Ferulic acid improves glucose homeostasis by modulation of key diabetogenic activities and restoration of pancreatic architecture in diabetic rats.

There are increasing concerns on the rising cases of diabetes mellitus with type 2 diabetes (T2D) being of major interest as well as the cost of its treatment. Plant phenolic compounds are natural and potent antioxidants that have been widely reported for their antidiabetic activities properties, one of which is ferulic acid. The effect of ferulic acid (FA) on major diabetogenic activities and pancreatic architecture linked to T2D was investigated in T2D rats. T2D was induced in male Sprague-Dawley rats using the fructose-streptozotocin model. Diabetic rats were treated with FA at 150 or 300 mg/kg bodyweight (bw). Normal control consisted of rats administered with food and water, while diabetic control consisted of untreated diabetic rats. Metformin was used as the standard drug. The rats were humanely sacrificed after 5 weeks of treatment. Their blood, liver, and pancreas were collected for analysis. Total glycogen content and carbohydrate metabolic enzymes activities were analyzed in the liver, while the pancreas and serum from blood were analyzed for oxidative stress biomarkers, purinergic and cholinergic enzyme activities, and amylase and lipase activities. The pancreatic tissue was further subjected to microscopic and histological examinations. FA caused a significant (p < 0.05) decrease in blood glucose level, with concomitant increase in serum insulin level. Treatment with FA also led to elevated levels of GSH, HDL-c, SOD, and catalase activities, while concomitantly suppressing malondialdehyde, cholesterol, triglyceride, LDL-c, NO, ALT, AST, creatinine, urea, and uric acid levels, acetylcholinesterase, ATPase, ENTPDase, 5'-nucleotidase, lipase, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-biphosphatase activities. Histology analysis revealed an intact pancreatic morphology in FA-treated diabetic rats. While transmission electron microscopy (TEM) analysis revealed an intact pancreatic ultrastructure and increased number of insulin granules in ß-cells. Taken together, these results portray that the antidiabetic potentials of ferulic acid involves modulation of major diabetogenic activities and maintenance of the pancreatic ultrastructure architecture.

Selenium and vitamin B6 cosupplementation improves dyslipidemia and fatty liver syndrome by SIRT1/SREBP-1c pathway in hyperlipidemic Sprague-Dawley rats induced by high-fat diet.

Previously, our group found that the dietary trace mineral element selenium and vitamin B6 (VitB6) alone was involved in lipid metabolism. However, the effects of selenium combined with VitB6 on hyperlipidemia and lipid metabolism have not been reported until now. We hypothesized that selenium and VitB6 cosupplementation would alleviate the hyperlipidemic and hepatic dysfunction and with minimum side effects in a Sprague-Dawley rat model of hyperlipidemia induced by a high-fat diet. Our results showed that selenium combined with VitB6 could improve dyslipidemia and displayed better in vivo hypocholesterolemic abilities at early intervention. Moreover, cosupplementation reduced atherogenic indexes (atherogenic index and atherogenic index of plasm) and the ratio of ApoB/ApoA1. The liver function index aspartate aminotransferase in serum was reduced, as was and total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol in liver. The intervention also increased the levels of ApoA1 in serum and high-density lipoprotein cholesterol of liver. In addition, the combination of selenium and VitB6 decreased liver lipid deposition and alleviated steatosis, reduced adipocyte size of white adipose tissue, increased the activities of hepatic lipase and total lipase and the hepatic 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) level, decreased the hepatic mRNA transcription of lipogenic and regulatory genes including Srebf1 and downstream fat synthesis-related enzymes (Acc and Fasn) and cholesterol synthesis speed limiting enzyme Hmgr, increased the mRNA abundance of Lcat and Cyp7a1, increased the protein expression of SIRT1 and PPARα, and up-regulated the protein expression of sterol regulatory element-binding protein 1c in the livers of hyperlipidemia rats. We first demonstrated that oral selenium and VitB6 cosupplementation exerted synergism in lowering blood and liver lipid profiles and antiatherosclerotic effects in hyperlipidemic rats by reducing endogenous cholesterol and lipid synthesis, enhancing the transport of cholesterol to hepatocytes and promoting fatty acid beta oxidation.

Zwitterionic meso-silica/polypeptide hybrid nanoparticles for efficient azithromycin delivery and photodynamic therapy for synergistic treatment of drug-resistant bacterial infection.

