Zinc inhibition of electron transfer: mechanism of beta receptor inhibition?

Zinc has been shown to inhibit β-receptor activation of adenylate cyclase at a post receptor site. We have postulated that the β-receptor is one of several receptors activated by reduction, followed by transmembrane elector transfer accelerated by GTP. GTP accelerates electron transfer in a model system and this accelerated electron transfer is inhibited by zinc. This could explain the mechanism of the post receptor inhibition by zinc of the adenylate cyclase stimulation which follows β-receptor activation.     

Growth inhibition of pathogenic yeast isolates by α-difluoromethylornithine: An inhibition of ornithine decarboxylase

A large body of evidence exists suggesting that polyamines can play essential roles in cellular growth and differentiation. We examined the ability of -difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, the major rate-limiting enzyme in polyamine biosynthesis, to inhibit the growth of Candida albicans, C. tropicalis, and C. parapsilosis. Substantial growth-inhibition was observed for all three species at DFMO concentrations ranging from 1 to 100 mM. C. tropicalis was significantly more susceptible to DFMO than C. albicans or C. parapsilosis. Depletion of cellular polyamine pools was seen in all 3 species following exposure to DFMO and polyamine depletion enhanced the susceptibility of the organisms to DFMO. The action of DFMO was specifically antagonized by exogenous polyamines. These data suggest that polyamines are important in the growth of Candida spp. and that inhibitors of polyamine biosynthesis may be useful as antifungal agents.     

Suppression of endothelial cell activity by inhibition of TNFα

Introduction

TNFα is a proinflammatory cytokine that plays a central role in the pathogenesis of rheumatoid arthritis (RA). We investigated the effects of certolizumab pegol, a TNFα blocker, on endothelial cell function and angiogenesis.

Methods

Human dermal microvascular endothelial cells (HMVECs) were stimulated with TNFα with or without certolizumab pegol. TNFα-induced adhesion molecule expression and angiogenic chemokine secretion were measured by cell surface ELISA and angiogenic chemokine ELISA, respectively. We also examined the effect of certolizumab pegol on TNFα-induced myeloid human promyelocytic leukemia (HL-60) cell adhesion to HMVECs, as well as blood vessels in RA synovial tissue using the Stamper-Woodruff assay. Lastly, we performed HMVEC chemotaxis, and tube formation.

Results

Certolizumab pegol significantly blocked TNFα-induced HMVEC cell surface angiogenic E-selectin, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression and angiogenic chemokine secretion (P < 0.05). We found that certolizumab pegol significantly inhibited TNFα-induced HL-60 cell adhesion to HMVECs (P < 0.05), and blocked HL-60 cell adhesion to RA synovial tissue vasculature (P < 0.05). TNFα also enhanced HMVEC chemotaxis compared with the negative control group (P < 0.05) and this chemotactic response was significantly reduced by certolizumab pegol (P < 0.05). Certolizumab pegol inhibited TNFα-induced HMVEC tube formation on Matrigel (P < 0.05).

Conclusion

Our data support the hypothesis that certolizumab pegol inhibits TNFα-dependent leukocyte adhesion and angiogenesis, probably via inhibition of angiogenic adhesion molecule expression and angiogenic chemokine secretion.     

Some aspects of mechanism of inhibition of cholesterol absorption by β-sitosterol

(1) Mixed bile salt micelle solubilized either cholesterol or β-sitosterol to a comparable extent. When added simultaneously, β-sitosterol restricted the micellar solubility of cholesterol. (2) β-Sitosterol also reduced the cholesterol content in the aqueous (micellar) phase of the intestinal contents of rats, the extent of reduction being comparable with that observed in vitro. The intestinal uptake of cholesterol in vivo was equivalent to the micellar incorporation of cholesterol both in vitro and in vivo. (3) β-Sitosterol had no inhibitory effect on cholesterol absorption from the micellar solution in jejunal loops in situ, whereas the rate of β-sitosterol uptake was only about one-fifth that of cholesterol. (4) The intestinal uptake of β-sitosterol intubated into the stomach of rats was about one-fifth that of cholesterol. The intestinal brush-border membrane discriminated these sterols. These results suggest that the restriction of the micellar solubility of cholesterol, rather than the inhibition of uptake from brush-border membrane, is the major determinant for the interference of β-sitosterol with cholesterol absorption.     

