Two regulatory isozymes of glutamine synthetase from Bacillus caldolyticus, an extreme thermophile.

Two glutamine synthetases (EC 6.3.1.2) have been purified to homogeneity from $\text{B. caldolyticus}$ strain YTP, grown at 70° on a minimal, defined medium. The enzymes are virtually identical in size and molecular weight (12 sub-units of MW 50,000), but differ in their isoelectric points, electrophoretic mobility, net charge, inherent thermal stability, affinity for substrates, and activity responses to metal ions and pH. Of primary interest is the observation that the more acidic form (pI = 5.2), E_I, is strongly feedback-regulated by certain amino acids derived from glutamine (Gly, L-Ala, L- and D-ser) but not by L-Glu or AMP, whereas the less acidic form (pI = 5.5), E_II, is inhibited most strongly by L-Gln and AMP, but not by the above amino acids. Both enzymes are inhibited strongly by ADP, CTP, NAD, glucosamine-6-P, less strongly by nucleotide diphosphates and L-Trp, and are activated by nucleotide monophosphates other than AMP. These results suggest that overall regulation of glutamine synthetase by the full spectrum of end product metabolites derived from L-Gln is accomplished by regulatory isozymes in this extremely thermophilic organism.     

Male copulatory behavior and the induction of ovulation in female voles: a quest for species specificity.

Two experiments were conducted to investigate species specificity in the neuroendocrine responsiveness of female prairie voles to the copulatory patterns of males. In Experiment 1, prairie vole males mated for one ejaculatory series were not significantly more effective in inducing ovulation in prairie vole females than montane voles mated with prairie vole females for one series, two series, or to satiety. Mating with conspecific males did result in significantly more implanted embryos than did heterospecific matings. In Experiment 2, it was found that, when the amount of vaginal stimulation was both low and equated across groups, prairie vole males were significantly more effective in triggering ovulation in female prairie voles than were either meadow voles or montane voles. Although there appears to be some species specificity to the “vaginal codes” of these congeneric species, its biological significance is unclear.     

Semiautomated processing of systolic time intervals.

Systolic time intervals are commonly used to identify changes in ventricular function. The method described facilitates measurement and calculation of many such intervals. This method utilizes a printed polygraph recording of electrocardiogram, phonocardiogram, and carotid pulse contour; a digitizing device for reading the necessary coordinates from the record; and a minicomputer for calculation of the intervals and for data analysis. Intervals related to approximately 100 pulse beats can be read, calculated, printed in chart and graph form, and subjected to some analyses in about an hour.     

Demographic response of tundra small mammals to a snow fencing experiment

Snow cover is a key environmental component for tundra wildlife that will be affected by climate change. Change to the snow cover may affect the population dynamics of high‐latitude small mammals, which are active throughout the winter and reproduce under the snow. We experimentally tested the hypotheses that a deeper snow cover would enhance the densities and winter reproductive rates of small mammals, but that predation by mustelids could be higher in areas of increased small mammal density. We enhanced snow cover by setting out snow fences at three sites in the Canadian Arctic (Bylot Island, Nunavut, and Herschel Island and Komakuk Beach, Yukon) over periods ranging from one to four years. Densities of winter nests were higher where snow depth was increased but spring lemming densities did not increase on the experimental areas. Lemmings probably moved from areas of deep snow, their preferred winter habitat, to summer habitat during snow melt once the advantages associated with deep snow were gone. Our treatment had no effect on signs of reproduction in winter nests, proportion of lactating females in spring, or the proportion of juveniles caught in spring, which suggests that deep snow did not enhance reproduction. Results on predation were inconsistent across sites as predation by weasels was higher on the experimental area at one site but lower at two others and was not higher in areas of winter nest aggregations. Although this experiment provided us with several new insights about the impact of snow cover on the population dynamics of tundra small mammals, it also illustrates the challenges and difficulties associated with large‐scale experiments aimed at manipulating a critical climatic factor.     

Cardio-Metabolic Disease Risks and Their Associations with Circulating 25-Hydroxyvitamin D and Omega-3 Levels in South Asian and White Canadians

Objectives

This study compared cardio-metabolic disease risk factors and their associations with serum vitamin D and omega-3 status in South Asian (SAC) and White Canadians (WC) living in Canada’s capital region.

Methods

Fasting blood samples were taken from 235 SAC and 279 WC aged 20 to 79 years in Ottawa, and 22 risk factors were measured.

Results

SAC men and women had significantly higher fasting glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR), apolipoprotein B (ApoB), ratios of total (TC) to HDL cholesterol (HDLC) and ApoB to ApoA1, leptin, E-selectin, P-selectin, ICAM-1 and omega-3 (p < 0.05), but lower HDLC, ApoA1, vitamin D levels than WC (p < 0.05). SAC women had higher CRP and VEGF than WC women. Adequate (50–74.9 nmol/L) or optimal (≥ 75 nmol/L) levels of 25(OH)D were associated with lower BMI, glucose, insulin, HOMA-IR, TG, TC, low density lipoprotein cholesterol (LDLC), ApoB/ApoA1 ratio, CRP, leptin, and higher HDLC, ApoA1, omega-3 index, L-selectin levels in WC, but not in SAC. Intermediate (>4%-<8%) or high (≥ 8%) levels of omega-3 indices were related to lower E-selectin, P-selectin, ICAM-1 and higher HDLC, 25(OH)D levels in WC, but not in SAC. The BMIs of ≤ 25 kg/m² were related to lower LDLC, ApoB, VEGF, creatinine and higher 25(OH)D in WC, but not in SAC.

Conclusions

The associations of vitamin D, omega-3 status, BMI and risk factors were more profound in the WC than SAC. Compared to WC, vitamin D status and omega-3 index may not be good predictive risk factors for the prevalence of CVD and diabetes in SAC.     

Development of a novel inducer for EBV lytic therapy

Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population that persists as a latent infection in various lymphoid and epithelial malignancies. The total number of EBV associated malignancies is estimated to exceed 200,000 new cancers per year. Current chemotherapeutic treatments of EBV-positive cancers include broad-spectrum cytotoxic drugs that ignore the EBV positive status of tumors and have limited safety and selectivity. In an effort to develop new and more efficacious molecules for inducing EBV reactivation, we have developed high-throughput screening assays to identify a class of small molecules (referred to as the C60 series) that efficiently activate the EBV lytic cycle in multiple latency types, including lymphoblastoid and nasopharyngeal carcinoma cell lines. In this paper we report our preliminary structure activity relationship studies and demonstrate reactivation of EBV in the SNU719 gastric carcinoma mouse model and the AGS-Akata gastric carcinoma mouse model.     

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.     

Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the "hoop" shape of MB cytoskeletons in unfixed suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 260,000, were decreased in "uncoiled" MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.     

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.