Interaction of sortase A and lipase 2 in the inhibition of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm formation

Recombinant sortase A (SrtA) was used to immune rabbit, and the inhibitory activity of anti-SrtA serum on Staphylococcus aureus biofilm formation was tested. Biofilm formation was inhibited by anti-SrtA rabbit serum in S. aureus ATCC25923 and two clinical isolated strains. The antiserum was separated into two fractions, and the main component with the inhibitory activity was demonstrated to be the IgG fraction. Two proteins interact with the IgG fraction were identified by using an in vitro pull-down assay and were confirmed to be lipase 2 and γ-hemolysin by mass spectrometry. Cross-interaction between SrtA and lipase 2 was further confirmed by Western blotting. Addition of anti-lipase 2 serum in the culture medium also showed inhibitory effect against biofilm formation. Together, our study suggests anti-SrtA serum inhibits S. aureus biofilm formation and lipase 2 is one of the targets of anti-SrtA serum in this inhibition process. This is the first study to demonstrate the roles of antisera against SrtA and lipase 2 in the inhibition of biofilm formation in S. aureus.     

The mechanism of heparin stimulation of rat adipocyte lipoprotein lipase

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.     

Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.     

入侵植物猫爪藤体外细胞毒活性成分的筛选分离

为了筛选分离入侵植物猫爪藤的细胞毒活性成分,采用MTT法以75%乙醇提取物的不同组分分别处理人肝癌细胞SMMC7721、Bel7402和正常肝细胞Chang Liver,对他们的体外增殖抑制率进行了研究。结果表明,总醇提物的氯仿组分对肝癌细胞表现出明显的体外增殖抑制作用,其次是石油醚组分。从氯仿萃取组分中分离出具有更强细胞毒活性的成分熊果酸。因此,入侵植物猫爪藤具有体外细胞毒活性,熊果酸是其体外细胞毒活性的主要成分之一。     

Aromatic Substances in Leaves of Populus tremula as Inhibitors of Mycorrhizal Fungi

The inhibitory principles of aqueous extracts of aspen leaves, acting upon mycorrhizal fungi of forest trees, were isolated by extraction with ethyl acetate and chromatography on silicic acid. Two inhibitors were identified as benzoic acid and catechol by gas chromatography and mass spectrometry. When added to a synthetic medium, these substances had a strong inhibitory effect on the growth of different Boletus-species and a weaker inhibitory effect on litter-decomposing Marasmius-species. When the fraction of the extract which was not soluble in ethyl acetate was included in the medium, the aromatic compounds still inhibited the mycorrhizal fungi while the growth of the litter-decomposers was stimulated.     

Sesquiterpene farnesol as a competitive inhibitor of lipase activity of Staphylococcus aureus

Staphylococcus aureus lipase (SAL) is known to possess broad substrate specificity for triacylglycerides. We found that a sub-minimum inhibitory concentration of farnesol (1000 mg L(-1)) inhibits this lipase activity on a Mueller-Hinton agar containing 1% Tween substrates. A quantitative lipase assay using p-nitrophenyl palmitate (pNPP) revealed that the inhibitory action of farnesol appears to be the result of the inhibition of lipase activity rather than of its secretion into the culture medium. The inhibition was observed in all the tested 8 methicillin-susceptible S. aureus and 31 methicillin-resistant S. aureus clinical isolates. Using homogeneous lipase purified by hydrophobic interaction chromatography, it was revealed that farnesol could competitively inhibit the lipase activity against the substrate pNPP.     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

Evidence for Naturally Occurring Inhibitors of Archegonial Differentiation in Lygodium japonicum

