Specificity of Neurotensin Metabolism by Regional Rat Brain Slices

Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.     

Degradation of Rat Brain Cholinergic Muscarinic Receptors In Vitro: Enhancement by Agonists and Inhibition by Antagonists

Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation.     

一株DMP降解菌的分离鉴定及其降解特性

从山东省潍坊市污染河流底泥中筛选到1株能够以酞酸酯(Phthalic acid esters, PAEs)为唯一碳源和能源生长的微生物, 命名为JDC-3, 根据形态学观察、生理生化指标测定和分子生物学鉴定结果, 将该菌株初步鉴定为戴尔福特菌属(Delftia sp.), 以一对简并引物, 首次在该属中扩增出编码邻苯二甲酸双加氧酶的基因片段。同时以邻苯二甲酸二甲酯(Dimethyl phthalate, DMP)为目标测试物, 利用高效液相色谱(HPLC)测定了JDC-3的降解性能, 得出该菌对DMP降解的最佳条件为: pH 7.0~8.0、温度30°C~35°C; 在不同DMP初始浓度下研究了该菌的降解动力学, 结果表明当浓度低于300 mg/L时的降解动力学方程为ln C = - 0.06837 t + A, 半衰期为12.48 h, 当初始浓度不断增加, DMP对JDC-3的抑制能力增强, JDC-3对DMP的降解速率不断下降, 半衰期增大。     

Metabolism of Angiotensin Peptides by Neuronal and Glial Cultures from Rat Brain

The degradation pattern and rate of [Ile5]-Angiotensin (Ang) I, II, and III were studied in neuron-enriched and glia-enriched cells in primary cultures from rat brain. Metabolites were separated by HPLC, and their identities were evaluated by comparison of their retention times with those of synthetic Ang peptide fragments and by analysis of their amino acid composition. Major metabolites were identified as des-Asp1-[Ile5]-Ang I, des-Asp1-[Ile5]-Ang II, [Ile5]-Ang II (3-8) hexapeptide, [Ile5]-Ang II (4-8) pentapeptide, and [Ile5]-Ang II (5-8) tetrapeptide. Glia-enriched cells degraded [Ile5]-Ang I and [Ile5]-Ang III significantly faster than neuron-enriched cells, whereas no difference between the two types of cells was found in the degradation rate of [Ile5]-Ang II. Although the half-lives of [Ile5]-Ang I and [Ile5]-Ang III in neuron-enriched cells from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were not significantly different, neuron-enriched cultures from WKY rats metabolized [Ile5]-Ang II about 2.6 times faster than neuron-enriched cells derived from SHR.     

植物的不同组织对氰化物降解的可行性研究

氰化物是目前世界范围内最常使用的提取黄金等贵重金属的沥滤剂,其对自然生态环境的污染和破坏以及对人畜的毒性作用是众所周知的,氰化物污染的治理也就成为了环境工作者关注的焦点课题。植物细胞的悬浮液通常用于研究污染物的降解及在植物体内的生化反应机制。本实验直接用三种杨柳科植物的不同组织(根须、嫩茎、老茎和叶片)来观察和测定植物细胞线粒体中的氰丙氨酸合成酶(β-cyanoalanine synthase)转化氰化物的可行性。实验是在一封闭的玻璃器皿(100mL)中进行的(100mL的氰化钾溶液中加入1.0g(鲜重)的植物组织,氰化钾溶液的浓度0.444～0.457 CN mg·L~(-1))。在为期28 h的时间内,水溶液中的氰化物22.13～67.04％被植物的不同组织去除。最快的氰化物去除率发现在美洲黑杨(Populus deltoides)的叶片实验组(1.9449 mg CN/kg(鲜重)·h),苏柳(Salix matssudana Koidz×Salix alba L)的叶片实验组次之(1.7259 mg CN/kg(鲜重)·h),最慢的氰化物去除率发现在苏柳的嫩茎实验组(0.4934 mg CN/kg(鲜重)·h)。实验结果表明,选用植物的组织同样可以观察和测定污染物在植物体内的转化,特别是植物的叶片表现的尤其敏感。     

Degradation of acetochlor by four microbial communities

Four microbial communities capable of degrading acetochlor, designated A, D, E, and J, were obtained from acetochlor-contaminated soil and sludge. Acetochlor at an initial concentration of 55mg/L was completely degraded by the four mixed cultures after 4 days. At 80 mg/L acetochlor, more than 99% degradation was observed with D, 84% with A and E, and 88% with J after 9 days. There are primary eight strains of bacteria in community A, three in community D, E, and J, respectively. No single isolate was able to degrade acetochlor efficiently. The acetochlor biodegradation products were identified by gas chromatography-mass spectrometry. The probable degradative pathways of acetochlor involved dechlorination, hydroxylation, deethoxymethylation, cyclization, carboxylation, and decarboxylation. Propachlor, alachlor, and metolachlor, which are also the main components of the chloroacetanilide herbicide, could be degraded by the four mixed cultures to some degree. Given the high degradation rates observed here, the four mixed cultures obtained may be useful in the degradation processes of acetochlor.     

Degradation of p-benzyloxyphenol by Acinetobacter sp.

Abstract Acinetobacter sp. utilized p -benzyloxyphenol as sole carbon source and degraded it to p -hydroxybenzaldehyde, p -hydroxybenzoic acid, protocatechuic acid and catechol. The intermediates were identified by paper chromatography, TLC, IR, GC and HPLC. Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase during the degradation of p -benzoloxyphenol.     

