In the course of screening inhibitors from the methanol (MeOH) extracts of 168 medicinal plants against lymphocyte cell-specific kinase (Lck) Src -homology 2 (SH2) binding to a synthetic phosphotyrosine-containing peptide (phosphopeptide), we isolated rosmarinic acid from the MeOH extract of Prunella vulgaris, which showed specific inhibitory activity. The IC 50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 microM. However, even at concentrations of 0.1 to 1000 microM, no significant inhibitions were observed against other SH2 domains binding such as the growth factor receptor binding protein 2 (Grb2) SH2 domain to phosphopeptide of Shc and phospholipase Cgamma1 (PLCgamma1) SH2 domain to translational elongation factor 1alpha (EF1alpha) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50 % at a concentration of 8 microM in Jurkat cells stimulated with anti-CD3 and anti-CD4 antibodies. FK506 and cyclosporin A (CsA) employed as positive controls showed less than 30 % inhibition at the same concentration. In addition, rosmarinic acid inhibited the intracellular [Ca 2+] i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 microM of rosmarinic acid, which is one of the earliest responses of antigen-specific T cell receptor (TCR) and of the upstream pathway of IL-2 expression. Taken together, these results suggest that rosmarinic acid has the potential to specifically inhibit Lck SH2 domain binding to its cognate ligand, including ZAP-70, Cbl, HS-1, and PLCgamma1, and Lck-dependent Ca 2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.

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Three flavonoids: norwogonin, dihydronorwogonin and baicalein, were isolated from the roots of Scutellaria baicalensis, as potential inhibitors of VHR dual-specificity protein tyrosine phosphatase (DS-PTPase). Norwogonin (IC 50 = 1.1 microM), dihydronorwogonin (IC 50 = 2.9 microM) and baicalein (IC 50 = 2.4 microM) showed potent inhibitory activity toward VHR, but had no inhibitory activity against T-cell protein tyrosine phosphatase or serine/threonine protein phosphatase 1. From comparisons to the inhibitory activities of other similar flavonoids, it could be suggested that the presence of a hydroxy group in the B ring of flavonoids interferes with the inhibitory activity toward VHR DS-PTPase.

The aim of this observational study was to determine the optimal timing of interleukin-6 receptor inhibitor (IL6ri) administration for coronavirus disease 2019 (COVID-19).Patients with COVID-19 were given an IL6ri (sarilumab or tocilizumab) based on iteratively reviewed guidelines. IL6ri were initially reserved for critically ill patients, but after review, treatment was liberalized to patients with lower oxygen requirements. Patients were divided into two groups: those requiring ≤45% fraction of inspired oxygen (FiO2) (termed stage IIB) and those requiring >45% FiO2 (termed stage III) at the time of IL6ri administration. The main outcomes were all-cause mortality, discharge alive from hospital, and extubation.A total of 255 COVID-19 patients were treated with IL6ri (149 stage IIB and 106 stage III). Patients treated in stage IIB had lower mortality than those treated in stage III (adjusted hazard ratio (aHR) 0.24, 95% confidence interval (CI) 0.08–0.74). Overall, 218 (85.5%) patients were discharged alive. Patients treated in stage IIB were more likely to be discharged (aHR 1.43, 95% CI 1.06–1.93) and were less likely to be intubated (aHR 0.43, 95% CI 0.24–0.79).IL6ri administration prior to >45% FiO2 requirement was associated with improved COVID-19 outcomes. This can guide clinical management pending results from randomized controlled trials.

To directly define the role of phospholipase Cγ1 (PLCγ1) in NF‐κB activation, NF‐κB promoted luciferase reporter gene plasmid (pNF‐κB‐Luc) was transfected into rat‐3Y1 fibroblasts that overexpress whole PLCγ1 (PLCγ1‐3Y1), src homology domains SH2‐SH2‐SH3 of PLCγ1 (SH223‐3Y1) and v‐src (Src‐3Y1). Transient transfection with pNF‐κB‐Luc remarkably increased the luciferase activity in all three transformants compared with normal rat‐3Y1 cells. Pretreatment with inhibitors of protein tyrosine kinase reduced this increase in luciferase activity, but U73122 (a PLC inhibitor) did not. While PD98059, an inhibitor of mitogen activated protein kinase (MAPK), significantly reduced the luciferase activity, there was no effect by wortmannin and Ro‐31‐8220, inhibitors of phosphatidylinositol 3‐kinase (PI3K) and protein kinase C (PKC), respectively. This study shows a direct evidence that the SH2‐SH2‐SH3 region of PLCγ1 contributes to the NF‐κB signaling and that MAPK, but not PI3K and PKC, is involved in SH2‐SH2‐SH3 mediated NF‐κB activation in these cells.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.

