产β-葡聚糖酶淀粉液化芽孢杆菌的选育

通过对一株淀粉液化芽孢杆菌进行紫外和^60Co逐级诱变，使该菌株产β-葡聚糖酶酶活从80U／mL提高到105U／mL；对突变株进行多次单菌落分离及传代，对其传代稳定性的研究表明：该菌株产酶性能稳定；在5L发酵罐中突变株的产酶水平比摇瓶要高，可达120U／mL，产酶周期比摇瓶缩短了15h．     

Thermobifida fusca WSH03-11突变株合成角质酶的发酵条件

以放线菌Thermobifida fusca WSH03—11——高产角质酶突变株为出发菌株，在摇瓶中考察了碳、氮源等条件以及接种量、装液量和初始pH值等环境条件对突变株细胞生长和产角质酶的影响。通过优化发酵条件，突变菌株的角质酶酶活达10．1U／mL，提高了15％。在此基础上，在5L发酵罐中进一步研究了碳源流加对突变株发酵产酶的影响，结果发现，分别在0、24、48h添加1％乙醇，角质酶酶活达到16．4U／mL，细胞干重（DCW）达到3．7g／L，而且发酵时间也由110h缩短到50h。     

高产中性纤维素酶菌株的诱变选育和筛选方法

设计了一种根据水解透明圈与菌落直径之比(D／d)以初步判断菌株产酶活力大小的方法,发现菌株的液体发酵酶活的对数(lgE)和菌落透明圈直径与菌落直径之比(D／d)基本上呈线性关系.采用紫外线和γ射线对中性纤维素酶产生菌木霉ZC(39 U／mL)进行了反复诱变和大量筛选,得到了一株高产中性纤维素高产突变菌株BE40-39(99 U／mL),并对出发株ZC和突变株BE40-39的性状进行了比较.     

高产纤维素酶菌株的诱变选育和筛选

用青霉（Penicillium sp．）HK-003为原始菌，经亚硝基胍（NTG）、羟胺复合诱变，根据变异菌株菌落形态变化与产酶高低的关系。结合酶活力检测理化指标，筛选出一株高产稳定的纤维素酶菌株LX-435，其产酶能力由200U／mL提高到415U／mL，较出发菌株提高2．08倍。对该菌种进行了连续8次继代培养，结果表明其遗传性状稳定。     

半纤维素酶高产菌种的选育及产酶条件

以黑曲霉WA301为出发株,经过紫外诱变获得一株遗传性状稳定的半纤维素酶的高产突变株WA9024.通过对培养基中碳源、氮源、水分和起始pH值的优化,突变株WA9204以麸皮为基质的固态发酵产半纤维素酶的能力进一步得到提高.在较适的条件下,WA9024的甘露聚糖酶酶活达到8984U/g,木聚糖酶酶活达到503U/g,分别比出发菌株提高了1160%和210%.     

新型果蔬加工用酶--粥化酶

以黑曲霉菌株A.niger103为出发株,通过紫外、亚硝基胍等诱变剂进行反复诱变处理,得到一株高产菌株A.niger537,果胶酶酶活提高了130%,同时测定了其纤维素酶、木聚糖酶、蛋白酶及淀粉酶的酶活,分别达到4.76,176.82,832.4,105.73 U/mL.并通过单因素试验研究了营养条件对改变各酶产酶量及其酶系组成的影响.     

霉菌酸性蛋白酶高产突变菌株9169的选育

对一株宇佐美曲霉进行紫外光、微波和60Co的逐级诱变育种,使酸性蛋白酶产量从2800U/mL提高到7200U/mL.突变株传代多次,产酶性能保持稳定;对突变株与出发株的发酵过程进行了比较研究,发现突变株最大产酶时间提前10h以上,而培养基pH值对产酶有一定影响.     

中性纤维素酶高产菌株的诱变选育及产酶条件

通过紫外线和γ射线交替诱变,筛选得到一株高产纤维素酶的突变菌株BE40 39.与出发株相比,其产酶能力提高1.3倍,发酵性状与出发菌株也有明显的差异.突变菌株发酵试验发现,Avicel是突变菌株产酶最合适的纤维素质碳源.通过对发酵培养条件的试验,得出最佳培养条件是:Avicel质量浓度为0.1g/dL,胰蛋白胨质量浓度为0.3g/dL,最适发酵温度为30℃,最佳培养基初始pH4.2,发酵5d达到产酶高峰.     

