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.     

Ileal Loop Fluid Accumulation and Production of Diarrhea in Rabbits by Cell-free Products of Clostridium perfringens

The ability of cell extracts and culture filtrates of various strains of C. perfringens to produce ileal loop fluid accumulation and overt diarrhea in rabbits was tested. Good correlation was obtained in the ability of whole cells and a toxic factor (present in cell extracts and concentrated culture filtrates) to produce both fluid accumulation in ileal loops and diarrhea when injected into the normal ileum of the rabbit. The toxic factor was present in cell-free preparations when cells were grown in a sporulation medium, but not when they were grown in an asporogenic medium. The factor was shown to be heat labile, nondialyzable, and was inactivated by Pronase but not by trypsin, lipase, or amylase. Loss of activity occurred at pH 1.0, 3.0, 5.0, and 12.0.     

Evidence for independent genetic regulation of heart and adipose lipoprotein lipase activity

The relationship between the genes controlling heart and adipose lipoprotein lipase in fasted animals has been studied. 32 inbred mouse strains were tested for variations in heart or adipose specific activity and thermolability. The survey revealed that specific activity of heart and adipose lipoprotein lipase varied as much as 3-fold and 20-fold, respectively. In thermolability, up to a 2-fold variation was observed in the lipase in each tissue. The correlation coefficient between variations in heart and adipose lipase was apparently not significant for both parameters studied. Additional studies were performed in two strains, BALB/c and C57BL/6, along with the recombinant inbred set derived from them. The two strains did not show genetic variation for lipoprotein lipase thermolability, although the inactivation rate of heart lipase was higher than that of adipose lipase. However, BALB/c and C57BL/6 displayed significant differences in their levels of lipoprotein lipase specific activity. Thus, strain C57BL/6 showed higher heart activity when compared to BALB/c, whereas the latter showed higher adipose lipase activity when compared to C57BL/6, i.e. an inverse relationship. The specific activity levels of heart and adipose lipoprotein lipase in the recombinant inbred strains derived from BALB/c and C57BL/6 exhibited independent inheritance. Thus, in adipose tissue, a single major gene seems to control the variation observed, while the inheritance pattern of heart activity could imply involvement of more than one gene. Moreover, two out of the seven recombinant strains showed distinct recombinant phenotypes, indicating that separate unlinked genes control the variations found in heart and adipose activity. We conclude that the expression of heart and adipose lipoprotein lipase activity is under independent genetic control.     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。     

傣药植物内生真菌的抑菌活性评价及发酵条件探索

从云南傣药植物中分离到180株内生真菌,选用9种培养基进行发酵。利用茄腐镰刀菌、尖孢镰刀菌、立枯丝核菌、玉米小斑病、稻梨孢菌等5种植物病原菌作为指示菌株,结合TLC检测对其发酵粗提物进行活性评价和化学多样性分析,以期寻找到具有开发潜力的活性菌株。研究结果表明活性菌株为36株,其中有7株菌活性好且产物多样性丰富。     

Antifungal and antibacterial activities of indigenous <Emphasis Type="Italic">Streptomyces</Emphasis> isolates from saline farmlands: prescreening,ribotyping and metabolic diversity

A culture collection of 110 indigenous Streptomyces strains originally isolated from saline farmlands (Punjab, Pakistan) using stringent methods was screened biologically and chemically to investigate their potential for the production of bioactive secondary metabolites. In a biological screening the crude extracts obtained from the culture broth of selected strains were analysed for their activity against a set of test organisms, including Gram-positive, Gram-negative bacteria, fungi and microalgae using the disk diffusion bioassay method. Additionally a cytotoxicity test was performed by means of the brine shrimp microwell cytotoxicity assay. In a chemical screening each of the crude extracts was analysed by TLC using various staining reagents and by HPLC-MS/MS measurements. The results depicted an impressive chemical diversity of crude extracts produced by these strains. The taxonomic status of the selected strains was confirmed by preliminary physiological testing and 16S rRNA gene sequencing.     

Isolation and characterization of some moderately halophilic bacteria with lipase activity

Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group of biocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from the Maharla salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 ± 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from the Maharla lake.     

Studies on the hormone-sensitive lipase of adipose tissue

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.     

Lipases in autolysed cultures of filamentous fungi

Fifty-one fungi from different genera and strains were checked in plate to determine lipase activity in protein precipitates from their autolysed cultures. Each of them was then analysed at 3·5, 6·5 and 9·2 pH units and, as a consequence, basic lipases with high activity at 9·2 pH were found after 1 h of incubation. Only 25% of the studied fungi showed this lipase activity, among them the best producers were fungi from genus Fusarium (47% of fungi had lipase activity). In addition to lipase activity, Fusaria showed a low hydrolytic activity on cutin and suberin. The genus Aspergillus produced lipase and cutinase activity to a similar extent. Aspergillus nidulans 2544 also showed suberinase activity in a considerable amount. Penicillium species had very low activities. Other species and strains from genus Trichoderma , order Mucorales and class Basidiomycetes, did not show lipase activity in their degradative processes.     

Comparative immunological studies on arylsulfatase in bacteria of the family Enterobacteriaceae: occurrence of latent arylsulfatase protein regulated by sulfur compounds and tyramine.