Archegonial differentiation in prothallia of Lygodium japonicum was inhibited when the filtrate of conditioned medium or the extracts of prothallia with organic solvents were added to the medium. By varying the timing of treatment with the methanol extract, archegonial differentiation was shown to start at least 4 days before microscopically detectable change. The inhibitory effect of methanol extract was nullified by transferring the treated plants to a fresh medium omitting the methanol extract, so that the archegonial formation became discernible 6 days after the transfer. The inhibitory activity was stable in both acidic and basic solutions at room temperature, and was partially lost by boiling at pH 3 or 11 for 30 min. The inhibitor, which could be retrieved from the filtrate and the methanol extract, was fractionated into the neutral ethyl acetate fraction, but was not found in the acidic ethyl acetate fraction and in the aqueous residue. At least two active zones were separated on thin layer chromatograms of the ethyl acetate extracts from the filtrate and the methanol extract, and the relative flow-rates of each active zone from these two sources were very similar. The evidence described above indicates that specific inhibitors of archegonial differentiation may be produced in the tissue of prothallia of Lygodium and eventually be secreted to the medium.     

Lipase Expression in Pseudomonas alcaligenes Is Under the Control of a Two-Component Regulatory System

Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.     

Lipoxygenase inhibitors derived from marine macroalgae

The solvent extracts from the algae Sargassum thunbergii (Sargassaceae) and Odonthalia corymbifera (Rhodomelaceae) were subjected to soybean lipoxygenase inhibitory screening. Two hydrophobic inhibitors were obtained from the extracts of S. thunbergii through inhibitory assay-guided fractionation. The inhibitors were identified as known exo-methylenic alkapolyenes (6Z,9Z,12Z,15Z)-1,6,9,12,15-henicosapentaene (1) and (6Z,9Z,12Z,15Z,18Z)-1,6,9,12,15,18-henicosahexaene (2). The alkapolyenes 1 and 2 showed higher inhibitory activity than the known inhibitor nordihydroguaiaretic acid (NDGA). Pheophytin a (3) was obtained from the extract of O. corymbifera. The inhibitor 3 also showed higher inhibitory activity than NDGA. This is the first report on lipoxygenase inhibition of exo-methylenic alkapolyenes and a chlorophyll a-related substance.     

Design,synthesis, biological evaluation and molecular modelling studies of indole glyoxylamides as a new class of potential pancreatic lipase inhibitors

A series of eighteen indole glyoxylamide analogues were synthesized, characterized and evaluated for their pancreatic lipase inhibitory activity. Porcine pancreatic lipase (Type II) was used with 4-nitrophenyl butyrate (as substrate) for the in vitro assay. Compound 8f exhibited competitive inhibition against pancreatic lipase with IC₅₀ value of 4.92 µM, comparable to that of the standard drug, orlistat (IC₅₀ = 0.99 µM). Compounds 7a-i and 8a-i were subjected to molecular docking into the active site of human PL (PDB ID: 1LPB) wherein compound 8f possessed a potential MolDock score of −153.037 kcal/mol. Molecular dynamics simulation of 8f complexed with pancreatic lipase, confirmed the role of aromatic substitution in stabilizing the ligand through hydrophobic interactions (maximum observed RMSD = 3.5 Å).     

Harmaline and hispidin from Peganum harmala and Inonotus hispidus with binding affinity to Candida rugosa lipase: In silico and in vitro studies

The inhibitory effect of phenolic compounds and alkaloids of Inonotus hispidus and Peganum harmala on Candida rugosa lipase was investigated, also, their antioxidant activities using DPPH, ABTS and phosphomolybdenum were studied in this paper. The phenolic extracts have shown a stronger antiradical activity than the alkaloids extracts. The enzymatic inhibition produced by these extracts is described here for the first time. The results have shown that the phenolic and the alkaloid extracts are good inhibitors of C. rugosa lipase. Thus, the inhibitor molecules (harmaline and hispidin) have been isolated from P. harmala and I. hispidus. Their structures were elucidated by ¹H NMR analysis. Molecular docking has been achieved using AutoDock Vina program to discuss the nature of interactions and the mechanism of inhibition. Therefore, these isolated molecules could be used in the treatment of candidiasis.     

