Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.     

High-level expression and characterization of a highly thermostable chitosanase from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.     

Hypotrypsinemia as a possible factor in the activation of motor function of the duodenum in pancreatic atrophy in rats]

The effect of pancreas atrophy on myoelectrical activity of the duodenum and serum trypsin, trypsin inhibitor, amylase, lipase activity has been described. The activation of the duodenum motor activity has been established. The changes in the motor activity were connected with trypsin and trypsin inhibitor activity of serum. The motor activity changes were compensated by trypsin therapy.     

高温胁迫下两种藓类植物过氧化物酶活性的变化

在不同的高温胁迫条件下 ,对湿地匍灯藓 (Plagiomnium acutum)和大羽藓 (Thuidium cymbifolium)过氧化物酶 (POD)活性及其与处理时间和处理温度的关系进行了初步研究。结果表明 ,在一定的温度范围内 ,随着温度的升高 ,POD活性增加 ,二者成线形关系。在一定温度条件下 ,一般随着处理时间的延长 ,POD活性增加。但是当超过一定的温度 (4 5～ 5 0°C)以及一定的处理时间 (4～ 6h) ,POD活性有所下降。结果还表明 ,湿地匍灯藓的POD活性显著高于大羽藓。而且在高温胁迫下 ,湿地匍灯藓 POD活性变化比大羽藓活跃 ,其变化幅度也比大羽藓大。     

Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium

Two different types of extracellular protease activity were identified in the culture fluid of Phanerochaete chrysosporium wild-type BKM-F grown in submerged batch culture on N-limited media. The first activity, which appears to be inherent to the active growth phase, displayed a maximum on day 2 and decreased to a very low level on day 4. The second activity, which appeared at day 8 following the peak of ligninase activity, seems to be characteristic of late secondary metabolism and is stimulated by carbon starvation. Cultures started with half the amount of glucose of other cultures showed a remarkably earlier development of secondary activity. In contrast, the fed-batch addition of glucose started when ligninase activity was at a maximum (day 6) completely repressed secondary protease activity and enhanced ligninase production. The addition of exogenous veratryl alcohol increased the level of secondary protease activity, whereas the oxygen supply pattern significantly affected both the time course and the level of overall proteolytic activity. The addition of phenylmethylsulfonyl fluoride to growing cultures (0, 1, or 6 days) diminished overall protease activity, while it significantly enhanced ligninase activity. In all cases, the time courses of protease and ligninase activities were negatively correlated, indicating that protease activity promotes the decline of ligninase activity in batch culture.     

Electroencephalographic study of the effect of neurotoxin DSP-4 in iron model of chronic focal epilepsy.

The effect of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on electroencephalographic activity (EEG) was studied in the model of chronic focal epilepsy induced by intracortical injection of FeCl3 in the rat. EEG activity was recorded from the epileptogenic focus (ipsilateral and contralateral) in chronic experiments before and after DSP-4 treatment. In some experiments EEG activity was also simultaneously recorded from the cortical epileptogenic focus and locus coeruleus before and after DSP-4 treatment to study the effect of iron-induced seizure activity and of DSP-4 on the locus coeruleus electrical activity. The results showed that DSP-4 aggravated the iron-induced epileptiform activity as well as the locus-coeruleus electrical activity. The data also showed that, induction of epilepsy by FeCl3 is accompanied by enhancement of the locus coeruleus electrical activity. Our study demonstrates that DSP-4 intensifies and modifies the epileptic activity in the iron-induced chronic epilepsy model and that the effects of toxin persist for a longer duration.     

Characterization of calmodulin-activated protein kinase activity of rat adipocyte endoplasmic reticulum fraction

Calmodulin-activated protein kinase activity in the endoplasmic reticulum fraction of rat adipocytes was identified and characterized. The major endogenous protein substrate of the calmodulin-activated kinase activity has an apparent molecular weight of 54,000 as determined by sodium dodecyl sulfate gel electrophoresis. The calmodulin-activated component of the activity was saturated at 10 microM ATP. Calcium or calmodulin alone did not increase the activity, but the simultaneous presence of calcium and calmodulin increased activity three to four-fold. Half-maximal activation of this activity occurred at 8 microM Ca2+. The addition of increasing amounts of calmodulin caused a concentration-dependent activation in the presence of calcium, which was saturable at high calmodulin concentrations. Magnesium was required for activity, with half-maximal activity occurring at 230 microM. The antipsychotic drug trifluoperazine inhibited the activation of the protein kinase activity by calmodulin, but had a negligible effect on the basal activity. Half-maximal inhibition occurred at 63 microM. Phosphorylation of the 54,000 mol. wt band was independent of cAMP, cGMP and the combination of cAMP and cAMP-dependent protein kinase. Calmodulin-activated protein kinase phosphorylated both phosphoserine and phosphothreonine residues in the 54,000 mol. wt substrate. These experiments have partially characterized a calmodulin-activated protein kinase activity from adipocytes, which appears to be a unique activity of unknown function.     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

Effect of acetylcholine on the cerebral microsomal Na, K-ATPase of rats of different ages

Na, K- and Mg-ATPase activity of the cerebral cortex microsomal fraction has been studied and compared in adult and old rats. The activity of Na, K-ATPase decreases while that of Mg-ATPase increases with age. The total ATPase activity remains unchanged. The effect of acetylcholine on ATPase activity has been found to be age-dependent.     

