Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Thymic irradiation inhibits the rapid recovery of TH1 but not TH2-like functions of CD4+ T cells after total lymphoid irradiation.

Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.     

Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus.

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.     

Expression of the PRC II avian sarcoma virus genome.

We found that the genomic RNA of the replication-defective avian sarcoma virus PRC II was 4.0 kilobases long. A Northern blot analysis of the viral RNAs present in PRC II-transformed cells showed that the PRC II genome was expressed as a single 4.0 kilobase mRNA species. In vitro translation of polyadenylic acid-containing 70S virion RNA yielded two highly related proteins of 110,000 and 105,000 daltons (P110 and P105), which were synthesized from messenger activity that sedimented as expected for the 4.0 kilobase PRC II genome (at 25 to 27S). P110 and P105 were identified as in vitro translation products of the PRC II genome by immunoprecipitation and tryptic peptide mapping and were the only PRC II-specific polypeptides detected by in vitro synthesis. In addition, we found that immune complexes prepared from PRC II 70S virion RNA in vitro translation products contained a tyrosine-specific protein kinase activity. A comparison of the in vitro- and in vivo-synthesized proteins revealed that PRC II-transformed cells also contained 110,000- and 105,000-dalton proteins, which were indistinguishable from in vitro-synthesized P110 and P105 by electrophoretic mobility and tryptic peptide analysis. Both P110 and P105 were present in producer cells and in seven individual nonproducer clones. A pulse-chase analysis showed that P105 was the primary translation product of the PRC II genome and that P110 was derived from P105 by post-translational modification. Under conditions of long-term labeling with [35S]methionine, P110 and P105 were present in a molar ratio of approximately 1:1. These results indicated that the transformation-specific product of the PRC II genome, previously referred to as a single component (P105), actually consists of two polypeptides related by post-translational modification.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

A nonsemen copulatory fluid influences the outcome of sperm competition in Japanese quail

Sperm competition is a powerful and widespread evolutionary force that drives the divergence of behavioural, physiological and morphological traits. Elucidating the mechanisms governing differential fertilization success is a fundamental question of sperm competition. Both sperm and nonsperm ejaculate components can influence sperm competition outcomes. Here, we investigate the role of a nonsemen copulatory fluid in sperm competition. Male Japanese quail possess a gland that makes meringue‐like foam. Males produce and store foam independent of sperm and seminal fluid, yet transfer foam to females during copulation. We tested whether foam influenced the outcome of sperm competition by varying foam state and mating order in competitive matings. We found that the presence of foam from one male decreased the relative fertilization success of a rival, and that foam from a given male increased the probability he obtained any fertilizations. Mating order also affected competitive success. Males mated first fertilized proportionally more eggs in a clutch and had more matings with any fertilizations than subsequent males. We conclude that the function of foam in sperm competition is mediated through the positive interaction of foam with a male's sperm, and we speculate whether the benefit is achieved through improving sperm storage, fertilizing efficiency or retention. Our results suggest males can evolve complex strategies to gain fertilizations at the expense of rivals as foam, a copulatory fluid not required for fertilization, nevertheless, has important effects on reproductive performance under competition.     

Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K⁺ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K⁺ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using ⁸⁶radioactive rubidium ion (⁸⁶Rb⁺) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.     

The influence of blood glucose on neutrophil function in individuals without diabetes

We assessed the association of neutrophil function with glycated hemoglobin (HbA1c) levels in a Japanese general population. Participants were 809 males and females who were over 20 years old living in the Iwaki region in Aomori Prefecture located in northern Japan. Lifestyle parameters (smoking, alcohol consumption, and exercise habits), HbA1c and neutrophil function such as reactive oxygen species (ROS) production capability and phagocytic activity (PA) were measured. ROS production capability was measured before and after phagocytic stimulus to obtain basal ROS production and stimulated ROS production. Level of HbA1c had a positive correlation with basal ROS production (p=0.053), a negative correlation with stimulated ROS production (p=0.072) and PA (p=0.059) only in post‐menopausal groups, and not in pre‐menopausal groups. However, there were no correlations between levels of HbA1c and neutrophil functions in male. In conclusion, in the present study, despite the presence of diabetes, chronic hyperglycemia was found to cause an increase in daily basal ROS production of neutrophils, and increased susceptibility to infection caused by reduced neutrophilic reaction in females in their menopause. Therefore, from the oxidative point of view, strict glycemic control is necessary to prevent post‐menopausal females from developing diabetic complications in spite of the presence of diabetes.     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.