The effect of repeated immobilization on the level of plasma corticosterone and on the activity of several liver enzymes in rats.

The effect of repeated stress on the level of plasma corticosterone and on the activity of several target enzymes for this hormone in the liver was studied. In adult male rats immobilized for 2.5 hrs daily, on day 7 the response of both plasma corticosterone and hepatic tyrosine aminotransferase is modified: After similar increases immediately after immobilization as in aminals stressed for the first time, in the conditioned rats precocious decreases to initial values take place. Moreover, on day 4, 24 hrs after a third immobilization, there are increases arise partly at least as a consequence of diminished food intake, as shown by comparing them with data from pair-fed rats. Partial fasting leading also to slight increase of hepatic glucose-6-phosphatase activity constitutes an important part of repeated stress with substantial impacts on metabolic processes.     

Pulmonary xanthine oxidase activity of rats exposed to prolonged immobilization stress

This study was designed to study xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity in the lung of rats exposed to prolonged restraining immobilization stress. Immobilization caused more than twofold increase of xanthine oxidase activity in the rat lung. The activity of xanthine oxidase decreased in lung homogenates incubated at -20 degrees C for 24 h. The same incubation of homogenates from control rats caused a non-significant increase of the activity. No measurable NAD(+)-dependent xanthine dehydrogenase activity could be established in the lungs of both control rats and rats subjected to immobilization. All rats revealed methylene blue-dependent xanthine dehydrogenase activity which was more than two-times higher in the immobilized animals. Incubation at -20 degrees C for 24 h increased the methylene blue-dependent xanthine dehydrogenase activity in homogenates from control rats and decreased the enzyme activity in homogenates from immobilized rats. A working hypothesis was proposed for the sequence of events explaining the results obtained: XO-catalyzed generation of activated oxygen species may take place in the initiation of lipid peroxidation in the lung of rats immobilized for prolonged periods of time.     

Characterization of immobilized enzymes in polyurethane foams in a dynamic bed reactor

-d-Galactosidase (E 3.2.1.23) from Aspergillus oryzae was immobilized with polyurethane foam (PUF). Among several immobilization methods attempted in this work, the immobilized enzyme preparation by in-situ co-polymerization between enzyme and prepolymer HYPOL 3000 showed the highest activity. The intrinsic kinetics of PUF-immobilized enzyme was determined in a dynamic bed reactor, used to increase transport rates. The immobilization mechanism in PUF was studied by measurements of immobilized enzyme kinetics and by using scanning electron microscopy combined with immuno-gold labeling techniques. The results showed that immobilization was predominantly by covalent bonding between primary amino groups of -d-galactosidase and isocyanate groups of the prepolymers. Entrapment in the PUF micropores assisted the immobilization of enzymes, and adsorption on the surface of macropores was not important for immobilization. The bicinchoninic acid method was applied for the determination of PUF loading capacity and specific enzyme activity and used to determine enzyme deactivation during immobilization.     

Interaction of the effects of glucose and stress hormones on the activity of key enzymes of liver glycogen metabolism in rats

Endogenous corticosterone released in protracted immobilization stress fails to increase the activity of liver glycogen synthase, perhaps because of the inhibition of synthase phosphatase by phosphorylase a. It was also found, that in rats subjected to acute immobilization stress the stimulation of the activity of both synthase a and total forms by glucose administered i.v. is depressed. Finally, in rats fasting for 24 h a paradoxical augmentation by glucose of the stimulatory effect of glycogenolytic hormones released in acute immobilization stress on phosphorylase a activity was observed.     

Effect of Retinol on Liver Lipid Metabolism of Rats

Effect of feeding 4.23, 16.94 and 27.53 mg of retinol daily for 10 days on the liver lipids of adult rats has been studied. Feeding of different amounts of retinol produced dose dependent toxicity symptoms in rats. Retinol feeding resulted in significant elevations of liver total lipids, total fatty acids, and glycerides, The amounts of liver esterified cholesterol were significantly raised in rats fed different amounts of retinol. Acetate-1-¹⁴C incorporation was increased in liver total cholesterol of rats fed 27.53 mg retinol and in free cholesterol of all retinol fed rats. Total ¹⁴C activity of hepatic triglycerides of retinol fed rats was the same as that of control, but their specific activity was decreased. Significant alterations were noted in phosphatidyl serine, lysophosphatidyl choline, lysophosphatidyl ethanolamine, sphingomyelin, phosphatidyl choline, phosphatidyl ethanolamine and phosphatidic acid and polyglycerophosphate fractions in liver rats fed different amounts of retinol.     

