analogs of string functions for integrable highest weight representations of the affine Kac-Moody algebra A(1)((1)). We obtain closed form formulas for certain t-string functions of levels 2 and 4. As corollaries, we obtain explicit identities for the corresponding affine Hall-Littlewood functions, as well as higher level generalizations of Cherednik's Macdonald and Macdonald-Mehta constant term identities.

Characterization of a 10- to 14-kilodalton protease-sensitive mycobacterium tuberculosis H37Ra antigen that stimulates human -yi T cells

gamma delta T-cell receptor-bearing T cells (gamma delta T cells) are readily activated by intracellular bacterial pathogens such as Mycobacterium tuberculosis. The bacterial antigens responsible for gamma delta T-cell activation remain poorly characterized. We have found that heat treatment of live M. tuberculosis bacilli released into the supernatant an antigen which stimulated human gamma delta T cells, gamma delta T-cell activation was measured by determining the increase in percentage of gamma delta T cells by flow cytometry in peripheral blood mononuclear cells stimulated with antigen and by proliferation of gamma delta T-cell lines with monocytes as antigen-presenting cells. Supernatant from heat-treated M. tuberculosis was fractionated by fast-performance liquid chromatography (FPLC) on a Superose 12 column. Maximal gamma delta T-cell activation was measured for a fraction of 10 to 14 kDa. Separation of the supernatant by preparative isoelectric focusing demonstrated peak activity at a pi of <4.0. On two-dimensional gel electrophoresis, the 10- to 14-kDa FPLC fraction contained at least seven distinct molecules, of which two had a pi of <4.5. Protease treatment reduced the bioactivity of the 10- to 14-kDa FPLC fraction for both resting and activated gamma delta T cells. Murine antibodies raised to the 10- to 14-kDa fraction reacted by enzyme-linked immunosorbent assay with antigens of 10 to 14 kDa in lysate of M. tuberculosis. In addition, gamma delta T cells proliferated in response to an antigen of 10 to 14 kDa present in M. tuberculosis lysate. gamma delta T-cell-stimulating antigen was not found in culture filtrate of M. tuberculosis but was associated,vith the bacterial pellet and lysate of M. tuberculosis. These results provide a preliminary characterization of a 10- to 14-kDa, cell-associated, heat-stable, low-pI protein antigen of M. tuberculosis which is a major stimulus for human gamma delta T cells.