Contact inhibition of what? An analytical review

Quite a number of phenomena having to do with cells' influences upon one another's movements have come to be regarded as expressions of “contact inhibition.” However, no single, central mechanism has been shown to underlie them all. Consequently, the term “contact inhibition” should not be used without operational modifiers. Inhibitions of individual cell movements imputed to be mediated by cell-cell contacts include inhibition of overlapping (which results in monolayering), of colony expansion, of cell speed (nuclear translocation), of ruffling, of orthogonal movement (proposed to explain spontaneous parallel alignment of cells), and of neighbor exchanges. The six inhibitions listed above are operationally distinct, and only two (overlapping and colony expansion) are known to result from a common mechanism. A seventh phenomenon, so-called “contact inhibition of cell division” (more operationally termed postconfluence inhibition of cell division) is in a separate category and is not considered here. Evidence eliminating action-at-a-distance is available only for the first three, and hence only these should at present be termed contact inhibitions. Inhibition of neighbor exchanges is yet hypothetical; at its extreme, it would immobilize cells in a confluent monolayer, but such immobilization has been found not to occur. Contact inhibition of overlapping, the most studied of the six, is not displayed by invasive cells with respect to normal cells; invasive tumor cells overlap freely upon normal cells, although not necessarily upon one another. Contact inhibition of overlapping, and its loss by invasive cells, can readily be interpreted, by means of the differential adhesion hypothesis, as consequences of cell-type-specific differences in cell-cell and cell-substratum “strengths of adhesion.” These strengths of adhesion are formulated as specific interfacial free energies, which are the only parameters of cellular adhesiveness that have been rigorously shown to determine equilibrium configurations of cell populations.     

Human tumour-induced inhibition of interferon action in vitro: Reversal of inhibition by β-carotene (pro-vitamin A)

Inhibitors of human interferon action that might be relevant to tumour resistance or escape mechanisms were investigated in a macrophage system. The effects of IFN on macrophage Fc gamma receptor expression were inhibited by three preparations: (1) a low-molecular-weight component of normal autologous serum; (2) a low-molecular-weight component of carcinoma supernatant; and (3) physiological concentrations of retinol and retinoic acid. Since human carcinoma tissue contains abnormally high levels of retinoic acid-binding protein, the possibility that a tumour-associated retinoid contributes to tumour-induced inhibition in vitro was investigated. Inhibition of IFN action in vitro by retinoic acid (vitamin A acid) was found to be reversed by beta-carotene (pro-vitamin A). When tested in the tumour system beta-carotene also reversed inhibition by the human-carcinoma-derived signal. These data are consistent with the view that at least one of the tumour-derived signals inhibitory towards IFN is a tumour-associated retinoid, although firm evidence for this must await further physicochemical characterization of the inhibitory signal(s). The present data clearly show, nevertheless, that human tumour-induced inhibition of IFN in vitro can be reversed by the pro-vitamin beta-carotene.     

Ejection of structural zinc leads to inhibition of γ-butyrobetaine hydroxylase

γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II) dependent oxygenase that catalyses an essential step during carnitine biosynthesis in animals. BBOX is inhibited by ejection of structural zinc by a set of selenium containing analogues. Previous structural analyses indicated that an undisrupted N-terminal zinc binding domain of BBOX is required for catalysis. Ebselen is a relatively potent BBOX inhibitor, an observation which may in part reflect its cardioprotective properties.     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

In vitro evaluation of single- and multi-strain probiotics: Inter-species inhibition between probiotic strains, and inhibition of pathogens

Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit Clostridium difficile, Escherichia coli and S. typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p < 0.05 or less) greater inhibition of pathogens in 12 out of 24 cases. Despite evidence that probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain.     

Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)_2–4, were applied. The k_cat/K_m values for (GlcNAc)_2–4 are 239.8, 111.3 and 79.8 s^?1 mM^?1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.     

