Regulation of the human autologous T cell proliferation by endogenously generated C5a.

The immunomodulating role of endogenously synthesized C5 and subsequently generated C5a was studied in a serum-free human autologous mixed leukocyte reaction (AMLR) using either separated T and non-T cell populations or unfractionated mononuclear leukocytes of human peripheral blood. Monoclonal mouse IgG or Fab fragments against human C5/C5a were used as probes for the evaluation of the biological effects of C5a. The reduction of DNA synthesis after the addition of nanogram amounts of anti-C5/C5a mAb was dose-dependent, reaching maximum levels of 30-50%. Of special importance was the availability of a mAb that recognizes a neoepitope present on C5a and not on serum-derived C5. The demonstration of the specificity of its inhibitory effect suggests that C5 is synthesized under the in vitro conditions employed and that the subsequently generated C5a exerts biological effects on T cell proliferation.     

The effect of pyrophosphate on the rigor fibers of rabbit m. psoas fibers

The effect of MgPPi on the rigor force of glycerinated fibres in the range of ionic strength 75-250 mM at two temperatures 18 degrees and 5 degrees C was studied. At 18 degrees C the maximum of this effect was above the range of average ionic strength. At 5 degrees C the greatest effect of MgPPi was observed at low ionic strength.     

Effect of insulin and cortisol on oxidation of (1-14C)glucose, (6-14C)glucose, (1-14C)palmitate and (1-14C)leucine in the tissues of swine during the neonatal period

The intensity of [1-14C]glucose, [6-14C]glucose, [1-14C]palmitate and [1-14C]leucine oxidation and the effect of insulin and hydrocortisone on this process were studied in the brain, duodenum mucosa, liver and skeletal muscle of 1- and 5-day old piglets in vitro. Most of the studied substrates are oxidized in the tissues of 5-day piglets more intensively. Insulin stimulates oxidation of [1-14C]glucose, [6-14C]glucose and [1-14C]leucine in the brain and duodenum mucosa in 1- and 5-day old piglets, while in the liver and skeletal muscle--only in 5-day old piglets. Hydrocortisone administration enhances oxidation of [1-14C]leucine in most of the studied tissues in 1-day piglets and oxidation of [1-14C]glucose and [6-14C]glucose--in 5-day piglets. Both hormones produce no essential influence on the intensity of [1-14C]palmitate oxidation in the studied tissues of piglets or somewhat weaken it.     

Preferential oxidative dehalogenation upon conversion of 2-halophenols by Rhodococcus opacus 1G

The regiospecificity of hydroxylation of C2-halogenated phenols by Rhodococcus opacus 1G was investigated. Oxidative defluorination at the C2 position ortho with respect to the hydroxyl moiety was preferred over hydroxylation at the non-fluorinated C6 position for all 2-fluorophenol compounds studied. Initial hydroxylation of 2,3, 5-trichlorophenol resulted in the exclusive formation of 3, 5-dichlorocatechol. These results indicate that, in contrast to all other phenol ortho-hydroxylases studied so far, phenol hydroxylase from R. opacus 1G is capable of catalyzing preferential oxidative defluorination but also oxidative dechlorination.     

The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.     

The processes of methane formation and oxidation in the soils of the Russian arctic tundra

Methane emission from the following types of tundra soils was studied: coarse humic gleyey loamy cryo soil, peaty gley soil, and peaty gleyey midloamy cryo soil of the arctic tundra. All the soils studied were found to be potential sources of atmospheric methane. The highest values of methane emission were recorded in August at a soil temperature of 8-10 degrees C. Flooded parcels were the sources of atmospheric methane throughout the observation period. The rates of methane production and oxidation in tundra soils of various types at 5 and 15 degrees C were studied by the radioisotope method. Methane oxidation was found to occur in bog water, in the green part of peat moss, and in all the soil horizons studied. Methane formation was recorded in the horizons of peat, in clay with plant roots, and in peaty moss dust of the bogey parcels. At both temperatures, the methane oxidation rate exceeded the rate of methane formation in all the horizons of the mossy-lichen tundra and of the bumpy sinkhole complex. Methanogenesis prevailed only in a sedge-peat moss bog at 15 degrees C. Enrichment bacterial cultures oxidizing methane at 5 and 15 degrees C were obtained. Different types of methanotrophic bacteria were shown to be responsible for methane oxidation under these conditions. A representative of type I methylotrophs oxidized methane at 5 degrees C, and Methylocella tundrae, a psychroactive representative of an acidophilic methanotrophic genus Methylocella, at 15 degrees C.     

Formation and structure of the C5b-7 complex of the lytic pathway of complement.

The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.     