The treatment of drug-resistant bacterial infections attributed to the overuse of antibiotics still remains a serious challenge globally. Herein, zwitterionic charge switchable meso-silica/polypeptide hybrid nanoparticles (MSPNs) were prepared for the synergistic chemo-photodynamic therapy in the treatment of drug-resistant bacterial infections. Subsequently, azithromycin (AZT) and methylene blue (MB) were loaded in the MSPNs to form the combined chemo-photodynamic therapeutic nanoparticles (MSPNs-AZT/MB) for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Remarkably, the as-prepared MSPNs-AZT/MB exhibited a negative surface charge of -5.2 mV at physiological pH while switching into positive surface charge of 24.7 mv in an acidic environment, leading to enhanced binding with bacterial surface. The lipase-triggered AZT release up to 77.9 % was achieved, and the loaded MB demonstrated efficient singlet oxygen (1O2) generation for photodynamic therapy. The in vitro experimental results displayed an excellent antibacterial effect against MRSA in both planktonic and biofilm phenotypes. Additionally, the as-prepared MSPNs-AZT/MB exhibited synergistic and enhanced antibacterial infection effect up to 94 % comparing to monotherapy in a mice model. Considering the above advantages, the as-prepared combined chemo-photodynamic therapeutic nanoparticles showed promising biocompatibility and clinical potential for the efficient therapy of drug-resistant bacteria.

Effect of exogenous lipase on subcutaneous adipose tissue in a porcine animal model.

BACKGROUND: Topical exogenous lipase has been approved for cosmetic use and has been used to mobilize fat from adipocytes. The objective of this study was to determine the effects of exogenous lipase in the subcutaneous adipose tissue. METHODS: Three different concentrations of exogenous lipase 1× (2 Units per ml), 5× (10 units per ml), and 10× (20 units per ml) were applied in a porcine model. Normal saline (NS) solution (as negative control) and phosphatidylcholine (as positive control) were also injected. Skin and subcutaneous tissue biopsies, up to the fascia, were obtained from each injection site on the 3rd day after injection. The number of cells per 20× field was counted as an indirect measurement of the size of the adipocytes. RESULTS: For 1× lipase, the number of cells per field was 47.80 (±7.63) versus 27.26 (±4.93), and 34.66 (±6.84) for NS, and phosphatidylcholine, respectively. For 5× lipase, the count was 36.06 (±4.74) versus 24.13 (±5.18), and 33.2 (±9.34). For 10× lipase, it was 40.06 (±4.35) versus 29.26 (±2.34) and 32.66 (±6.30) (p < .05 for all groups). CONCLUSIONS: A higher number of cells per field were observed in the lipase samples, inferring a decreased volume of adipocytes. No inflammation and/or loss of cell architecture were evidenced in the exogenous lipase groups.

Aqueous extract of Psidium guajava leaves: phenolic compounds and inhibitory potential on digestive enzymes.

Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.

Optimisation of combi-lipases from Aspergillus niger for the synergistic and efficient hydrolysis of soybean oil.

The enzymatic properties of four lipases (A, B, C and D) from different strains of Aspergillus niger, were investigated, and a 3-factor mixture design and triangular surface analysis were performed to screen the optimal combi-lipase by observing synergistic effects. Lipases B and D differed in optimal pH, temperature and substrate specificity. A combi-lipase with 31.2% lipase B and 68.8% lipase D (w/w, equal to units of 30.36% and 69.64%) exhibited optimal hydrolytic activity on soybean oil, which exceeded the sum of the combined activities of individual lipases (P < 0.05). Free fatty acid from the hydrolyzed soybean oil indicated that the synergistic effect of the combi-lipase resulted from the different fatty acid specificities of the two lipases. Overall, combi-lipase afforded an effective route for the application of lipase enzymes to animal feeds.

Aqueous extract of Psidium guajava leaves: phenolic compounds and inhibitory potential on digestive enzymes

ABSTRACT Leaves of Psidium guajava L. (guava) have been widely used in the popular way for prevention and treatment of various diseases. Thus, the objective of this study was to evaluate the inhibitory potential of leaves aqueous extract from three cultivars of P. guajava (Pedro Sato, Paluma and Século XXI) on α-amylase, α-glycosidase, lipase, and trypsin enzymes, in the presence or not of simulated gastric fluid and to determine the content of phenolic compounds by high performance liquid chromatography. All cultivars presented the same composition in phenolic compounds, but in different proportions. The compounds identified are gallic acid, epigallocatechin gallate, syringic acid, o-coumaric acid, resveratrol, quercetin, and catechin (which was the major compound in all the cultivars evaluated). In the absence of simulated gastric fluid, it was observed different inhibitions exercised by the leaves aqueous extracts from three cultivars of P. guajava on each enzyme. In presence of simulated gastric fluid, all cultivars showed increase in the inhibition of lipase and α-glycosidase, and decrease in inhibition of α-amylase and trypsin enzymes. These results indicate that P. guajava leaves aqueous extracts from all cultivars evaluated possess potential of use as an adjuvant in the treatment of obesity and other dyslipidemias.