The arylsulfatases of 21 strains of the family Enterobacteriaceae were compared by measuring their enzymatic activities and immunological reactivities. Enzyme formation under repressing, nonrepressing, and derepressing conditions was tested. Antiserum prepared against pure arylsulfatase from Klebsiella aerobgenes W70 was tested against the enzyme extracts from the strains using double diffusion, quantitative precipitation, and immunoelectrophoresis. No close relationship was found between arylsulfatase activity and immunological cross-reactionship was found between arylsulfatase activity and immunological cross-reactivity. The strains in the family Enterobacteriaceae could be divided into two groups on the basis of the immunological properties of their enzyme. Antisera formed a precipitin band with both active and inactive enzyme proteins from Escherichia, Citrobacter, Salmonella, Klebsiella, and Enterobacter, but not with the proteins from Serratia, Proteus, and Erwinia, even though some strains of these species had enzyme activity. It was also found that the formation of arylsulfatase proteins, irrespective of whether they had enzyme activity, were under regulation by sulfur compounds and tyramine.     

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca²⁺, Zn²⁺, Mg²⁺) - with the exception of Co²⁺— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.     

Inhibitors of lipase activities in soybean and other oil seeds

In the cotyledon extracts of seedlings of many oil seeds, including soybean, sunflower, cucumber, and peanut, the in vitro lipase activity was too low to account for the observed in vivo lipolysis. The low in vitro lipase activity was due to the presence of lipase inhibitors in the extracts. The inhibitors from soybean were characterized based on their effects on the hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases. The inhibitors were not dialyzable and unaltered by RNase and β-galactosidase treatment. However, they were sensitive to heating and protease digestion. The inhibitory effect of the inhibitors was expressed irrespective of the sequence of the addition of lipase, substrate, and inhibitors to the assay medium. The inhibitory effect was equally expressed when the inhibitors were added either before or after the lipase reaction had been in progress. The inhibitory effect of the inhibitors was independent of the amount of lipase present in the assay, but was dependent on the amount of substrate added. High substrate concentration eliminated totally the inhibitory effect of the inhibitors. Most of the inhibitors were recovered in the soluble fraction in subcellular fractionation. They were present in the 2-4S and not in the 7S, and 11S (storage proteins) protein fraction. There was a gradual decrease of the inhibitors in the cotyledons in the postgerminative growth. We suggest that the inhibitors are proteins which bind to the surface of the substrate micelles. The binding prevents the normal functioning of lipase which acts on the interfacial area between the aqueous medium and the micelle surface.     

Inhibition of Candida rugosa lipase by berberine and structurally related alkaloids, evaluated by high-performance liquid chromatography.

It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478-1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with beta-naphthyl laurate, and beta-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of beta-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10-16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.     

Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid

Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

In vitro antibacterial activity of Chilean red wines against Helicobacter pylori

The antibacterial activity of sixteen Chilean red wines (Cabernet Sauvignon, Cabernet Merlot, Cabernet Organic and Pinot Noir), and the active extracts of two randomly selected wines were assayed for their antibacterial activity on six strains of Helicobacter pylori isolated from gastric biopsies. The active fraction of the wines was obtained by dichloromethane extraction, and the antibacterial activity of the wines and extracts was evaluated by an agar diffusion method. All the red wines studied showed some antibacterial activity on the six strains of H. pylori, although the strains were heterogeneous in their susceptibility to each particular wine. The active fraction of the two wines selected also showed good activity against the strains tested. The main active compound was identified as resveratrol. The results presented indicate that Chilean red wines have antibacterial activity against H. pylori, which depends mainly on the presence of resveratrol.     

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Acid Lipase of Castor Bean Lipid Bodies : Isolation and Characterisation

The acid lipase of castor endosperm lipid bodies has been studied using colorimetric assay based on the measure of the hydrolytic activity of p-nitrophenyl ester of palmitate and other acyl derivatives. These substrates are compatible with the natural triacylglycerols for the measure of lipolytic activities. The subcellularly-surveyed acid lipolytic activity in the germinated castor bean endospermal tissue was found to be enhanced in the lipid bodies. The lipase, which is partially latent and tightly associated with lipid bodies, is an exceptionally stable enzyme with an optimum activity at pH 4.5 and displays an inverse relationship between its activity and the acyl chain length of its substrate. To facilitate isolation of the acid lipase, a procedure has been developed to solubilise the membrane-bound enzyme in an active form. The detergent-solubilised acid lipase after two chromatographic steps yielded an eight-fold active preparation which after gel permeation resolved as heterogeneous aggregate in excess of 500 kD. Lipase-enriched preparations showed consistent presence of 14 and 60 kD proteins which constituted the most abundant species of the lipid bodies. Although it has not been possible to obtain an active lipase preparation in a state free of either the 14 or 60 kD protein, the lipase activity in the detergent extracts of lipid bodies was immunoprecipitable with antibodies raised against the 60 kD component.     

Production of thermostable lipolytic activity by thermus species

A quantitative screening for intra- and extracellular lipolytic activity was performed in submerged cultures of four Thermus strains using two different media (named T or D medium). Major differences in the extracellular lipolytic activity were observed in T medium, the highest values being for Thermus thermophilus HB27 and Thermus aquaticus YT1 strains (18 and 33 U/L, respectively). Two enzymes with lipase/esterase activity were identified in the four Thermus strains by zymogram analysis, with molecular weights of 34 and 62 kDa. No kinetic typification of the enzymes as primary metabolites was possible for any of the Thermus strains, because of the lack of a good fitting of the experimental lipolytic activity production rates to the Luedecking and Piret model. However, a linear relationship was found between the absolute values of biomass and total lipase/esterase activity (sum of intracellular and extracellular). For T. thermophilus HB27, an increase in the aeration rate caused the increase in the production of biomass and, particularly, intracellular lipolytic activity but the extracellular lipolytic activity was not affected except for the series with the strongest oxygen limitation. Transmission electronic microscopy revealed that T. thermophilus HB27 formed rotund bodies surrounded by a common membrane in cultures in the early stationary phase. The results suggest the occurrence of a specific mechanism of lipase/esterase secretion that might be due to the different composition and permeability of the cell membranes and those surrounding the rotund bodies.