CCK26–33 Degrading Activity in Brain and Nonneural Tissue: A Metalloendopeptidase

Cholecystokinin octapeptide (CCK26-33) is metabolized by neural membranes with an initial cleavage to CCK29-33 and subsequent breakdown to CCK31-33 and CCK32-33; this pattern of proteolysis occurs on incubation with either P2 or purified lysed synaptosomal membranes. To determine whether the pattern of CCK26-33 proteolysis is unique to the brain and whether regional brain differences in its pathway or rate exist, we analyzed the proteolysis of CCK by synaptic membranes of various brain areas and cellular membranes of peripheral tissue. The pattern of degradation in brain did not differ among the regions studied. The overall proteolysis rate, as measured by the formation of tryptophan, was higher in the striatum than in the cortex, although CCK29-33 was formed at the same rate in both areas. In nonneural tissue, the rate of degradation was highest in liver membranes and lowest in pancreatic acinar cell preparations. Thus, it appears that degradative peptidases are not necessarily colocalized with CCK receptors. The pattern of product formation is the same in peripheral compared with CNS membranes; thus, the degradative pathway does not appear to be unique to brain tissue. The enzyme present in synaptic membranes that is responsible for CCK29-33 formation requires a metal ion and sulfydryl groups for the catalysis and thus is a metalloendopeptidase. Furthermore, its activity is inhibited by Ac-Gly-Phe-Nle-al, a peptide aldehyde whose sequence bears some homology to the amino acid sequence in the region of CCK26-33 that is cleaved by this enzyme.     

Degradation of cytokinins by cytokinin oxidases in plants

The degradation metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This review attempts to collate the limited information available about this enzyme and introduce new facts, obtained in our laboratory, concerning the mechanism of degradation of cytokinins bearing unsaturated isoprene side chains. However, complete clarification of the effects of cytokinin oxidase on cytokinin regulation and its molecular and biochemical properties will be dependent upon the purification of the protein with cytokinin oxidase activity to homogeneity and progress in the development of requisite molecular probes.     

Degradation of carboxin and oxycarboxin by microorganisms

Summary Algae, protozoa and photosynthetic bacteria which occur in considerable number in wet soils were examined for their ability to degrade the fungicides in broth culture. Blue green algae, namely, species of Anabaena, Nostoc and Tolypothrix brought about extensive degradation of the fungicides as revealed by thin layer chromatography. Green alga,Chlorella vulgaris also degraded the fungicides. The photosynthetic bacteriumRhodospirillum sp., failed to degrade carboxin beyond sulphoxide stage but degraded oxycarboxin to a greater extent. A protozoan species, Colpoda on the other hand, brought about extensive degradation of carboxin but not of oxycarboxin. These organisms have not been examined before for the detoxification of either carboxin or oxycarboxin.Part of Ph.D. Thesis, submitted to USA, Bangalore-65 under the guidance of the second author.     

Degradation of lactoferrin by periodontitis-associated bacteria

Abstract The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena , slow by Capnocytophaga ochracea , Actinobacillus actinomycetemcomitans and Prevotella intermedia , and very slow or absent by Prevotella nigrescens , Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros . All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.     

Degradation of Arachidonoyl-Labeled Phosphatidylinositols by Brain Synaptosomes

Abstract: Incubation of synaptosomes together with 1-acyl-2-[¹⁴C]arachi-donoyl-sn-glycerophosphoinositols (GPI) and sodium deoxycholate yielded diacylglycerols and free arachidonic acid. Diacylglycerol formation is attributed to hydrolysis by the diacyl-GPI-specific phospholipase C (EC 3.1.4.10), and this reaction requires sodium deoxycholate for optimal activity. The free arachidonic acid formed is attributed to hydrolysis of diacyl-GPI by phospholipase A (EC 3.1.1.5). Free fatty acid release was observed during incubation, even in the absence of bile salts, but this process was preferentially stimulated by sodium taurocholate. The release of fatty acids was not specific for diacyl-GPI, as similar release was obtained during incubation with other phosphoglycerides. In the presence of deoxycholate (2 mg/ml), the release of diacylglycerols was maximal at a diacyl-GPI concentration around 1.0 mM. However, the free fatty acid release was linear with respect to the substrate at least up to 1.4 mM. The rate of diacylglycerol release from diacyl-GPI was more rapid in the initial 30 min, whereas the free fatty acid release was linear with time up to 2 h. Under this incubation condition, calcium was found to stimulate both types of hydrolytic action, although the concentration needed to achieve this stimulation was rather high. This type of labeled precursor is potentially useful for studies of the different modes of diacyl-GPI degradation by enzymes in brain subcellular membranes.     

PLGA支架降解对血管化功能影响的体外研究

目的:研究聚乳酸-羟基乙酸(PLGA)支架材料降解产物对血管内皮细胞增殖、迁移和小管样结构形成的影响。方法:将PLGA支架材料放入磷酸盐缓冲液(PBS)中体外无菌降解1、2、4周。用降解液处理人脐静脉内皮细胞株HUVEC,采用Brdu ELISA法、Transwell小室法和小管形成实验检测PLGA支架材料降解液对血管内皮细胞增殖、迁移和小管样结构形成的影响。结果:PLGA支架材料1周降解液对内皮细胞的迁移和小管形成无明显影响,对内皮细胞增殖有一定的促进作用。随着降解时间的延长,2周降解液抑制内皮细胞的迁移和小管形成,4周的降解液对内皮细胞增殖、迁移和小管形成均有抑制作用。结论:PLGA支架材料降解初期有对血管内皮细胞的增殖有促进作用,降解后期可能由于降解过程中产生的酸性物质累积增多,影响了血管内皮细胞的生长和功能,从而抑制新生血管的形成。