Vitamin D Supplementation Modulates Blood and Tissue Zinc, Liver Glutathione and Blood Biochemical Parameters in Diabetic Rats on a Zinc-Deficient Diet

Previous studies suggest a protective effect of vitamin D3 on zinc deficiency-induced insulin secretion and on pancreas β-cell function. The aim of this study was to investigate the effect of vitamin D on blood biochemical parameters, tissue zinc and liver glutathione in diabetic rats fed a zinc-deficient diet. For that purpose, Alloxan-induced diabetic rats were divided into four groups. The first group was fed a zinc-sufficient diet while the second group was fed a zinc-deficient diet. The third and fourth groups received zinc-sufficient or zinc-deficient diets plus oral vitamin D3 for 27 days. At the end of the experiment, blood, femur, pancreas, kidney and liver samples were taken from all rats. The serum, femur, pancreas, kidney and liver zinc concentrations, liver glutathione, serum alkaline phosphatase activity, daily body weight gain and food intake were lower in the zinc-deficient rats in comparison with those receiving adequate amounts of zinc. These values were increased in the zinc-deficient group that was supplemented with vitamin D3. The serum total cholesterol, triglycerides, total protein, urea, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and blood glucose values were higher in rats fed a zinc adequate diet, but their concentrations were decreased by vitamin D3 supplementation. The serum total protein levels were not changed by zinc deficiency and vitamin D3 supplementation. These results suggest that vitamin D3 modulates tissue zinc, liver glutathione and blood biochemical values in diabetic rats fed a zinc-deficient diet.     

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats.

A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.     

Effect of short-term exercise training on muscle glycogen in resting conditions in rats fed a high fat diet.

It has been reported that exercise training increases muscle glycogen storage in rats fed a high carbohydrate (CHO) diet in resting conditions. The purpose of this study was to examine whether a 3-week swimming training programme would increase muscle glycogen stores in rats fed a high-fat (FAT) diet in resting conditions. Rats were fed either the FAT or CHO diet for 7 days ad libitum, and then were fed regularly twice a day (between 0800 and 0830 hours and 1800 and 1830 hours) for 32 days. During this period of regular feeding, half of the rats in both dietary groups had swimming training for 3 weeks and the other half were sedentary. The rats were not exercised for 48 h before sacrifice. All rats were killed 2 h after their final meal (2030 hours). The glycogen contents in red gastrocnemius muscle, heart and liver were significantly higher in sedentary rats fed the CHO diet than in those fed the FAT diet. Exercise training clearly increased glycogen content in soleus, red gastrocnemius and heart muscle in rats fed the CHO diet. In rats fed the FAT diet, however, training did not increase glycogen content in these muscles or the heart. Exercise training resulted in an 87% increase of total glycogen synthase activity in the gastrocnemius muscle of rats fed the CHO diet. However, this was not observed in rats fed the FAT diet. The total glycogen phosphorylase activity in the gastrocnemius muscle of the rats of both dietary groups was increased approximately twofold by training.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic response to short periods of starvation in hypo- and hyper-thyroid male rats.

1) Thyroidectomized rats were fed with a low iodine diet, injected daily with 0, 0.1, 1.8 or 25 microgram of L-thyroxine/100 g body wt., and compared with intact controls. 2) Plasma protein-bound iodine was decreased in the rats given the 0 and 0.1 microgram doses, unchanged in those given the 1.8 microgram doses, unchanged in those given the 1.8 microgram dose increased in those given the 25 microgram one. 3) The liver content of DNA-P, phospholipid-P, proteins and fatty acids was decreased in the rats that did not receive thyroxine, practically recuperated in those receiving 0.1 microgram and normal in those given 1.8 or 25 microgram of thyroxine. 4) 3 h of starvation produced a reduction in the liver content of total fatty acids that disappeared after 24 h. 5) When fed, liver glycogen concentration was low in the rats given 25 microgram of thyroxine. 6) With starvation, the fall in liver glycogen and blood glucose, and the rise in liver acetyl-CoA and citrate and blood glycerol concentrations were faster in the thyroidectomized rats that did not receive thyroxine than in the other groups. 7) The rise in plasma free fatty acid and blood ketone bodies concentrations were similar in all the groups, the greater level of the first parameter being observed after 6 h of starvation in the rats given 25 microgram of thyroxine and in the second one after 24 h in the rats given either 0.1, 1.8 or 25 microgram of thyroxine. 8) The rapid decrease in the availability of carbohydrate stores with starvation in the thyroidectomized rats could be responsible for their fast call for lipid utilization. The slower response to fasting in the hyperthyroid animals is probably a consequence of their reduced amount of endogenous substrates to be mobilized.     

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states

Summary Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2–3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200–800 ng dry weight. At saturating cyclic AMP concentrations (46 M) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 M) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 M) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.     

The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats.

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.     

Metabolic effects of ethanol in the rat liver.

The NADPH is one of the cofactors in ethanol metabolism. The aim of the study was to investigate the effect of ethanol on a NADPH generating enzyme (G6P-DH) and on some metabolic parameters of the liver. After a 2-day starvation period rats were fed a lipid free diet for three days. During this refeeding period the animals were divided into three groups; they received a single daily dose of 4 g per kg b.w. ethanol, isocaloric aqueous glucose solution or water by gastric tube. In response to ethanol the activity of hepatic G6P-DH decreased. The amount of triglyceride remained unchanged, certain changes occurred in the fatty acid composition of total lipid. The liver glycogen content was elevated. In female rats treated with ethanol the activity of glucose-6-phosphatase increased.     

