Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover

Tracer methods for VLDL-TG kinetics vary in their ability to account for the effect of tracer recycling, which can influence the calculation of VLDL-TG fractional catabolic rates (FCRs). We evaluated a novel approach, involving stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling, for measuring VLDL-TG kinetics in normolipidemic human subjects. When administered as a bolus simultaneously, both tracers provided identical VLDL-TG FCRs when the data were analyzed by a compartmental model that accounted for hepatic lipid tracer recycling, but not by non-compartmental analysis. The model-derived FCR was greater than that determined using a non-compartmental approach, and was 2- to 3-fold higher than that usually reported by using a bolus of radioactive [3H]glycerol. When palmitate tracer was given as a constant infusion, VLDL-TG turnover appeared 5-fold slower, because tracer recycling through hepatic lipid pools could not be resolved with the infusion protocol. We conclude that accounting for tracer recycling, particularly the contribution of hepatic glycerolipid pools, is essential to accurately measure VLDL-TG kinetics, and that bolus injection of stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling analysis offers a reliable approach for measuring VLDL-TG kinetics.     

Phytases: microbial sources,production, purification,and potential biotechnological applications

The review deals with phytase-producing microorganisms along with optimum conditions for its production. Various methods used for purifying phytases and their characteristics are discussed. Heterologous gene expression, cost-effective large-scale phytase production, and various biotechnological applications of the enzyme in animal feed and food industries are also discussed.     

Purification and kinetics of a raw starch-hydrolyzing,thermostable, and neutral glucoamylase of the thermophilic mold Thermomucor indicae-seudaticae

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.     

Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells

Telomere shortening limits the regenerative capacity of primary cells in vitro by inducing cellular senescence characterized by a permanent growth arrest of cells with critically short telomeres. To test whether this in vitro model of cellular senescence applies to impaired organ regeneration induced by telomere shortening in vivo, we monitored liver regeneration after partial hepatectomy in telomerase-deficient mice. Our study shows that telomere shortening is heterogeneous at the cellular level and inhibits a subpopulation of cells with critically short telomeres from entering the cell cycle. This subpopulation of cells with impaired proliferative capacity shows senescence-associated beta-galactosidase activity, while organ regeneration is accomplished by cells with sufficient telomere reserves that are capable of additional rounds of cell division. This study provides experimental evidence for the existence of an in vivo process of cellular senescence induced by critical telomere shortening that has functional impact on organ regeneration.     

Enhanced secretion and low temperature stabilization of a hyperthermostable and Ca2+-independent alpha-amylase of Geobacillus thermoleovorans by surfactants

AIMS: Selection of suitable surfactants for enhancing and stabilizing alpha-amylase of Geobacillus thermoleovorans. METHODS AND RESULTS: Geobacillus thermoleovorans was cultivated in shake flasks containing 50 ml of starch-yeast extract-tryptone (SYT) medium with/without surfactants. Titres of the enzyme in media were monitored. The enzyme was also preserved at 4 degrees C with/without surfactants and enzyme activities were determined. Among polyethylene glycol (PEGs) of different molecular weights, PEG 8000 (0.5%, w/v) caused a slight increase in the enzyme titre, while Tween-20, Tween-40 and Tween-60 (0.03%, w/v) exerted a significant stimulatory effect on enzyme secretion. In the presence of SDS, Tween-80 and cholic acid (0.03%, w/v), the enzyme production was nearly twofold higher than that in the control. The anionic (SDS, cholic acid) and non-ionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced alpha-amylase secretion. Furthermore, anionic surfactants exhibited stabilizing effect on the enzyme during preservation at 4 degrees C. CONCLUSIONS: PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide). Further, these surfactants were found to stabilize the enzyme at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The secretion of Ca2+-independent hyperthermostable alpha-amylase was enhanced in the presence of certain anionic and non-ionic detergents in the medium. Furthermore, the surfactants stabilized the enzyme during preservation at 4 degrees C. The use of this enzyme in starch hydrolysis eliminates the addition of Ca2+ in starch liquefaction and its subsequent removal by ion exchange from sugar syrups.     

Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.     

Preparation, characterization, and in vitro and in vivo evaluation of lovastatin solid lipid nanoparticles

The purpose of this research was to study whether the bioavailability of lovastatin could be improved by administering lovastatin solid lipid nanoparticles (SLN) duodenally to rats. Lovastatin SLN were developed using triglycerides by hot homogenization followed by ultrasonication. Particle size and zeta potential were measured by photon correlation spectroscopy. The solid state of the drug in the SLN and lipid modification were characterized. Bioavailability studies were conducted in male Wistar rats after intraduodenal administration of lovastatin suspension and SLN. Stable lovastatin SLN having a mean size range of 60 to 119 nm and a zeta potential range of −16 to −21 mV were developed. More than 99% of the lovastatin was entrapped in the SLN. Lovastatin was dispersed in an amorphous state, and triglycerides were in {ieE162-1} form in the SLN. In vitro stability studies showed the slow release and stability of lovastatin SLN. The relative bioavailabilities of lovastatin and lovastatin hydroxy acid of SLN were increased by ∼173% and 324%, respectively, compared with the reference lovastatin suspension. Published: March 23, 2007     

Influence of selenium (antioxidant) on gliclazide induced hypoglycaemia/anti hyperglycaemia in normal/alloxan-induced diabetic rats

Oxidative stress is involved in diabetes mellitus and its complications. Since diabetes is a stress-related disorder, supplementation with antioxidants may improve the condition. The purpose of this study is to know the effect of oral administration of selenium on blood glucose and its influence on gliclazide induced hypoglycaemia/antihyperglycaemia in normal and alloxan-induced diabetic rats. Albino rats of either sex were divided into three groups of six each. Group-I/II/III were treated with selenium 1/2 TD (0.9 μg/200 g rat)/TD (1.8 μg/200 g rat)/2TD (3.6 μg/200 g rat), respectively. Later group II was treated with gliclazide TD (1.44 mg/200 g rat)/selenium TD + gliclazide TD with a washout period of 1 week between the treatments. Diabetes was induced by alloxan monohydrate 100 mg/kg body weight i.p. A group of six rats showing fasting blood glucose levels ranging from 175–250 mg/dl were selected for the study. Rats were treated with selenium TD, gliclazide TD and selenium TD + gliclazide TD with a washout period of 1 week between the treatments. Selenium 1/2 TD and TD produced hypoglycaemia while 2TD produced hyperglycaemia. The combination of selenium TD with gliclazide TD, significantly enhanced the glucose lowering effect of gliclazide in normal and diabetic rats.