氮离子束诱变提高黄嘌呤氧化酶产生菌产酶能力

通过氮离子束诱变节杆菌(Arthrobacter sp.g-1984)筛选到黄嘌呤氧化酶高产菌株X7,产酶活力为24.49 U/g,是出发株产酶活力(8.81 U/g)的2.78倍,经发酵条件优化后,产酶活力达到36.67 U/g,是出发菌株产酶活力的4.16倍,产酶高峰缩短了21 h.对细菌黄嘌呤氧化酶酶学性质进行了初步研究,其最适反应条件为37℃、pH 6.5、Fe2 对酶活力有较强的抑制作用,EDTA对酶活力有明显的激活作用.     

突变黑曲霉高产β-葡萄糖苷酶的培养基优化

对实验室筛选出产β- 葡萄糖苷酶的黑曲霉进行辐射诱变处理得到高产酶突变菌株,同时采用单因素和正交实验对黑曲霉产酶培养基进行优化研究.结果表明:经过X射线辐射诱变的黑曲霉产的β- 葡萄糖苷酶活力提高了18.86 U/mL.该菌株最适宜产酶的培养基为(质量分数%): 麸皮与玉米粉(1∶1)1.5、 (NH 4 ) 2 SO 4 0.15、 KH 2 PO 4 0.2、吐温80 0.1,所得酶活力最大可达到59.86 U/mL.     

pH对衰老相关β-半乳糖苷酶染色的影响

目的观察正常老化及应激诱导老化（stress—inducedprematuresenescence,SIPS）的小鼠成纤维细胞在不同pH值条件下,衰老相关β-半乳糖苷酶（senescence—associatedp—galactosidase,SA—β-Gal）染色效果的变化规律。方法取新生1～3dC57BL／6小鼠背部皮肤成纤维细胞进行传代培养,UVB照射应激诱导老化细胞,以不同代次的细胞和应激诱导老化细胞为实验对象,进行衰老相关蛋白检测,并在不同pH条件下进行衰老相关β-半乳糖苷酶染色,对其着色情况进行观察和分析。结果在P1代细胞、P6代细胞、应激诱导老化细胞和P12代细胞中,p53／p21蛋白表达依次增多;正常P1代细胞衰老相关β-半乳糖苷酶染色阴性,其余细胞组随着pH值的由低到高（6．0～8．0）,着色情况均呈现由强到弱的变化趋势,P6细胞、SIPS细胞、P12细胞分别在pH7．0、7．5和8．0时阳性染色消失。结论随着细胞衰老程度的增加,衰老相关p一半乳糖苷酶染色将在较高pH时才会呈现假阴性,提示染色时安全pH范围应根据细胞代次而定。     

缺血再灌注损伤后肾小管上皮细胞的衰老演变及其意义

目的观察肾脏缺血再灌注损伤（IRI）后正常和衰老肾小管上皮细胞的演变，探讨细胞衰老在衰老相关性肾脏病理变化中的作用。方法以低龄（2月龄）和高龄（12月龄）野生鼠为研究对象。建立左肾IRI模型。于IRI后0d、1d、3d、7d、1月、3月、6月取肾组织，用HE染色观察肾小管组织学变化；免疫组织化学检测肾小管上皮细胞增殖细胞核抗原（PCNA）的表达；组织化学染色观察肾小管上皮细胞衰老相关β-半乳糖苷酶（SA-β-gal）的活性；TUNEL法检测凋亡肾小管上皮细胞。结果肾脏IRI后0d，肾小管以坏死为主，高龄鼠比低龄鼠更为明显（P〈0．05）。IRI1d后出现肾小管上皮细胞凋亡，7d凋亡达到高峰（P〈0．05），且在同一时间点，高龄鼠比低龄鼠严重（P〈0．05）。低龄鼠IRI肾1月时出现肾小管上皮细胞衰老，而对侧肾没有出现，3月、6月点衰老细胞显著增多（P〈0．05）；高龄鼠IRI后0d双肾均可见大量衰老的肾小管上皮细胞，但IRI肾的衰老细胞在IRIld后明显减少（P〈0．05），1月后又逐渐增多。6月后高龄鼠双肾衰老的肾小管上皮细胞几乎又达到同一水平。PCNA阳性染色细胞出现的几率两组相比差异无统计学意义（P〉0．05），但低龄组细胞增殖能力要强于高龄组。对高龄鼠IRI后1d点肾小管上皮细胞凋亡与衰老之间的相关分析显示，二者存在显著负相关（r=-0．82,P〈0．001）。结论IRI可促进正常肾小管上皮细胞衰老的进程。已经进入衰老状态的肾小管上皮细胞在遭受IRI刺激后，更易走向死亡[坏死和（或）凋亡]。肾小管上皮细胞的这种演变，在老化相关性肾脏病理变化发生和进展中可能发挥着重要作用。     

