Proline reduces brain cytochrome c oxidase: prevention by antioxidants

In the present study, we initially investigated the in vivo (acute and chronic) and in vitro effects of proline on cytochrome c oxidase (complex IV) activity in rat cerebral cortex to test the hypothesis that proline might alter energy metabolism and that this alteration could be provoked by oxidative stress. The action of alpha-tocopherol and ascorbic acid on the effects produced by proline was also evaluated. For acute administration, 29- and 60-day-old rats received one subcutaneous injection of proline (18.2 micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1h later. For chronic treatment, proline was injected subcutaneously twice a day at 10h intervals from the 6(th) to the 28(th) day of age. Rats were sacrificed 12h (29(th)) or 31 days (60(th)) after the last injection. Results showed that acute administration of proline significantly diminished the activity of cytochrome c oxidase in the cerebral cortex of 29- and 60-day-old rats. On the other hand, chronic hyperprolinemia reduced this complex activity only on day 29, but not on the 60(th) day of life. In another set of experiments, 22-day-old rats or 53-day-old rats were pretreated for 1 week with daily intraperitoneal administration of alpha-tocopherol (40 mg/kg) and ascorbic acid (100mg/kg) or saline. Twelve hours after the last antioxidant injection, rats received a single injection of proline or saline and were killed 1h later. In parallel to chronic treatment, rats received a daily intraperitoneal injection of alpha-tocopherol and ascorbic acid from the 6(th) to the 28(th) day of life and were killed 12h after the last injection. Results showed that the pretreatment with alpha-tocopherol and ascorbic acid before acute proline administration or concomitant to chronic proline administration significantly prevented these effects. We also observed that proline (3.0 microM-1.0 mM) when added to the incubation medium (in vitro studies) did not alter cytochrome c oxidase activity. Data suggest that the inhibitory effect of proline on cytochrome c oxidase activity is possibly associated with oxidative stress and that this parameter may be involved in the brain dysfunction observed in hyperprolinemia.     

Schizophrenia-like neurophysiological abnormalities in 22q11.2 deletion syndrome and their association to COMT and PRODH genotypes

22q11.2 deletion syndrome (22q11.2DS) is a common genetic risk factor for the development of schizophrenia. We investigated two neurophysiological endophenotypes of schizophrenia – P50 sensory gating and mismatch negativity in 22q11.2DS subject and evaluated their association with catechol O-methyltransferase (COMT) and proline dehydrogenase (PRODH) genetic variants. We also assessed the association of neurophysiological measures with schizophrenia-like symptomatology in 22q11.2DS. Fifty-nine subjects, 41 with 22q11.2DS and 18 typically developing controls, participated in the study. The participants with 22q11.2DS were genotyped for the COMT Val¹⁵⁸Met (rs4680) and PRODH Gln¹⁹Pro (rs2008720) and Arg¹⁸⁵Trp (rs4819756) polymorphisms. Following psychiatric evaluation, all the participants underwent neurophysiological recordings and executive function assessment. The 22q11.2DS group showed poorer sensory gating of the P50 response than the controls. Within the 22q11.2DS group, the COMT Met allele was associated with poorer sensory gating, while both the COMT Met allele and the PRODH Pro-Arg haplotype were associated with smaller mismatch negativity amplitudes. Smaller mismatch negativity amplitudes predicted greater impairment of executive functions and greater severity of schizophrenia-like negative symptoms in 22q11.2DS. The current study demonstrates that sensory gating impairments that are typical of schizophrenia are found in 22q11.2DS subjects. Our results further suggest that COMT and PRODH genetic variations contribute to sensory gating and mismatch negativity schizophrenia-like impairments in 22q11.2DS, possibly via dopaminergic/glutamatergic networks. The associations of mismatch negativity impairments with increased severity of schizophrenia-like negative symptoms and poorer executive functions performance in our 22q11.2DS sample suggest that mismatch negativity is a potential endophenotype for schizophrenia in 22q11.2DS.     