A Novel Lipase as Aquafeed Additive for Warm-Water Aquaculture.

A novel Acinetobacter lipase gene lipG1 was cloned from DNA extracted from intestinal sample of common carp (Cyprinus carpio), and expressed in E. coli BL21. The encoded protein was 406 amino acids in length. Phylogenetic analysis indicated that LipG1 and its relatives comprised a novel group of true lipases produced by Gram-negative bacteria. LipG1 showed maximal activity at 40℃ and pH 8.0 when pNP decanoate (C10) was used as the substrate, and remained high activity between 20℃ and 35℃. Activity of the lipase was promoted by Ca2+ and Mg2+, and inhibited by Zn2+ and Cu2+. Moreover, LipG1 is stable with proteases, most commercial detergents and organic solvents. Substrate specificity test indicated that LipG1 can hydrolyse pNP esters with acyl chain length from C2 to C16, with preference for medium-chain pNP esters (C8, C10). Lastly, LipG1 was evaluated as an aquafeed additive for juvenile common carp (Cyprinus carpio). Results showed that supplementation of LipG1 significantly improved the gut and heptaopancreas lipase activity of fish fed with palm oil diet. Consistently, improved feed conversion ratio and growth performance were recorded in the LipG1 feeding group, to levels comparable to the group of fish fed with soybean oil diet. Collectively, LipG1 exhibited good potential as an aquafeed additive enzyme, and deserves further characterization as the representative of a novel group of lipases.

A piglet with surgically induced exocrine pancreatic insufficiency as an animal model of newborns to study fat digestion.

The maldigestion and malabsorption of fat in infants fed milk formula results due to the minimal production of pancreatic lipase. Thus, to investigate lipid digestion and absorption and mimic the situation in newborns, a young porcine exocrine pancreatic insufficient (EPI) model was adapted and validated in the present study. A total of thirteen EPI pigs, aged 8 weeks old, were randomised into three groups and fed either a milk-based formula or a milk-based formula supplemented with either bacterial or fungal lipase. Digestion and absorption of fat was directly correlated with the addition of lipases as demonstrated by a 30% increase in the coefficient of fat absorption. In comparison to the control group, a 40 and 25% reduction in total fat content and 26 and 45% reduction in n-3 and n-6 fatty acid (FA) content in the stool was observed for lipases 1 and 2, respectively. Improved fat absorption was reflected in the blood levels of lipid parameters. During the experiment, only a very slight gain in body weight was observed in EPI piglets, which can be explained by the absence of pancreatic protease and amylase in the gastrointestinal tract. This is similar to newborn babies that have reduced physiological function of exocrine pancreas. In conclusion, we postulate that the EPI pig model fed with infant formula mimics the growth and lipid digestion and absorption in human neonates and can be used to elucidate further importance of fat and FA in the development and growth of newborns, as well as for testing novel formula compositions.

Combination of enzymes and flow perfusion conditions improves osteogenic differentiation of bone marrow stromal cells cultured upon starch/poly(epsilon-caprolactone) fiber meshes.

Previous studies have shown that alpha-amylase and lipase are capable of enhancing the degradation of fiber meshes blends of starch and poly(epsilon-caprolactone) (SPCL) under dynamic conditions, and consequently to promote the proliferation and osteogenic differentiation of bone marrow stromal cells (MSCs). This study investigated the effect of flow perfusion bioreactor culture in combination with enzymes on the osteogenic differentiation of MSCs. SPCL fiber meshes were seeded with MSCs and cultured with osteogenic medium supplemented with alpha-amylase, lipase, or a combination of the two for 8 or 16 days using static or flow conditions. Lipase and its combination with alpha-amylase enhanced cell proliferation after 16 days. In addition, the flow perfusion culture enhanced the infiltration of cells and facilitated greater distribution of extracellular matrix (ECM) throughout the scaffolds in the presence/absence of enzymes. A significant amount of calcium was detected after 16 days in all groups cultured in flow conditions compared with static cultures. Nevertheless, when alpha-amylase and lipase were included in the flow perfusion cultures, the calcium content was 379 +/- 30 microg/scaffold after as few as 8 days. The highest calcium content (1271 +/- 32 microg/scaffold) was obtained for SPCL/cell constructs cultured for 16 days in the presence of lipase and flow. Furthermore, von Kossa staining and tetracycline fluorescence of histological sections demonstrated mineral deposition within the scaffolds for all groups cultured for 16 days under flow. However, all the data corroborate that lipase coupled with flow perfusion conditions improve the osteogenic differentiation of MSCs and enhance ECM mineralization.