Fibroblasts cultured from venous ulcers display cellular characteristics of senescence

Purpose: A well-recognized characteristic of venous ulcers is impaired healing. Fibroblasts cultured from venous ulcers (wound-fb) have been shown to have reduced growth rates and are larger than normal fibroblasts (normal-fb) from the ipsilateral limb. Reduced growth capacity and morphologic changes are 2 well-known traits of cellular senescence. Other molecular changes are overexpression of matrix proteins, such as cellular fibronectin (cFN), and enhanced activity of β-galactosidase at pH of 6.0 (senescence associated β-Gal, or SA-β-Gal). Senescence, an irreversible arrest of cell proliferation with maintenance of metabolic functions, may represent in vivo aging and thus may be related to impaired healing. Methods: Cultured normal-fb and wound-fb from 7 venous ulcer patients (average age, 51 years) were obtained by taking punch biopsies of the perimeter of the ulcer and from the ipsilateral thigh of the same patient. Growth rates, SA-β-Gal activity, and level of cFN protein (immunoblot) and message (Northern blot) were measured. Results: In all patients, wound-fb growth rates were significantly lower than those of normal-fb (P = .006). A higher percentage of SA-β-Gal positive cells were found in all wound-fb (average, 6.3% vs. 0.21%; P = .016). The level of cFN, was consistently higher in all wound-fb tested. Also, in 4 patients, the level of cFN messenger RNA (mRNA) was increased. Conclusion: Fibroblasts cultured from venous ulcers exhibited characteristics associated with senescent cells. Accumulation of senescent cell in ulcer environment may be associated with impaired healing. (J Vasc Surg 1998;28:876-83.)     

Collagen degrading activity associated with Mycobacterium species

BACKGROUND: The mechanism of Mycobacterium tuberculosis penetration into tissues is poorly understood but it is reasonable to assume that there is a contribution from proteases capable of disrupting the extracellular matrix of the pulmonary epithelium and the blood vessels. A study was undertaken to identify and characterise collagen degrading activity of M tuberculosis. METHODS: Culture filtrate protein extract (CFPE) was obtained from reference mycobacterial strains and mycobacteria isolated from patients with tuberculosis. The collagen degrading activity of CFPE was determined according to the method of Johnson-Wint using 3H-type I collagen. The enzyme was identified by the Birkedal-Hansen and Taylor method and its molecular mass determined by SDS-PAGE and Sephacryl S-300 gel filtration chromatography using an electroelution purified enzyme. RESULTS: CFPE from Mycobacterium tuberculosis strain H37Rv showed collagenolytic activity that was four times higher than that of the avirulent strain H37Ra. The 75 kDa enzyme responsible was divalent cation dependent. Other mycobacterial species and those isolated from patients with tuberculosis also had collagen degrading activity. CONCLUSIONS: Mycobacterium species possess a metalloprotease with collagen degrading activity. The highest enzymatic activity was found in the virulent reference strain H37Rv.     

Paradoxical increase in nitric oxide synthase activity in hypercholesterolaemic rats with impaired renal function and decreased activity of nitric oxide.