Lipase production of Aspergillus oryzae

Aspergillus oryzae produced a small amount of lipase (0.05–0.8 U/wet-g of solid medium) in solid cultures, in contrast to the larger amount (0.46 U/ml) in a shake-flask culture in a modified GYP medium containing 2% glucose, 1% yeast extract and 2% Polypepton. Optimum conditions of lipase production in the submerged culture of A. oryzae were determined in terms of pH, composition of medium, and temperature. In a shake-flask culture at 28°C, the maximum amount of lipase increased to 0.78 U/ml upon the addition of 3% soybean oil to the modified GYP medium. In a jar fermentor culture, 30 U/ml lipase activity was obtained after 72 h at 28°C under appropriate conditions. Lipase production was greatly influenced by the culture temperature, and the optimum temperature for lipase production was about 24°C with a narrow temperature range, which was 10 degrees lower than that for the growth. In the submerged cultures, two kinds of lipase at least exhibiting different substrate specificities were also suggested.     

Lipase inhibitory activity of ethyl acetate fraction from Ecklonia cava extracts

Obesity is a key contributing risk factor to cardiovascular disease, certain cancers, and diabetes. Much effort has being made to investigate potential inhibitors against lipase from natural products. The ethyl acetate (EA) extract of Ecklonia cava (EC) were tested for their ability to inhibit pancreatic lipase activity in vitro. The 22 sub-fractions from EA extract were separated using silica gel column chromatography. Among the sub-fractions, the EA6 sub-fraction exhibited the highest inhibitory activity. Dieckol compound was isolated from the EA6 sub-fraction, which inhibited the lipase activity in a concentrationdependent manner with IC₅₀ value at 0.26 mg/mL. These results suggest that EC has potential as a natural antiobesity agent.     

A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

A simple radioactive assay system is elaborated for the measurement of dihyrofolate synthetase activity in Escherichia coli. It is also applicable to Neisseria gonorrhoeae and N. meningitidis extracts. Eight oxidized and reduced pteroate analogs have been examined for inhibitory activity. The most active inhibitor was dihydrohomopteroic acid followed by dihydro-10-thiopteroic acid, dihydrofolic acid, and dihydroisopteroic acid. The enzyme appears to be incapable of binding with substrate and any of the inhibitors in their oxidized forms.     

Measles virus inhibits acquisition of lymphocyte functions but not established effector functions

The effect of measles-virus infection on effector activities of human lymphocytes and on the generation of certain effector activities was studied in vitro. Addition of measles virus to allogeneic mixed lymphocyte cultures resulted in a strongly depressed cytolytic activity in a subsequent cell-mediated lympholysis assay. Late addition of measles virus did not inhibit cytotoxic effector function, although effector cells were probably infected. Similarly, measles-virus infection did not affect the ability of lymphocytes to mediate antibody-dependent cellular cytotoxicity. Addition of measles virus to lymphocytes with, or shortly after, exposure of the cells to the polyclonal activator pokeweed mitogen resulted in abolition of the synthesis of immunoglobulins in vitro. When the virus was added late, the rate of Ig secretion was only partially inhibited. Finally, when lymphocytes were cultured without stimulus in medium supplemented with fetal bovine serum, a population of inhibitory cells was generated. Measles virus was able to prevent the generation of such inhibitory cells. In conclusion, measles virus inhibited acquisition of various effector functions, but the activities of committed lymphocytes were generally not affected.     

A comparative kinetic study on human pancreatic and Thermomyces lanuginosa lipases: Inhibitory effects of tetrahydrolipstatin in the presence of lipid substrates