“Glial inhibition” of memory in Alzheimer's disease

<正>Two recent studies on Nature Medicine and Nature Communications reported the unusual roles of astrocytes in the brain,that is,release gamma-aminobutyric acid(GABA)and impair hippocampal memory in Alzheimer’s disease(AD).AD represents one common form of neurodegenerative diseases,and is characterized by progressive dementia.The pathological feature of an AD brain includes the appearance     

Substrate inhibition and affinities of the glyoxysomal β-oxidation of sunflower cotyledons

During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C₄ substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999     

Lysine inhibition of Saccharomyces cerevisiae: role of repressible L-lysine ε-aminotransferase

Lysine added to grain mashes under nitrogen-limiting conditions (as in most industrial fermentations) inhibited growth of Saccharomyces cerevisiae. This inhibition was relieved by raising the assimilable nitrogen content. Lysine-induced inhibition is not mediated through accumulation of -oxoadipic acid, an intermediate of lysine metabolism which accumulates by a back up of intermediates in de novo synthesis. Lysine degradation is regulated by the synthesis of L-lysine -aminotransferase, an enzyme that catalyses the first step in one of three possible routes of lysine degradation (not previously reported in S. cerevisiae). Synthesis is repressed under nitrogenlimiting conditions, but derepressed when excess assimilable nitrogen is available. Derepression results in degradation of lysine and decreases inhibitory effects on growth. The toxic compound appears to be lysine itself.The authors are with the Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0W0. Canada     

Mechanism of inhibition of mitochondrial ATP synthase by 17β−Estradiol

17β-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling (“slipping”) of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and V_FCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and V_olig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the F_o component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F₁-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.     

Structure–activity relationships of the thujaplicins for inhibition of human tyrosinase

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, β, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC₅₀ of 1.15 μM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC₅₀ = 571.17 μM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.     

Effect of protein synthesis inhibition on recovery of UV- and γ-irradiated Schizosaccharomyces pombe from repair inhibition by caffeine

Summary The progress of repair in Schizosaccharomyces pombe may be followed during post-irradiation incubation by measuring, after various intervals, the ability of UV- or -irradiated cells to avoid enhanced lethality when exposed to the repair inhibitor caffeine (Gentner and Werner, 1975). This technique has now been used to investigate the effect of inhibition of protein synthesis on repair of UV- and -irradiation-induced damage in this organism.When protein synthesis was inhibited with cycloheximide in UV-irradiated wild-type cells, only a small amount of recovery from caffeine inhibition occurred; this indicated that post-irradiation protein synthesis was required for repair, and in particular for the recombinational repair pathway, which is a major mechanism for repair of UV damage in this organism.In -irradiated wild-type cells, inhibition of post-irradiation protein synthesis reduced the rate of recovery from repair inhibition by caffeine, but full recovery from caffeine-sensitive damage did occur at longer incubation times. We attribute the reduction in rate to the effect of protein synthesis inhibition on the recombinational repair pathway, because this pathway is known to be involved in the repair of both -ray and UV damage. The recovery that took place at the slower rate must reflect a caffeine-sensitive pathway which is involved only in repair of -ray damage and which does not require post-irradiation protein synthesis for activity.AECL Reference No. 5402     

Synthesis and α-glucosidase inhibition activity of dihydroxy pyrrolidines

A new series of Deacetylsarmentamide A and B derivatives, amides and sulfonamides of 3,4-dihydroxypyrrolidines as α-glucosidase inhibitors were designed and synthesized. The biological screening test against α-glucosidase showed that some of these compounds have the positive inhibitory activity against α-glucosidase. Saturated aliphatic amides were more potent than the olefinic amides. Among all the compounds, 5o/6o having polar –NH₂ group, 10f/11f having polar –OH group on phenyl ring displayed 3–4-fold more potent than the standard drugs. Acarbose, Voglibose and Miglitol were used as standard references. The promising compounds 6i, 5o, 6o, 10a, 11a, 10f and 11f have been identified. Molecular docking simulations were done for compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.