Anomeric specificity of glucose effect on cAMP, fructose 1,6-bisphosphatase, and trehalase in yeast

The addition of beta-D-glucose (final concentration, 50 mM) to a cell suspension of Saccharomyces cerevisiae in stationary phase caused a rapid 4-fold increase in the concentration of cAMP, while a 2-fold increase of cAMP was observed by the addition of alpha-D-glucose. beta -D-Glucose was also more effective than alpha-D-glucose in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase. These results, taken together with the previous report that alpha-D-glucose is transported more rapidly than beta-D-glucose in Saccharomyces cerevisiae, do not support the view currently proposed by some investigators that cotransport of D-glucose with protons causes the depolarization of the cell membrane, resulting in the activation of adenylate cyclase. The present data, however, provides supporting evidence for the view that cAMP-dependent protein kinase is implicated in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase.     

Effects of in vivo monoclonal anti-I-A antibody treatment in neonatal mice on intrathymic and peripheral class II antigen expression

Intrathymic, Ia-bearing antigen-presenting cells (APC) are believed to play an important role in the development of a mature, functional T-cell repertoire. We used chronic in vivo treatment of neonatal mice with anti-I-A monoclonal Ab (MAb) to examine the expression of I-A and I-E antigens on intrathymic and peripheral APC. Three weeks after continuous treatment with anti-I-A MAb, FACS analysis of unfractionated spleen cells revealed a 75-90% reduction in the number of I-A bearing cells. Splenic antigen-presenting capacity measured by the ability of unseparated or density gradient-enriched APC to stimulate I-A- or I-E-reactive T-cell hybridomas was also greatly reduced. In contrast to the expression of I-A and I-E molecules in the splenic APC, anti-I-A MAb treatment resulted in decreased thymic APC I-A expression without significant changes in I-E as measured by FACS analysis. This was confirmed in functional studies in which allo-I-A- or auto-I-A-reactive T-cell hybridomas could not be stimulated by treated thymic APC. Unlike splenic APC, anti-I-A-treated thymic APC did not differ significantly from normals in their ability to stimulate allo-I-E-reactive T hybridomas. This lack of linkage or comodulation of I-A and I-E expression on thymic but not splenic APC may allow us to study the role of I-A molecules and I-E molecules on the development and expansion of functional, mature T-cell repertoires.     

Catabolite inactivation of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase in yeast

Catabolite inactivation of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase was studied using the protease-deficient and vacuole-defective yeast strain pep4-3. The catabolite inactivation of fructose 1,6-bisphosphatase in pep4-3 was found to have a normal first inactivation step but with a defective second proteolytic step. In contrast, catabolite inactivation of cytoplasmic malate dehydrogenase was normal in pep4-3. These results suggest that the proteolytic pathways utilized in the hydrolysis of the two enzymes may be different and that proteolysis of fructose 1,6-bisphosphatase may require functional vacuoles while proteolysis of cytoplasmic malate dehydrogenase may not.     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .     

Guanosine 5'-triphosphate, 3'-diphosphate 5'-phosphohydrolase. Purification and substrate specificity

The regulatory nucleotide guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) and its precursor guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) are accumulated during stringent response in bacterial cells. The enzyme pppGpp-5'-phosphohydrolase, which catalyzes the conversion of pppGpp to ppGpp, was partially purified from Escherichia coli. It has Mr = 140,000 and an apparent Km of 0.11 mM for pppGpp. It requires Mg2+ and a monovalent cation. NH4+ is preferred over K+, while Na+ is inactive. The enzyme does not hydrolyze GTP, ATP, pppApp, or ppGpp. It is also not effectively inhibited by these nucleotides. pppGpp-5'-phosphohydrolase hydrolyzes the 3'-monophosphate analog pppGp equally well (apparent Km of 0.13 mM), yielding the recently identified MS III nucleotide (ppGp). pppGpp-5'-phosphohydrolase does not have RNA 5'-terminal gamma-phosphatase activity; however, 5'-terminal phosphates are released by pppGpp-5'-phosphohydrolase when the GTP-terminated RNA chains are first converted into oligonucleotides by RNase A treatment. pppGpp-5'-phosphohydrolase was found to actively hydrolyze the dinucleotide fragment pppGpNp but exhibited very low activity toward longer chain fragments. The 3'-unphosphorylated dinucleotide pppGpN was, however, not hydrolyzed. The ability of pppGpp-5'-phosphohydrolase to hydrolyze pppGpp, pppGp, and pppGpNp, but not pppG and pppGpN, indicates that pppGpp-5'-phosphohydrolase is rather nonspecific toward the 3'-OH substitutions of the substrates although a free, unsubstituted phosphate group at the 3'-OH position is essential.     

Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium

PMA causes rapid down-modulation of CD4 molecules on murine immature thymocytes, human PBL, and CD4-positive human tumor cell lines, but not on murine peripheral lymphocytes. The mechanisms of phorbol ester-induced down modulation of CD4 molecules, however, have not been elucidated. To determine how PMA down-modulates CD4 expression by T lymphocytes, we studied the ability of inhibitors of protein kinase C, calmodulin, actin, and tubulin to block PMA-induced modulation of CD4 in several murine and human cell types. We also tested the ability of intracellular and extracellular calcium chelators to block CD4 internalization. There was marked variability in the degree of PMA-induced down-modulation of CD4 among various cell types. The effects of PMA on CD4 expression were greater for murine thymocytes, for human PBL, and for the human lymphoblastic leukemia cell line, MOLT-3, than for any of the other cell types studied. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked phosphorylation but not internalization of CD4 molecules induced by PMA. Therefore, phosphorylation of CD4 molecules by protein kinase C is not required for the internalization of the molecules. Internalization was blocked by both inhibitors of calmodulin, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide, and trifluoperazine. PMA-induced internalization of CD4 was blocked by Quin-2 AM, which chelates intracellular calcium. EGTA, which chelates extracellular calcium, did not block internalization. Inhibitors of actin or tubulin did not block internalization. These results suggest that PMA-induced modulation of CD4 can occur in the absence of phosphorylation of the CD4 molecules and is calmodulin and intracellular calcium dependent.     

Autoantibody in MRL/Mp-lpr/lpr mice. A monoclonal antibody specific for the abnormal T cells from mice bearing the lpr/lpr gene

We generated mAb from an unimmunized autoimmune MRL/Mp-lpr/lpr mouse. One of these mAb A108, reacted with cell surface Ag present on abnormal T cells from MRL/Mp-lpr/lpr, C3H-lpr/lpr, and C57BL/6-lpr/lpr mice. We failed to detect significant numbers of A108 bearing cells in the lymph nodes of MRL-Mp/+/+, normal C3H or normal C57BL/6 mice. Therefore, the expression of A108 correlates with the presence of the lpr/lpr gene. A108 binds to a variety of murine T cell tumor lines (e.g., EL4, BW5147, and YAC-1) and human T cell tumor lines (e.g., MOLT-3, Sup T1, and Jurkat). A108 does not bind to normal human PBL. Immunoprecipitation of surface iodinated EL-4 and BW5147 with A108 identified one major protein with a Mr of about 17.5-kDa. The significance of these findings with respect to the development of lymphoid proliferation and autoimmune disease in mice bearing the lpr/lpr gene will be discussed.     

On the applicability of adaptive bioprocess state estimators

This article presents an industrial case study, examining the application of a novel adaptive biomass estimator to an industrial microfungi production process. It is our intention that this contribution should focus upon the implementation issues of the algorithm, in preference to a rigorous theoretical development. The novel algorithm adopted is developed from Adaptive Inferential Estimation studies of Guilandoust and co-workers. The technique utilizes input-output process measurements obtained at different frequencies, thereby providing more frequent estimates of biomass concentration than are otherwise available from off-line laboratory analyses. The algorithm is particularly suited to the biotechnology industry, as it is capable of utilizing irregular assay measurements with varying delays.Although this article demonstrates the encouraging industrial implications of the adaptive algorithm, like all adaptive techniques currently developed, it is restricted by the inability to perform robust on-line system identification. The ultimate selection of a "suboptimal" "fixed parameter" algorithm for on-line implementation, is therefore directly attributable to these inadequacies. Aspects of data acquisition, data pretreatment, and data quality are critical for real process applications, and while some practical approaches are adopted here, many important implementation problems remain unresolved. (c) 1993 John Wiley & Sons, Inc.     

Division of Listeria monocytogenes serovar 4b strains into two groups by PCR and restriction enzyme analysis.

Altogether, 133 strains of Listeria monocytogenes serovar 4b were investigated. A segment of 2,916 bp containing parts of the two genes inlA and inlB in L. monocytogenes was amplified by the PCR technique. The PCR product obtained was cleaved with the restriction enzyme AluI, and the fragments generated were separated by gel electrophoresis, leading to two distinct groups: PCR-restriction enzyme analysis groups I and II, containing 37 and 96 strains, respectively. The PCR-restriction enzyme analysis method described in this paper could be a useful tool for the subtyping of L. monocytogenes serovar 4b strains.