In tsRSV-infected NRK (tsNRK) cells, pp60(v-src) reactivation bytemperature-shift from a nonpermissive temperature, 39 degrees C, to a permissive one, 32 degrees C, induced the production of inositol phosphates (IPt) and phosphatidylethanol (PEt), This was accompanied by an increase in membrane-associated protein kinase C (PKC) activity in the absence of exogenous growth factors. However, with serum-stimulation, the amounts of IPt and PEt at 32 degrees C were less than those at 39 degrees C, Pretreatment with PKC inhibitors, Re-31-8220 and staurosporine, enhanced the accumulation of IPt but not of PEt at 32 degrees C. The tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) was increased either by serum or by pp60(v-src) reactivation. These results suggest that serum transduces its signal through PLC gamma 1 mediation, and that pp60(v-src), possibly through the PKC mediation, negatively affects serum-induced PLC gamma 1 activation.

Sporulation-specific glucoamylase (SGA) gene was isolated from genomic library of Saccharomyces diastaticus 5114-9A by using glucoamylase non-producing mutant of S. diastaticus as a recipient. When the glucoamylase activities of culture supernatant, periplasmic, and intracellular fraction of cells transformed with hybrid plasmid containing SGA gene were measured, the highest activity was detected in culture supernatant. SG;A produced by transformant and extracellular glucoamylase produced by S. diastaticus 5114-9A differed in enzyme characteristics such as optimum temperature, thermostability, and resistance to SDS and urea. But the characteristics of SGA produced by sporulating yeast cells and vegetatively growing transformants were identical.

In our screening to search inhibitors of phosphoinositide(PI)-specific phospholipase C (PI-PLC), two inhibitors, MT965-A and -B were isolated from a culture broth of an actinomycetes. MT965-A and -B were identified as fatty acid derivatives, 14-methylpentadecanoic acid and 16-methyllinoleic acid methyl ester, respectively, based on the spectral data including NMR and MS. Both inhibitors directly inhibited not only in vitro PLC gamma 1 activity but also the platelet-derived growth factor (PDGF)-induced inositol phosphates (IPt) formation in NIH 3T3 gamma 1 cells overexpressing PLC gamma 1. However, the inhibitors enhanced in, vitro protein kinase C (PKC) activity. On examination of the effects of various fatty acids (FAs) on activities of PLC, PKC, and PDGF-induced IPt formation, the unsaturated FAs (UFAs) showed the same activities like the inhibitors, but the saturated FAs (SFAs) did not show similar activities. It was inferred that the chain length, degree of unsaturation, methyl esterification, branching with a methyl group, and cis-configuration were important for their activity.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Amentoflavone was isolated as an inhibitor of phospholipase C gamma 1 (PLC gamma 1) and phosphoinositides (PI)-turnover in PLC gamma 1 overexpressing NIH3T3 fibroblasts (NIH3T3 gamma 1) from Selaginella tamariscina (Selaginellaceae) together with other related biflavonoids, isocryptomerin and cryptomerin B. Only amentoflavone inhibited the PLC gamma 1 activity with an IC50 of 29 mu M and the formation of total inositol phosphates (IPt) in PDGF-stimulated NIH3T3 gamma 1 with an IC50 of 9.2 mu M but did not show inhibitory activity against protein kinase C. Isocryptomerin and cryptomerin B did not show inhibitory activity against PLC gamma 1 at the concentration of 1 50 mu M, and did not inhibit IPt production in PDGF-induced NIH3T3 gamma 1 at the concentration of 180 mu M.

For the secretion of Bacillus stearothermophilus α ‐amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus α‐amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamylase I gene (STA1). The secretion of the heterologous α‐amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total α‐amylase activity was detected in the extracellular culture medium. The secreted α‐amylase was glycosylated and its molecular weight increased from 61 kDa to 75 kDa. The thermostability of the glycosylated α‐amylase was markedly enhanced, compared with that of the non‐glycosylated enzyme from E. coli.

Herbimycin A, an inhibitor of protein tyrosine kinases, dose-dependently reduced PDGF-induced inositol phosphates (IPt) accumulation without effect on phosphatidylethanol (PEt) formation in PLC-λl-overexpressing NIH 3T3 (NIH 3T3γ1) cells. The compound also reduced tyrosine phosphorylations of some proteins including PLC-λl in response to PDGF. On the other hand, phorbol 12-myristate 13-acetate (PMA)-induced phospholipase D (PLD) activation was reduced by herbimycin A in the cells, indicating that the pathways for PLD activation by PDGF and PMA are different from each other. Also, these results suggest that PLC-y l activation is not always an upstream event for PLD activation and that tyrosine phosphorylation of one or more proteins not affected by herbimycin A should be indispensable for PLD activation in PDGF-stimulated NIH 3T3λ1 cells.

A mouse brain cDNA library was screened with a radioisotope-labeled probe of human placental protein tyrosine phosphatase. The isolated clone (MBPTP1B) was found to contain an open reading frame of 1,296 nucleotides as well as 5' (709 nucleotides) and 3' (341 nucleotides) non-coding regions. This cDNA encodes a PTP of 432 amino acids having a mass of 49,563 daltons and exhibiting 83% and 93.5% sequence identity to that of human PTP1B and rat PTP1, respectively. The expression of the cDNA in yeast was identified by western blot analysis and PTP activity assay.

The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN′ and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60°C for 30 min in the presence of 2 mM calcium chloride.