[Research and development of enzymes used in feed].

Enzymes can degrade the anti-nutrient factors in feedstuff, increase nutrient digestibility, and reduce pollution to environment, and have been widely supplemented in animal feedstuff. However, the use of enzymes is limited because of their undesirable properties, such as thermoliability and susceptibility against protease digestions. And its commercialization is also limited by low production efficiency and high cost. Therefore, the focuses for future enzyme development will be: (1) to obtain novel enzymes with better properties by high-throughput screening of enzyme encoding genes, especially those from extreme and special environments; (2) to improve enzyme properties using directed mutagenesis and protein engineering methods; (3) to achieve high-level fermentation of enzymes by heterogonous expression and optimization of codons, vectors and fermentation conditions; (4) to determine the effect of enzymes to animals and utilize enzymes efficiently.

Inhibitory activity of rabbit milk and medium-chain fatty acids against enteropathogenic Escherichia coli O128.

Colibacillosis represents a major cause of diarrhea in young rabbits. In order to elucidate protective effect of milk, in vitro and in vivo experiments were carried out. In the in vitro experiment, rabbit milk treated with lipase significantly decreased the number of viable cells in cultures of Escherichia coli, O128 serotype, from 10.3 to 6.2-7.3log(10)/(cfuml). The lipase effect was the same with heat-treated (100 degrees C/10min) and raw milk. Raw milk without lipase decreased the number of E. coli only marginally. In the in vivo experiment, weaned rabbits received feed contaminated with the same bacterium. The course of the infection was moderate, only 2 out of 36 infected rabbits died. Seven days after inoculation, caprylic acid at 5g/kg feed and triacylglycerols of caprylic and capric acid at 10g/kg feed decreased faecal output of E. coli from 10.2log(10)/(cfug) to 5.8 and 6.1log(10)/(cfug), respectively. The number of E. coli in faeces of non-infected rabbits averaged 4.0log(10)/(cfug). The growth of infected rabbits was slow for 2 weeks after infection. In the third week a compensatory growth was apparent. At the end of the experiment, average body weights of rabbits receiving caprylic acid and those of non-infected rabbits were not significantly different. It can be concluded that (i) lipids rather than proteins seem to be responsible for the antimicrobial activity of rabbit milk; and (ii) this activity was lipase-dependent. Caprylic acid or oils with high a concentration of it may be used as feed supplements for weanlings.

Disposition of lipid-based formulation in the intestinal tract affects the absorption of poorly water-soluble drugs.

Solvent Green 3 (SG), a model poorly water-soluble compound, was orally administered to rats with soybean oil emulsion or the Self-microemulsifying drug delivery system (SMEDDS) composed of Gelucire44/14. The bioavailability of SG after oral administration with SMEDDS was 1.7-fold higher than that with soybean oil emulsion. The intestinal absorption of lipid-based formulations themselves was evaluated by the in situ closed loop method. The effect of lipase and bile salt on their absorption was also evaluated. SMEDDS itself was rapidly absorbed in the intestine even in the absence of lipase and bile salt, and the absorption was increased by the addition of lipase and bile salt. On the other hand, no soybean oil emulsion was absorbed in the absence of lipase and bile salt. However, mixed micelle prepared from emulsion by incubating soybean oil emulsion with lipase and bile salt was rapidly absorbed through the intestine. Without lipase and bile salt, SG was not absorbed after administration with soybean oil emulsion. Therefore, we concluded that the degradation of soybean oil emulsion was needed for SG to be absorbed through the intestine. Furthermore, we investigated the intestinal absorption of SG after oral administration to rats whose chylomicron synthesis were inhibited by pretreatment with colchicine. Colchicine completely inhibited the intestinal absorption of SG after administration with each lipid-based formulation, suggesting that SG was absorbed from the intestine via a lymphatic route. Absorption of the dosage formulation should be paid attention when poorly water-soluble drugs are orally administered with lipid-based formulation.

Effects of pancreatic enzyme preparations on erythrocyte glutathione peroxidase activities and plasma selenium concentrations in cystic fibrosis.

