Lipase modification by various synthetic polymers for use in chloroform

Summary Lipase was modified with several hydrophilic and hydrophobic synthetic polymers. The modified lipase was solubilized into chloroform by. The catalytic esterification activity of modified lipase increased linearly with the increase of its solubility in chloroform.     

Deduced primary structure of a Xenopus proteasome subunit XC3 and expression of its mRNA during early development

Proteasome is a non-lysosomal proteinase complex ubiquitously distributed in eukaryotic cells. We isolated here the cDNA clone for one of the proteasome subunits (XC3) from Xenopus ovary cDNA libraries using rat RC3 cDNA as a prove. The cDNA is 885 bp long and encodes 234 amino acids. The deduced amino acid sequence is highly homologous (95.3%) to those of rat RC3 and human HC3 subunits. The mRNA for XC3 is one of the maternal mRNAs and detected at all the embryonic stages investigated, but its level changes in a characteristic way especially at the gastrula stage. We suggest that the highly conserved XC3 subunit plays an essential role in proteasome function and also that during Xenopus embryogenesis mRNA for XC3 subunit is replaced from maternal to newly-synthesized one probably around the gastrula stage.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings

Previous studies indicated that acute exposure of adrenal cells to adrenocorticotropic hormone (ACTH) markedly stimulates steroidogenic capacity in vitro but also inhibits cell proliferation. However, in vivo, ACTH is known to stimulate adrenal cell growth. To address this discrepancy, we determined the effect of long-term (9-11 days) continuous or intermittent exposure to ACTH on human fetal adrenal cell proliferation and steroidogenesis. Adrenal glands from fetuses 18-22 wk gestation were studied. Fetal zone cells were plated either on plastic or on an extracellular matrix (ECM) in the presence and absence of basic fibroblast growth factor (bFGF) (0.5 ng/ml) and 1 or 10 nM ACTH. As determined by cell counting, bFGF stimulated cell proliferation during 9 days in culture. In the presence of bFGF, the average doubling time decreased from 44 to 30 h on plastic and from 37 to 26 h on ECM. Under these conditions, ACTH did not inhibit cell proliferation. Proliferation of fetal adrenal corticosteroid-producing cells in the ACTH-treated cultures also was assessed by histochemical staining for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). The number of positive cells increased more than 4-fold between Days 5 and 9 in culture. Continuous treatment with 1 nM ACTH increased dehydroepiandrosterone sulfate (DHAS) production 5- to 10-fold during the first 5 days in culture. Thereafter, the stimulated hormone production decreased over time, although there was still a difference of almost 100-fold between the control and ACTH-treated cultures at the end of 9 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of neuromedin B and GRP-10 on gastrin and insulin release from cultured tumor cells of a malignant gastrinoma

A study relating to gastrin release from gastrinoma cells by neuromedin B and C-terminal decapeptide of gastrin releasing peptide (GRP-10) has not yet been reported. Therefore, we studied the effects of neuromedin B and GRP-10 on gastrin release from cultured dispersed cells prepared from both the primary tumor in the pancreas and the metastatic tumor in the liver from a case of malignant Zollinger-Ellison syndrome. Both the primary and metastatic tumors obtained by a curative operation contained similar concentrations of gastrin and glucagon, whereas the primary tumor contained 10 times more insulin than the metastatic tumor. Gastrin release from cultured cells of both tumors was suppressed by 0.1 and 10 nM neuromedin B and tended to be suppressed by 0.1-10 nM GRP-10. However, insulin release from cultured cells of the pancreatic tumor was stimulated by GRP-10, but not by neuromedin B. These results might suggest that receptor function for the bombesin family peptides is abnormal in gastrinoma cells in both primary and metastatic tumors, and that a major source of insulin secretary cells is the contaminated normal islet cells in the primary tumor.     

Plasma levels of 16 alpha-hydroxypregnenolone, 16 alpha-hydroxyprogesterone and 16 alpha-hydroxydehydroepiandrosterone in the fetus and neonates.

Simultaneous determination of unconjugated 16 alpha-hydroxypregnenolone (16 alphaOH-Preg), 16 alpha-hydroxyprogesterone (16 alphaOH-Prog) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOH-DHEA) in fetal and neonatal plasma was performed utilizing a newly developed radioimmunoassay. In all neonates, the three 16 alpha-hydroxysteroid levels were consistently higher in umbilical cord plasma than in the maternal peripheral circulation. 16 alpha-OH-Preg in the umbilical arterial plasma increased from 11.2 +/- 3.1 at 24 weeks to 29.7 +/- 12.0 ng/ml at term, 16 alphaOH-Prog from 15.5 +/- 3.2 to 34.3 +/- 11.0 ng/ml and 16 alphaOH-DHEA from 5.1 +/- 1.2 to 5.9 +/- 1.0 ng/ml. In the anencephalic neonates, only 16 alphaOH-Preg showed an increase pattern under ACTH priming. 16 alpha-OH-Preg levels for normal full term neonates remain relatively constant at the first 24 hr and show a slight decrease at 3 days post partum. In small full term neonates, 16 alphaOH-Preg levels in umbilical arterial plasma are considerably higher than in normal neonates and remain at roughly equivalent levels for the first 5 days post partum. 16 alphaOH-Prog and 16 alphaOH-DHEA levels in umbilical arterial plasma in normal and small full term neonates are almost equal and both groups show a rapid decrease during the first 24 hr. Comparison with findings of the three 16 alpha-hydroxysteroids in fetal and neonatal plasma is discussed.     

A role of membranes in the activation of a new multifunctional protein kinase system.

A new multifunctional protein kinase, which normally exists as an inactive form in the soluble fraction in mammalian tissues, attaches to membranes to exhibit full enzymatic activity. A low concentration of Ca2+ is absolutely necessary for this activation. This process is reversible. cAMP shows no effect. The active factors in membranes are phosphatidylinositol, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol, and phosphatidylethanolamine in that order. Phosphatidylcholine and sphingomyelin are far less effective. Cytoplasmic as well as other membrane fractions from various tissues are active in supporting the enzymatic activity. A possible role of this Ca2+ and phospholipid-activated protein kinase system in transmembrane control is proposed.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.