BACKGROUND: We have shown that acute exposure of oxidized low-density lipoprotein (OX-LDL) induces vasoconstriction in renal vessels and reduces glomerular filtration rate (GFR) in an isolated perfused rat kidney model by decreasing the activity of nitric oxide (NO). L-arginine has a protective role against OX-LDl-induced vasoconstriction. Micropuncture studies have demonstrated that short-term diet-induced hypercholesterolaemia is associated with decreased GFR and renal blood flow and increased glomerular capillary pressure. This may be mediated by decreased activity of NO. METHODS: Rats were made hypercholesterolaemic by supplementing the standard chow with 4% cholesterol and 1% sodium cholate. A group of rats on hypercholesterolaemic diet also received L-arginine in the drinking water. After 4 and 6 weeks, blood samples and 24-h urine samples were collected for the measurement of biochemical parameters. After 6 weeks, all rats were subjected to isolated perfusion of kidneys at a constant pressure of 100 mmHg. During isolated perfusion, the unused contralateral kidney was taken for morphological studies and for assessing the activity of nitric oxide synthase enzyme by beta-NADPH diaphorase histochemistry. RESULTS: Rats fed a high-cholesterol diet had LDL levels 3-6 times greater than the rats fed standard chow. Rats that received L-arginine in the drinking water had serum L-arginine levels 5-6 times greater than control rats. At 6 weeks, creatinine clearance was significantly lower in the rats on the high-cholesterol diet compared to the rats on standard chow and rats on high-cholesterol diet plus L-arginine. Twenty-four-hour urinary total nitrate and nitrite excretion in the hypercholesterolaemic rats was 1.5-2 times greater than that of control rats. Twenty-four-hour urinary cGMP excretion was significantly lower in the rats on a high-cholesterol diet, but in the rats on high-cholesterol diet and L-arginine, 24-h urinary cGMP excretion was not significantly different from that of control rats. During isolated perfusion of kidneys, renal perfusate flow was found to be significantly reduced in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation in the drinking water almost completely reversed the effect of a high-fat diet. Inulin clearance was also significantly reduced in kidneys on a high-fat diet in contrast to controls but not in kidneys on high fat-diet and L-arginine. Basal cGMP excretion in urine was significantly lower in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation restored the basal cGMP excretion in these kidneys. NO synthase (NOS) enzyme activity as assessed by NADPH diaphorase activity showed that kidney sections taken from the rats on a high-fat diet showed more intense staining, indicating increased activity compared to the kidney sections taken from the rats on a normal diet. CONCLUSION: Though activity of NO is diminished in hypercholesterolaemic rats with impaired renal function, there is a paradoxical increase in NO production and NOS activity. L-arginine reverses the effects of a high-fat diet.     

Isolation, culture and characterization of human renal tubular cells

Tubular cells have been isolated, characterized and cultured from more than 70 adult cadaver kidneys (postmortem time less than or equal to 12 hr.). Confluent monolayers were observed at 7 days after seeding (10(6) cells/ml.) and cells demonstrating normal human karyotypes have been passaged up to 6 times. Primary isolates and monolayer cultures were negative for Factor VIII activity, and strongly positive for gamma-glutamyltransferase activity and keratin. Ultrastructurally primary isolates consisted of cells with numerous mitochondria, microvilli, cytoplasmic filaments and well-developed endocytotic apparati. Monolayer cultures examined at 7, 14, 21 and 72 days demonstrated less prominent microvilli and the additional structures of desmosomes and cell junctions. Membrane-associated and cytosolic enzyme activities were measured up to 28 days in culture. The membrane-associated enzymes gamma-glutamyltransferase and alkaline phosphatase both exhibited approximately 10-fold decreases in activity during the 1st 7 days in culture. There was an approximately 5-fold increase in pyruvate kinase activity during the same time period, while fructose-1,6-bisphosphatase activity exhibited a 5-fold decrease. Glucose-6-phosphatase activity did not change during the 28 day culture period examined. From 7 to 28 days no further changes were noted in any of the enzyme activities measured. Decreased membrane-associated enzyme activity corresponded to the ultrastructural observation of less prominent microvilli. Increases in glycolytic enzyme activity and decreases in gluconeogenic enzyme activity may reflect the presence of glucose in the culture medium. The morphologic and biochemical evidence suggests that primary isolates and cultures are proximal tubule cells which should provide a well-defined in vitro human system for future studies.     

Inhibition of 1,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures.

This study used the ionophore, A23187, to examine the hypothesis that the regulation of alkaline phosphatase and phospholipase A2 activity by vitamin D3 metabolites in cartilage cells is mediated by changes in calcium influx. Confluent, fourth-passage cultures of growth zone and resting zone chondrocytes from the costochondral cartilage of 125 g rats were incubated with 0.01-10 microM A23187. Specific activities of alkaline phosphatase and phospholipase A2 were measured in the cell layer and in isolated plasma membranes and matrix vesicles. There was an inhibition of alkaline phosphatase specific activity at 0.1 microM A23187 in resting zone cells and at 0.1 and 1 microM in growth zone chondrocytes. At these concentrations of ionophore, the 45Ca content of the chondrocytes was shown to increase. Both the plasma membrane and matrix vesicle enzyme activities were inhibited. There was no effect of ionophore on matrix vesicle or plasma membrane phospholipase A2 in either cell type. In contrast, alkaline phosphatase activity is stimulated when growth zone chondrocytes are incubated with 1,25-(OH)2D3 and in resting zone cells incubated with 24,25-(OH)2D3. Phospholipase A2 activity is differentially affected depending on the metabolite used and the cell examined. Addition of ionophore to cultures preincubated with 1,25-(OH)2D3 or 24,25-(OH)2D3 blocked the stimulation of alkaline phosphatase by the vitamin D3 metabolites in a dose-dependent manner. The effects of ionophore were not due to a direct effect on the membrane enzymes since enzyme activity is isolated membranes incubated with A23187 in vitro was unaffected. These results suggest a role for calcium in the action of vitamin D metabolites on chondrocyte membrane enzyme activity but indicate that mechanisms other than merely Ca2+ influx per se are involved.     

Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents

A reduced capacity for mitochondrial fatty acid oxidation in skeletal muscle has been proposed as a major factor leading to the accumulation of intramuscular lipids and their subsequent deleterious effects on insulin action. Here, we examine markers of mitochondrial fatty acid oxidative capacity in rodent models of insulin resistance associated with an oversupply of lipids. C57BL/6J mice were fed a high-fat diet for either 5 or 20 weeks. Several markers of muscle mitochondrial fatty acid oxidative capacity were measured, including (14)C-palmitate oxidation, palmitoyl-CoA oxidation in isolated mitochondria, oxidative enzyme activity (citrate synthase, beta-hydroxyacyl CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and carnitine palmitoyl-transferase 1), and expression of proteins involved in mitochondrial metabolism. Enzyme activity and mitochondrial protein expression were also examined in muscle from other rodent models of insulin resistance. Compared with standard diet-fed controls, muscle from fat-fed mice displayed elevated palmitate oxidation rate (5 weeks +23%, P < 0.05, and 20 weeks +29%, P < 0.05) and increased palmitoyl-CoA oxidation in isolated mitochondria (20 weeks +49%, P < 0.01). Furthermore, oxidative enzyme activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, uncoupling protein (UCP) 3, and mitochondrial respiratory chain subunits were significantly elevated in fat-fed animals. A similar pattern was present in muscle of fat-fed rats, obese Zucker rats, and db/db mice, with increases observed for oxidative enzyme activity and expression of PGC-1alpha, UCP3, and subunits of the mitochondrial respiratory chain. These findings suggest that high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance in rodents by decreasing muscle mitochondrial fatty acid oxidative capacity.     

Glomerular angiotensinase A in the rat: increase of enzyme activity following renal ablation

Angiotensinase A (aminopeptidase A; ATA) is an angiotensin II splitting exopeptidase, which is localized in endothelial and epithelial cells of the glomerular tuft. In order to investigate the influence of a reduction in renal mass on enzyme activity, ATA activity was measured in isolated rat glomeruli five and 14 weeks after 1-1/3 nephrectomy. Glomerular ATA activity in remnant kidneys increased significantly after five weeks following ablation compared with glomeruli of two kidney control rats (5.34 +/- 4.02 vs. 1.71 +/- 1.96 mU/mg protein, P less than 0.05). After 14 weeks, however, this difference was no longer present. Treatment of rats with enalapril or saralasin inhibited the increase of ATA seen five weeks after renal ablation, whereas indomethacin had no effect on enzyme activity. Furthermore, normal two kidney rats, treated with furosemide, revealed a higher glomerular ATA than two kidney controls (5.5 +/- 2.64 vs. 2.1 +/- 1.7 mU/mg protein, P less than 0.05). In vitro superfusion of isolated glomeruli with enalaprilate or furosemide from rats after renal ablation did not influence enzyme activity, however, superfusion with 0.05 mM angiotensin II or 0.05 mM saralasin significantly reduced ATA. Our results suggest that glomerular ATA might be involved in the early regulation of the intrarenal renin-angiotensin system and could modify glomerular adaptation to reduce renal mass by affecting angiotensin II degradation.     

Enzyme activities in regenerating epithelium during wound healing. IV. Aminotransferases and NADP-dependent enzymes

Alanine aminotransferase activity decreased gradually in marginal and regenerating epithelium during the first 4 days of wound healing. A decreasing gradient of alanine aminotransferase activity was observed from the marginal epithelium to the migrating epithelial tip; the actively migrating epithelial tip exhibited only 10% of normal activity. In contrast to alanine aminotransferase activity, aspartate aminotransferase activity increased slightly and did not exhibit a significant differential distribution gradient within regenerating epithelium.The epithelium at the wound margin exhibited three-fold increases in glucose-6-phosphate dehydrogenase and malic enzyme activities, little alteration in 6-phosphogluconate dehydrogenase activity, and a 30% decrease in isocitrate dehydrogenase activity, together with an increased NADPH concentration. Regenerating epithelium demonstrated a five-fold increase in glucose-6-phosphate dehydrogenase and malic enzyme activities and a 60% increase in 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase activities, together with an elevated level of NADPH concentration.