To substitute for exocrine pancreatic insufficiency, patients with cystic fibrosis (CF) take pancreatic enzymes (PE) originating from porcine pancreas. Five different pancreatic enzyme preparations used by our patients contained 0.5-1.4 microg selenium per g tablet. In patients taking PE in doses that were gradually increased to improve fat absorption during a 48-month period, the effects of PE dose on erythrocyte selenium-dependent glutathione peroxidase (SeGSH-Px) activities and plasma selenium concentrations were studied. At baseline, erythrocyte SeGSH-Px activities were significantly lower in patients (p=.01), while plasma selenium concentrations did not differ between patients and healthy subjects. When PE dose and, consequently, selenium intake from PE was increased, erythrocyte SeGSH-Px activities (p < .001) and plasma selenium concentrations (p=.02) increased. Changes in SeGSH-Px activities during the initial 8 months correlated with those in selenium intake from PE (r=0.67, p < .001). Plasma selenium concentrations plateaued at 12 months and erythrocyte SeGSH-Px activities did so at 36 months, when patients had reached SeGSH-Px activities similar to those of healthy subjects. At 48 months, patients took an average lipase dose of 17400 U x kg(-1) x d(-1) and selenium dose from PE of 0.53 microg x kg(-1) x d(-1). We conclude that selenium content of PE preparations has a significant effect on SeGSH-Px activity in patients with CF. This form of selenium supply needs to be taken into account when selenium supplements are given to patients with CF.

Synergistic effects of lipid transfers and hepatic lipase in the formation of very small high-density lipoproteins during incubation of human plasma.

Studies have been performed to determine the involvement of very-low-density lipoproteins (VLDL), cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) in the formation of very small HDL particles. Human whole plasma has been incubated for 6 h at 37 degrees C in the absence and in the presence of various additions. There was minimal formation of very small HDL in incubations of non-supplemented plasma or in plasma supplemented with either VLDL, CETP or HL alone; nor were small HDL prominent after incubating plasma supplemented with mixtures of VLDL plus CETP, VLDL plus HL or CETP plus HL. By contrast, when plasma was supplemented with a mixture containing all three of VLDL, CETP and HL, incubation resulted in an almost total conversion of the HDL fraction into very small particles of radius 3.7 nm. The appearance of these very small HDL was independent of activity of lecithin: cholesterol acyltransferase. It was, however, dependent on both duration of incubation and on the concentrations of the added VLDL, CETP and HL. The effects of these incubations was also assessed in terms of changes to the concentration and distribution of lipid constituents across the lipoprotein spectrum. It was found that not only did lipid transfers and HL exhibit a marked synergism in promoting a reduction in HDL particle size but also that HL, although deficient in intrinsic transfer activity, enhanced the CETP-mediated transfers of cholesteryl esters from HDL to other lipoprotein fractions.

[Selectivity of inhibition of pancreatic enzyme secretion after lipase administration into the duodenum]. / Ob izbiratel'nosti tormozheniia sekretsii fermentov podzheludochnoi zhelezy pri vvedenii v dvenadtsatiperstnuiu kishku lipazy.

Administration of lipase into the duodenum inhibits pilase secretion and, to a lesser extent, secretion of proteinases in dogs. The inhibitory effect is prevented by simultaneous administration of cotton oil and hydrolysine acidized up to pH-2. The lipolytic activity of duodenal chyme seems to play a major part in correction of pancreatic juice enzyme range in urgent adaptation of pancreatic secretion to a type of consumed food.

Lipase-induced alterations of fatty acid synthesis by subcellular fractions from germinating pea (Pisum sativum L.).

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.

Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum).

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.

Subsynaptosomal localization and biochemical characterization of serotonin binding sites in the brain.

The subsynaptosomal distribution of the borohydride stabilizable binding of serotonin (5-HT) in the brain was investigated using various enzyme markers, such as NAD glycohydrolase (NADase), Na+, K+-activated ATPase for synaptic membranes, and monoamine oxidase (MAO) for outer mitochondrial membranes. The gross distribution of the activity of NADase and Na+, K+-activated ATPase in various membrane fractions was found to parallel the distribution of 5-HT binding in these fractions. Radioactivity bound to brain fractions was extractable with chloroform-methanol (2:1). The membranous material was solubilized by chloroform-methanol (2:1) and the recovered material, suspended in 0.32 M sucrose was found to retain its 5-HT binding capacity. The protein-phospholipid nature of the binding subcellular macromolecule was demonstrated with proteolytic and lipolytic enzymes.