The diversity of the proline-rich domain of pneumococcal surface protein A (PspA): Potential relevance to a broad-spectrum vaccine

Pneumococcal surface protein A (PspA) is a surface exposed, highly immunogenic protein of Streptococcus pneumoniae. Its N-terminal α-helical domain (αHD) elicits protective antibody in humans and animals that can protect mice from fatal infections with pneumococci and can be detected in vitro with opsonophagocytosis assays. The proline-rich domain (PRD) in the center of the PspA sequence can also elicit protection. This study revealed that although the sequence of PRD was diverse, PRD from different pneumococcal isolates contained many shared elements. The inferred amino acid sequences of 123 such PRDs, which were analyzed by assembly and alignment-free (AAF) approaches, formed three PRD groups. Of these sequences, 45 were classified as Group 1, 19 were classified as Group 2, and 59 were classified as Group 3. All Group 3 sequences contained a highly conserved 22-amino acid non-proline block (NPB). A significant polymorphism was observed, however, at a single amino acid position within NPB. Each of the three PRD groups had characteristic patterns of short amino acid repeats, with most of the repeats being found in more than one PRD group. One of these repeats, PKPEQP as well as the NPB were previously shown to elicit protective antibodies in mice. In this study, we found that sera from 12 healthy human adult volunteers contained antibodies to all three PRD groups. This suggested that a PspA-containing vaccine containing carefully selected PRDs and αHDs could redundantly cover the known diversity of PspA. Such an approach might reduce the chances of PspA variants escaping a PspA vaccine’s immunity.     

人工模拟干旱胁迫下金银花幼苗的生理适应性反应

目的研究人工模拟干旱胁迫条件下金银花幼苗的生理适应性反应,为揭示金银花植株的抗旱机制以及抗旱品种的选育提供理论依据。方法采用聚乙二醇（PEG-6000）模拟干旱胁迫处理金银花幼苗,检测幼苗体内的丙二醛（MDA）含量;超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）等保护酶活性,渗透调节物质脯氨酸（Pro）和可溶性糖含量。结果 10%、20%和30%PEG处理幼苗后120 h MDA含量分别高出对照组20.31%、28.12%和36.72%,随着PEG浓度升高,MDA含量逐渐增加。SOD、POD、CAT和APX活性总体呈现先升高后降低的趋势,各种酶对干旱胁迫的响应速率不同,CAT于干旱胁迫后立即开始启动,SOD、POD于干旱胁迫后24 h启动,而APX于干旱胁迫后48 h开始启动。Pro和可溶性糖含量均于处理后24 h开始增高,于处理后72 h达到峰值。结论随着干旱程度的增加和胁迫的延长,金银花体内膜质过氧化严重,通过增加Pro、可溶性糖的含量以及保护酶系统来抵御干旱胁迫,保护酶启动顺序为CAT、SOD、POD、APX。     

合成β-内酰胺酶抑制剂阿维巴坦的关键中间体的研究进展

β-内酰胺类抗生素耐药性是对目前全球卫生、食品安全和发展的重大威胁之一。由于新型抗生素开发难度不断增加,采用复方型抗生素就成为解决这一难题的重要手段。阿维巴坦是一类β-内酰胺酶抑制剂,其抑酶谱广,与其他β-内酰胺类抗生素联用可有效恢复或增强其活性,极大地缓解了针对耐药菌无药可用的局面。在几乎所有的合成路线中,都经历了一个重要的中间体：顺式-5-羟基哌啶-2-甲酸,然后由其再进一步衍生化后获得目标化合物。本文对顺式-5-羟基哌啶-2-甲酸的化学合成和酶促合成两种方式的研究现状和发展前景进行概述和总结,从而对其在医药合成领域中的应用提供参考。     

