Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog.

Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.     

A G-Protein Subunit Translocation Embedded Network Motif Underlies GPCR Regulation of Calcium Oscillations

G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of G_βγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the G_βγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential G_βγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component.     

An Eighteen Serum Cytokine Signature for Discriminating Glioma from Normal Healthy Individuals

Glioblastomas (GBM) are largely incurable as they diffusely infiltrate adjacent brain tissues and are difficult to diagnose at early stages. Biomarkers derived from serum, which can be obtained by minimally invasive procedures, may help in early diagnosis, prognosis and treatment monitoring. To develop a serum cytokine signature, we profiled 48 cytokines in sera derived from normal healthy individuals (n = 26) and different grades of glioma patients (n = 194). We divided the normal and grade IV glioma/GBM serum samples randomly into equal sized training and test sets. In the training set, the Prediction Analysis for Microarrays (PAM) identified a panel of 18 cytokines that could discriminate GBM sera from normal sera with maximum accuracy (95.40%) and minimum error (4.60%). The 18-cytokine signature obtained in the training set discriminated GBM sera from normal sera in the test set as well (accuracy 96.55%; error 3.45%). Interestingly, the 18-cytokine signature also differentiated grade II/Diffuse Astrocytoma (DA) and grade III/Anaplastic Astrocytoma (AA) sera from normal sera very efficiently (DA vs. normal–accuracy 96.00%, error 4.00%; AA vs. normal–accuracy 95.83%, error 4.17%). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using 18 cytokines resulted in the enrichment of two pathways, cytokine-cytokine receptor interaction and JAK-STAT pathways with high significance. Thus our study identified an 18-cytokine signature for distinguishing glioma sera from normal healthy individual sera and also demonstrated the importance of their differential abundance in glioma biology.     

Effect of the Peroxisome Proliferator Ciprofibrate on Lipid Peroxidation and 8-Hydroxydeoxyguanosine Formation in Transgenic Mice with Elevated Hepatic Catalase Activity

Peroxisome proliferators are a group of non-genotoxic hepatic carcinogens which have been proposed to act by increasing oxidative damage in the liver. To test this hypothesis, we have produced a transgenic mouse line that has elevated catalase activity specifically in the liver. In this study, we have examined if catalase overexpression influences the induction of lipid peroxidation or oxidative DNA damage, two mechanisms which have been hypothesized to be important in the carcinogenesis by peroxisome proliferators. Transgenic mice or non-transgenic litter mates were fed either 0.01% ciprofibrate or a control diet for 21 days. The activities of fatty acyl CoA oxidase and lauric acid hydroxylase were not significantly affected by catalase overexpression, although the ratio of fatty acyl CoA oxidase to catalase was significantly decreased in transgenic animals. Hepatic lipid peroxidation was estimated by quantifying the concentrations of malondialdehyde and conjugated dienes. Ciprofibrate treatment did not affect either endpoint, but catalase overexpression increased the concentrations of malondialdehyde (in untreated mice only) and conjugated dienes (in both untreated and ciprofibrate-fed mice). Oxidative DNA damage was estimated by quantifying 8-hydroxydeoxyguanosine (8-OHdG) by high-performance liquid chromatography/electrochemical detection. Ciprofibrate treatment significantly increased hepatic 8-OHdG concentrations, in agreement with several previous studies, but catalase overexpression did not significantly affect them, although 8-OHdG concentrations were decreased 50% in untreated mice. These results imply that the metabolism of hydrogen peroxide by catalase is not an important factor in the development of hepatic lipid peroxidation. The decrease in hepatic 8-OHdG in untreated transgenic mice and the increase seen after ciprofibrate administration imply that hydrogen peroxide is important in the formation of 8-OHdG. While the lack of decreased 8-OHdG levels in ciprofibrate-treated transgenic mice does not support this conclusion, it is possible that catalase levels were not sufficiently high to affect this endpoint. Transgenic mice with higher hepatic catalase activities may be required to resolve this issue.     

Studies on 1-methyl adenine transfer RNA methyltransferase of Mycobacterium smegmatis

The presence of 1-methyl adenine in transfer RNA is a feature that Mycobacterium smegmatis shares with only a few other prokaryotes. The enzyme 1-methyl adenine tRNA methyl transferase from this source has been purified and the preliminary results show the presence of two activity peaks with different substrate specificity.List of abbreviations tRNA Transfer Ribonucleic acid - EDC 1-ethyl-3(3-dimethyl)amino Propyl Carbodiimide - PCA Perchloric acid - SAH S-adenosyl-homocystine - SIBA 5-deoxy-5-S-isobutylthioadenosine - SAM S-adenosyl methionine     

Studies on the effects ofin vitro methyiation on aminoacylation of transfer RNA

In vitro methyiation ofEscherichia coli transfer ribonucleic acid by cell free extracts ofMycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S. (1978)J. Bact., 137, 1085]. We have studied the effect of this modification on aminoacylationof Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylationin vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation     

The Embryonically Active Gene, Unkempt, of Drosophila Encodes a Cys(3)his Finger Protein

The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys3His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development.     

Occurrence of 1-methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis.

The minor base composition of Mycobacterium smegmatis tRNA has been studied. Thin-layer chromatographic patterns of a ribonuclease T2 digest of mycobacterial tRNA indicated the presence of appreciable amounts of 1-methyladenosine (which is commonly present only in eucaryotic tRNA), dihydrouridine, and 7-methylguanosine. Ribothymidine was absent. The S-adenosylmethionine-dependent tRNA methylases of M. smegmatis catalyzed the formation of 1-methyladenosine when Escherichia coli tRNA was used as acceptor. Similarly, E. coli extracts methylated the tRNA of M. smegmatis, forming ribothymidine.