A smart bioconjugate of chymotrypsin

alpha-Chymotrypsin was immobilized on Eudragit S-100 via covalent coupling with 93% retention of proteolytic activity. The conjugate behaved as a smart biocatalyst and functioned as a pH-dependent reversibly soluble-insoluble biocatalyst. The pH optimum of chymotrypsin broadened on immobilization, and the immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme. The immobilized enzyme showed a slight shift in the temperature optimum and enhanced thermal stability retaining 70% of its original activity after 1 h of exposure to 40 degrees C as compared to the 25% residual activity for the free enzyme under identical conditions. K(m) and V(max) values did not change on immobilization. Also, the immobilized preparation was quite stable to reuse, it retained almost 85% of its original activity even after a fifth precipitation cycle. UV spectroscopy and circular dichroism were used to probe structural changes in the enzyme upon immobilization.     

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.     

The effect of corticotrophin on liver-type lipase activity in adrenals, liver and high-density lipoprotein subfractions in the rat

Hypercortisolism was induced in rats by the administration of a corticotrophin analogue (Synacthen depot). The effect of this treatment during different periods was studied in normally fed and overnight-fasted rats. The activity of liver-type lipases, i.e., of lipases similar to the heparin-releasable lipase of rat liver (liver lipase), was determined in the adrenal gland and in the liver. Short-term (16 h) treatment had no effect on the lipase activity in the adrenal gland. During prolonged treatment, however, the lipase activity rose to 600-700% of control values in 10 days and from then on remained constant. The effect was similar in fed and overnight-fasted rats. The lipase activity in the liver decreased upon Synacthen administration. In the fed rats a decrease of 25% of the initial value was found after 16 h, 40% after 3 days and 50% after 20 days of treatment. In overnight-fasted rats the lowering of the lipase activity was less marked than in fasted controls. Serum lipid levels and high-density lipoprotein (HDL) subclass concentrations were also measured. The cholesterol concentration in the lipoproteins with a density greater than 1.050 g/ml (HDL) was elevated in rats treated for 3-20 days. If the rats were treated for longer than 10 days, overnight fasting led to a normalization of the HDL-cholesterol levels. After separation of the HDL into two subfractions, a relatively 'light' apolipoprotein E-rich fraction and a more 'heavy' apolipoprotein A-I-rich fraction, in fed and fasted animals treated with Synacthen for 3 days both HDL subfractions were elevated. After 10 days treatment only the apolipoprotein A-I-rich HDL fraction was still enhanced in both fed and fasted rats.     

磁珠固定化结核分枝杆菌二氢叶酸还原酶及其表征

比较Ni~(2+)-NTA磁珠和羧基磁珠固定结核分枝杆菌二氢叶酸还原酶(Mycobacteriumtuberculosis dihydrofolate reductase,Mt DHFR),探索适合小分子配体混合物库筛选的Mt DHFR固定化方法。重组表达带6×His标签Mt DHFR,纯化后表征酶学性质,比较用Ni~(2+)-NTA磁珠和羧基磁珠固定化时相应固定化容量、保留活性、稳定性及对抑制剂响应。结果表明,Ni~(2+)-NTA磁珠对Mt DHFR固定化容量为(93±12)mg/g磁珠(n=3),但酶比活保留不超过32%,Ni~(2+)明显抑制酶活性,EDTA与Ni~(2+)呈协同抑制效应,Fe~(3+)无显著干扰。羧基磁珠活化固定Mt DHFR的容量(8.6±0.6) mg/g磁珠(n=3),固定化酶比活保留(87±4)%(n=3)。在含50 mmol/L KCl的100 mmol/L HEPES (pH 7.0)中,游离和固定化Mt DHFR在0℃保存16 h活性都无显著改变,但在25℃保存16 h,游离酶活性下降近60%而羧基磁珠固定化Mt DHFR活性下降仅35%。甲氨喋呤对游离Mt DHFR和固定化Mt DHFR的IC50无显著差异(P0.05)。综上,Ni~(2+)-NTA磁珠不适合固定化Mt DHFR;羧基磁珠固定化Mt DHFR能保留活性、热稳定性及对抑制剂的响应,该固定化方法有望用于快速筛选其配体混合物库。     

蒜氨酸酶的固定化及其酶学性质研究

为了提高蒜氨酸酶的稳定性并实现酶的反复利用,研究了影响蒜氨酸酶固定化的因素及固定化蒜氨酸酶的酶学性质。蒜氨酸酶的固定化以壳聚糖微球为载体,戊二醛为交联剂,固定化的最适条件为：戊二醛浓度4%,给酶量20.2U,交联时间2h。固定化蒜氨酸酶的最适pH值7.0,最适温度35℃,米氏常数Km 7.9 mmol/L,操作稳定性比较好,连续使用10次后酶活力损失低于10%。