秸秆纤维素分解菌的酶活力测定

目的:测定秸秆纤维素分解菌的酶活力。方法:从土壤中分离出具有分解纤维素能力的菌株,采用刚果红染色法进行粗选,得到7株透明圈较大的菌株。将这7株菌株液体发酵培养6d,再分别用滤纸分解度观察、羧甲基纤维素酶活法(CMC)、滤纸酶活法(FPA)和天然纤维素酶活法测定其酶活力。结果:在7株菌株中,F-1、F-2、F-3、F-5的酶活力测定结果与其溶解圈的测定结果、滤纸分解结果基本相同。且天然纤维素酶活力高的菌株,其CMC酶活、FPA酶活也高,滤纸分解效果也比较明显。结论:CMC法、FPA法和天然纤维素酶活法适于测定秸秆纤维素分解菌的酶活力。     

棘腹蛙消化道脂肪酶的分布及pH和温度对脂肪酶活力的影响

采用聚乙烯醇橄榄油乳化液水解法,测定了棘腹蛙消化道不同部位的脂肪酶活力,研究了pH和温度对脂肪酶活力的影响。结果显示,棘腹蛙消化道不同部位脂肪酶活力依次为回肠>直肠>十二指肠>胃>食道。pH和温度显著影响脂肪酶的活力,二者对脂肪酶活力影响的关系曲线均呈现为单峰型,食道、胃和肠道脂肪酶活力的最适pH值分别为5.0、5.0和7.5,最适温度均为35℃。     

A kinetic description of antifreeze glycoprotein activity

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

Characteristics of the effect of cestodes parasitizing the fish intestine on the activity of the host proteinases

The activity and spectrum of proteinases in the intestines of host fishes change upon infestation with cestodes. Serine proteinases are found to make a greater contribution to the total proteolytic activity. The reduction of proteolytic activity is associated with adsorption of the enzymes of the host on the surface of cestodes, and the increase in the activity is caused by the injury of the intestinal mucosa by the attachment apparatuses of cestodes. The inhibition of proteainase activity indicates the possible participation of microbiota enzymes in protein hydrolyses.     

Anticoagulant Activity of Sulfated Schizophyllan

Sulfated schizophylians with lower anticoagulant activity and higher anti-HIV activity were prepared. Those with sulfur contents above 6% showed anticoagulant activity irrespective of molecular weight. The activity was correlated with sulfur content. The triple helical structure of sulfated schizophylians did not affect their anticoagulant activity. A sulfated schizophyllan with a sulfur content of 5.0% showed low anticoagulant activity and good anti-HIV activity. These results indicate that the latter sulfated schizophyllan (sulfur content, 5%) would be useful as an anti-HIV agent for treatment of HIV-infected hemophiliacs.     

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

Summary The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

Proof of adenylate cyclase activity in the coronary artery of cattle]

In two fractions obtained from the bovine A. coronaria adenylate cyclase activity was identified and characterized. The adenylate cyclase activity of the 75,000 X g sediment shows a pH optimum at 7.4. The temperature dependence of this adenylate cyclase activity is linear when represented in the Arrhenius plot, and an Arrhenius activation energy of 13.2 kcal Mol-1 can be calculated for the enzyme reaction. The Km-value of the enzyme to ATP is 6 +/- 0.6 - 10(-4) M. The adenylate cyclase activity of the 75,000 X g sediment can be stimulated by NaF. 5'AMP and adenosine inhibit the adenylate cyclase activity of the 75,000 X g sediment. With regard to the enzyme activity, Mn++ and Co++ replace Mg++, but not Ca++. The monovalentcations Na+ and K+ do not influence the adenylate cyclase activity. In a particulate fraction containing plasma membranes, adenylate cyclase activity was also identified. This adenylate cyclase activity can be stimulated by catecholamines, noradrenaline, and isoproterenol. This stimulation can, however, only be proved for the enzyme in the coronaries of 9-week-old and 2-year-old animals. The adenylate cyclase activity from the coronaries of adult animals is not affected by catecholamines. These findings are discussed with regard to hypertension frequently found in adult animals.     

Effect of pentoxyphylline on certain indicators of the proteinase-proteinase inhibitor system in rats upon administration of cycloheximide

The pentoxifylline influence on neutral proteinase, alpha-2-macroglobulin, trypsin-alpha-1-proteinase inhibitor and elastaseinhibitory activity under cycloheximide injection has been investigated. Two hours after cycloheximide injection the activity of neutral proteinases increases in rats serum, lungs, heart, liver and kidneys. The preliminary injection of pentoxifylline prevents increase of neutral proteinases activity. Cycloheximide also decreases alpha-2-macroglobulin activity in serum and liver and trypsin-, elastaseinhibitory activity of alpha-1-proteinase inhibitor in all investigated organs. At using pentoxifylline the alpha-2-macroglobulin activity doesn't change in liver and increases in serum in comparison with only cycloheximide and there are no observed any alpha-1 inhibitor proteinase activity changes in rats serum and organs.