骨碎补总黄酮对去卵巢大鼠骨超微结构及脯氨酸羟化程度的影响

目的：研究应用骨碎补总黄酮后骨超微结构及脯氨酸羟化程度的变化,从骨胶原的超微结构来研究骨碎补总黄酮对骨强度的影响。方法：40只10月龄SD雌性大鼠（体重350～380g）,随机分成假切除组（空白组）、模型组、药物组、对照组,每组10只。假切除组只切除腹部的少许脂肪,其余各组采用双侧卵巢切除的方法进行动物造模。药物组每天灌服中药骨碎补总黄酮浓缩剂2ml（相当于骨碎补总黄酮0.02g）,共6个月;对照组每天灌服培美力悬剂2ml（相当于倍美力0.017mg）,共6个月。灌胃6个月后取L2进行扫描电镜观察,并以碱水解法测单位重量脯氨酸羟化程度。结果：骨碎补总黄酮能明显改善骨胶原的排列,减少断裂的骨胶原。药物组脯氨酸羟化程度为（58.77±0.56）%,对照组为（60.90±0.53）%,模型组为（47.34±0.52）%,药物组和对照组比较差异无统计学意义,但较模型组脯氨酸羟化程度提高（P〈0.05）。结论：骨碎补总黄酮能改善松质骨的超微结构及脯氨酸羟化程度,从而为药物防治骨质疏松的研究提供思路。     

The intracellular mobility of NPY and a putative mitochondrial form of NPY in neuronal cells

Preproneuropeptide Y is a precursor peptide to mature neuropeptide Y (NPY), which is a universally expressed peptide in the central and peripheral nervous system. NPY is normally routed to endoplasmic reticulum and secretory vesicles in cells, which secrete NPY. In our previous studies, we found a functional Leucine7 to Proline7 (L7P) polymorphism in the signal peptide sequence of preproNPY. This polymorphism affects the secretion of NPY and causes multiple physiological effects in humans. The sequence of NPY mRNA contains two in frame kozak sequences that allow translation initiation to shift, and translation of two proteins. In addition to mature NPY_1–36 also a putative truncated NPY_17–36 with mitochondrial targeting signal is produced. The purpose of this study was to investigate the protein mobility of the putative mitochondrial fragment and the effect of the L7P polymorphism on the cellular level using GFP tagged constructs. The mobility was studied with fluorescence recovery after photobleaching technique in a neuronal cell line. We found that the mobility of the secretory vesicles with NPY_1–36 in cells with L7P genotype was increased in comparison to vesicle mobility in cells with the more abundant L7L genotype. The mobility in the cells with the putative mitochondrial construct was found to be very low. According to the results of the present study, the mitochondrial truncated peptide stays in the mitochondrion. It can be hypothesized that this could be one of the factors affecting energy balance of the membranes of the mitochondrion.     

Changes in proline synthetic and degradative enzymes during matrix-induced cartilage and bone formation

Summary Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3 days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.     

Inherited factor VII deficiency: identification of two novel mutations (A191V and T239P) in the catalytic domain

We describe here five F7 mutations found in four patients without bleeding history, despite constitutional coagulation Factor VII (FVII) deficiency. All five mutations are missense and affect the catalytic domain of FVII (A191T, A191V, T239P, R224Q and M298I). The A191V and T239P mutations are novel and were found in homozygous patients with no clinical bleeding tendency. The patient diagnosed with the A191V mutation had a phenotype corresponding to a moderate type 1 FVII deficiency (FVII:C 4%, FVII:Ag 5%). The T239P mutation was found in a patient with mild type 2 FVII deficiency (FVII:C 25%, FVII:Ag 95%). Novel mutations are both in close vicinity to the charge-stabilizing system of FVII. Modeling studies allow understanding in part the molecular basis for the loss of function.     

The activities of alanine aminopeptidase,leucine aminopeptidase,proline dipeptidase and prolyl dipeptidase in the mucosa of the small intestine

Alanine aminopeptidase (EC 3.4.11.2), leucine aminopeptidase (EC 3.4.11.1), proline dipeptidase (EC 3.4.13.9), and prolyl dipeptidase (EC 3.4.13.8) have been investigated in small intestinal mucosa homogenates of normal children and children suffering from different degrees of villous damage. The activities of proline dipeptidase and prolyl dipeptidase could be shown to be significantly decreased in cases of subtotal and total villous atrophy, whereas the activities of alanine aminopeptidase and leucine aminopeptidase were not influenced. The results are discussed in view of the subcellular distribution of these enzymes.