The inhibitory effects of tetrahydrolipstatin (THL) on the hydrolytic activity of human pancreatic lipase (HPL) and T. lanuginosa lipase (TLL) on various lipidic substrates ‘poisoned’ with THL as previously described was studied, using either the pH-stat, monomolecular film or oil drop technique.Prior to adding lipase (method C), an ethanolic solution of THL was injected in a tributyrin (TC4) or a purified soybean oil (PSO) emulsion prepared in a pH-stat vessel. Under these conditions, THL was found to be a potent HPL inhibitor. After being dissolved in the pure triglyceride phase (method D), THL also strongly inhibited HPL. However, with TC4 as substrate TLL was efficiently inhibited by THL only when method C was used and not method D. The very different inhibitory effects on HPL and TLL recorded with method D and PSO as substrate were confirmed using the monomolecular film and oil drop techniques.With a monomolecular film of dicaprin (di-C10) as substrate, 1 molecule of THL embedded in 400 000 molecules of di-C10 sufficed to reduce the HPL activity to half of its initial value.HPL was therefore efficiently inhibited by THL with all the methods and substrates tested here. Paradoxically, TLL was inhibited by THL molecules transiently present in the aqueous phase and not by the THL molecules present at the triglyceride/water interface. It should therefore be stressed that the inhibitory effects of THL on each lipase depend strongly on the method and the substrate used.     

Antibacterial and phytochemical studies on Calotropis gigantia (L.) R. Br. latex against selected cariogenic bacteria

In vitro antibacterial potential of the chloroform, ethyl acetate, hexane, methanol and aqueous extracts of Calotropis gigantia (L.) R. Br. was evaluated by using five cariogenic bacteria, Actinomyces viscosus, Lactobacillus acidophilus, Lactobacillus casei, Streptococcus mitis and Streptococcus mutans. Agar well diffusion method and minimum inhibitory concentration (MIC) were used for this purpose. The chloroform extracted fraction of latex showed inhibitory effect against S. mutans and L. acidophilus with MIC value of 0.032 and 0.52 mg/mL, respectively. Qualitative investigation on structure elucidation of bioactive compound using IR, NMR and GC–MS techniques revealed the presence of methyl nonanoate, a saturated fatty acid.     

Development of a reproducible method to determine minimum inhibitory concentration (MIC) of plant extract against a slow-growing mycoplasmas organism

Mycoplasma species are fastidious bacteria that require a specialized medium for their growth, isolation and identification. There are no standardized tests to evaluate the in vitro susceptibility of mycoplasmas to medicinal plant extracts. A widely used in-broth, microtitre plate, minimum inhibitory concentration (MIC) assay was adapted and evaluated using acetone extracts of Anoigeissus leiocarpus on the isolates of Mycoplasma mycoides subsp. mycoides small colony variants (MmmSC). Several problems were encountered including the contamination of the medium by Bacillus species found in plants and the fact that the slow-growing mycoplasmas proved to be poor reducers of the indicator tetrazolium salt or resorcinol. We then examined a pH indicator-dependant technique to detect the acid production caused by the growth of the organism after glucose utilization from the broth medium. The method gives a clear cut-off point that was easy to read and interpret and was also reproducible.The MIC value for acetone extract of A. leiocarpus was 0.16 mg/ml. The development of this method now makes it possible to evaluate extracts of several plant species for antimycoplasmal activity.     

Purification and properties of adenosine 3′,5′-monophosphate phosphodiesterase from baker's yeast

Cyclic AMP phosphodiesterase from Saccharomyces cerevisiae was purified about 20,000-fold to homogeneity. The purified enzyme had a molecular weight of about 60,000 as estimated by gel filtration.The enzyme activity was optimal at pH 8.5–9.0 and was not stimulated by imidazole. Among cyclic 3′,5′-nucleotides, cyclic AMP was the most active substrate for the purified enzyme (K_m = 0.25 mM), but it was inhibitory at concentrations above 4 mm. N⁶,O^2′-dibutyryl cyclic AMP was not hydrolyzed at all.Unlike other cyclic AMP phosphodiesterases from various sources, the purified yeast enzyme did not require divalent metal ions for maximal activity and was rather inhibited in various degrees by added metal ions. The enzyme was not very sensitive to thiol inhibitors.The purified yeast enzyme was strongly inhibited by theophylline and slightly by caffeine. In contrast to the enzyme from S. carlsbergensis, the enzyme from S. cerevisiae was not inhibited at all by ATP or PP_i.The enzyme activity was not released into the growth medium, and the intracellular distribution studies indicated that the enzyme was located mainly in the cytosol fraction.