Ablation of CD8 and CD4 T cell responses by high viral loads

To evaluate the impact of sustained viral loads on anti-viral T cell responses we compared responses that cleared acute lymphocytic choriomeningitis virus infection with those that were elicited but could not resolve chronic infection. During acute infection, as replicating virus was cleared, CD8 T cell responses were down-regulated, and a pool of resting memory cells developed. In chronically infected hosts, the failure to control the infection was associated with pronounced and prolonged activation of virus-specific CD8 T cells. Nevertheless, there was a progressive diminution of their effector activities as their capacity to produce first IL-2, then TNF-alpha, and finally IFN-gamma was lost. Chronic lymphocytic choriomeningitis virus infection was also associated with differential contraction of certain CD8 T cell responses, resulting in altered immunodominance. However, this altered immunodominance was not due to selective expansion of T cells expressing particular TCR Vbeta segments during chronic infection. High viral loads were not only associated with the ablation of CD8 T cell responses, but also with impaired production of IL-2 by virus-specific CD4 T cells. Taken together, our data show that sustained exposure to high viral loads results in the progressive functional inactivation of virus-specific T cell responses, which may further promote virus persistence.     

Gravity and light effects on the circadian clock of a desert beetle,Trigonoscelis gigas

Circadian function is affected by exposure to altered ambient force environments. Under non-earth gravitational fields, both basic features of circadian rhythms and the expression of the clock responsible for these rhythms are altered. We examined the activity rhythm of the tenebrionid beetle, Trigonoscelis gigas, in conditions of microgravity (microG; spaceflight), earth's gravity (1 G) and 2 G (centrifugation). Data were recorded under a light-dark cycle (LD), constant light (LL), and constant darkness (DD). Free-running period (tau) was significantly affected by both the gravitational field and ambient light intensity. In DD, tau was longer under 2 G than under either 1 G or microG. In addition, tauLL was significantly different from tauDD under microG and 1 G, but not under 2 G.     

Glycosphingolipids of the model fungus Aspergillus nidulans: characterization of GIPCs with oligo-alpha-mannose-type glycans

Aspergillus nidulans is a well-established nonpathogenic laboratory model for the opportunistic mycopathogen, A. fumigatus. Some recent studies have focused on possible functional roles of glycosphingolipids (GSLs) in these fungi. It has been demonstrated that biosynthesis of glycosylinositol phosphorylceramides (GIPCs) is required for normal cell cycle progression and polarized growth in A. nidulans (Cheng, J., T.-S. Park, A. S. Fischl, and X. S. Ye. 2001. Mol. Cell Biol. 21: 6198-6209); however, the structures of A. nidulans GIPCs were not addressed in that study, nor were the functional significance of individual structural variants and the downstream steps in their biosynthesis. To initiate such studies, acidic GSL components (designated An-2, -3, and -5) were isolated from A. nidulans and subjected to structural characterization by a combination of one-dimensional (1-D) and 2-D NMR spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), ESI-MS/collision-induced decomposition-MS (MS/CID-MS), ESI-pseudo-[CID-MS]2, and gas chromatography-MS methods. All three were determined to be GIPCs, with mannose as the only monosaccharide present in the headgroup glycans; An-2 and An-3 were identified as di- and trimannosyl inositol phosphorylceramides (IPCs) with the structures Man alpha 1-->3Man alpha 1-->2Ins1-P-1Cer and Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->2Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester, and Cer = ceramide). An-5 was partially characterized, and is proposed to be a pentamannosyl IPC, based on the trimannosyl core structure of An-3.     

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).     

Emergence of adult northern and western corn rootworms (Coleoptera: Chrysomelidae) following reduced soil insecticide applications

A 3-yr investigation was conducted in commercial corn, Zea mays (L.), fields in eastern South Dakota to determine how reduced application rates of planting-time soil insecticides would influence temporal emergence patterns and survival of northern and western corn rootworms, Diabrotica barberi Smith and Lawrence, and D. virgifera virgifera LeConte, respectively. Beetle emergence was monitored at 2-d intervals throughout the entire adult emergence period of three growing seasons from corn plots treated with planting-time applications of labeled (1X) and reduced (0.5 and 0.75X) application rates of terbufos, tefluthrin, and chlorethoxyfos. No consistent insecticide- or rate-related impacts on mean total emergence per trap were recorded for any of the compounds investigated. However, terbufos applications resulted in a 52% reduction in the number of beetles captured per trap, 53% reduction in maximum rate of adult emergence, and a 59% reduction in overall rate of emergence over time for male D. virgifera during 1994. Terbufos also significantly extended the time required for emergence to peak and linear emergence of female D. virgifera to end in 1994. Tefluthrin applications delayed onset, end, and time of maximum emergence of female D. barberi by 9.9, 14.1, and 12 d, respectively, during 1993. Tefluthrin also reduced emergence rates over time for male (38%) and female (46%) D. barberi during 1994. Overall, application rate was inconsequential regarding total emergence, seasonal emergence pattern, or level of plant protection provided for all insecticides we tested in this 3-yr investigation. Our findings demonstrate that, if properly applied, the reduced application rates used in this study provide adequate root protection and will not significantly impact the biology of these pest species.     

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.     

Acid-sensing ion channels in malignant gliomas

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.