Sulfaphenazole derivatives as tools for comparing cytochrome P450 2C5 and human cytochromes P450 2Cs: identification of a new high affinity substrate common to those enzymes

The inhibitory effects of a series of sulfaphenazole (SPA) derivatives were studied on two modified forms of rabbit liver cytochrome P450 2C5 (CYP2C5), CYP2C5dH, and structurally characterized CYP2C5/3LVdH and compared to the previously described effects of these compounds on human CYP2C8, 2C9, 2C18, and 2C19. SPA and other negatively charged compounds that potently inhibit CYP2C9 had very little effect on CYP2C5dH, whereas neutral, N-alkylated derivatives exhibited IC50 values between 8 and 22 microM. One of the studied compounds, 4, that derives from SPA by replacement of its NH(2) substituent with a methyl group and by N-methylation of its sulfonamide moiety, acted as a good substrate for all CYP2Cs used in this study. Hydroxylation of the benzylic methyl of 4 is the major reaction catalyzed by all of these CYP2C proteins, whereas hydroxylation of the N-phenyl group of 4 was observed as a minor reaction. CYP2C5dH, 2C5/3LVdH, 2C9, 2C18, and 2C19 are efficient catalysts for the benzylic hydroxylation of 4, with K(m) values between 5 and 13 microM and k(cat) values between 16 and 90 min(-1). The regioselectivity observed for oxidation of 4 by CYP2C5/3LVdH was easily interpreted on the basis of the existence of two different binding modes of 4 characterized in the experimentally determined structure of the complexes of CYP2C5/3LVdH with 4 described in the following paper [Wester, M. R. et al. (2003) Biochemistry 42, 6370-6379].     

猪瘟病毒中国兔化弱毒疫苗株在原代牛睾丸细胞中增殖特性的研究

以猪瘟病毒中国兔化弱毒疫苗株 (CSFVC株 )为实验材料 ,研究该病毒株在原代牛睾丸细胞中增殖的基本特性与规律。找到了一株能够使用免疫荧光技术进行检测的C株猪瘟病毒 ,并对病毒滴度的测定方法进行了改进 ,发展完善了荧光斑技术 (F PFU)。使用改进的病毒滴度测定方法 ,对接毒后培养上清中病毒滴度的变化趋势进行检测。在原代牛睾丸细胞中 ,接毒后 5d ,几乎所有的细胞都可被病毒感染 ;释放到培养液中有活性的病毒粒子达到 10 5F PFU/mL以上 ,后维持在该水平。对原代细胞中细胞种类随代次增加的变化趋势进行了观察 ,并对CSFV的增殖特点及生产中出现的一收毒价不稳定、多次收获病毒等现象的机制进行探讨。并对影响猪瘟病毒增殖的因素进行了实验 ,不仅加深了对猪瘟病毒C株在原代牛睾丸细胞中增殖规律的了解 ,还为疫苗生产中猪瘟病毒产量的提高提供新的思路     

Polymorphism of human blood serum cholinesterase in populations of Evenks and Yakuts in Krasnoyarsk Territory

Polymorphism for E2 locus of human serum cholinesterase was studied in populations of evenks and yakuts of Krasnoyarsky region by the method of starch gel electrophoresis. Thee frequencies of C5+ phenotype correspond to C5+ frequencies in other mongoloid populations of Siberia. The activity of cholinesterase was determined by semiquantitative technique. Three individuals with a sharply decreased cholinesterase activity have been revealed.     

Complement Split Product C5a Mediates the Lipopolysaccharide-Induced Mobilization of Cfu-S and Haemopoietic Progenitor Cells, But Not the Mobilization Induced By Proteolytic Enzymes

Abstract. Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU-s) as well as granulocyte-macrophage progenitor cells (GM-CFU) and the early progenitors of the erythroid lineage (E-BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5-deficient mice. the mobilization by C activators in these mice could be restored by injection of C5-sufficient serum, suggesting a critical role for C5.
The manner in which C5 was involved in the C activation-mediated stem cell mobilization was studied using a serum transfer system. C5-sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5-sufficient and C5-deficient mice. C5-deficient serum was not able to do so. the resistance of the mobilizing principle to heat treatment (56°C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5-deficient mice also induced mobilization of CFU-s.     

Complement split product C5a mediates the lipopolysaccharide-induced mobilization of CFU-s and haemopoietic progenitor cells, but not the mobilization induced by proteolytic enzymes

Intravenous (i.v.) injection of mice with lipopolysaccharide (LPS), and the proteolytic enzymes trypsin and proteinase, mobilizes pluripotent haemopoietic stem cells (CFU-s) as well as granulocyte-macrophage progenitor cells (GM-CFU) and the early progenitors of the erythroid lineage (E-BFU) from the haemopoietic tissues into the peripheral blood. We investigated the involvement of the complement (C) system in this process. It appeared that the early mobilization induced by LPS and other activators of the alternative complement pathway, such as Listeria monocytogenes (Lm) and zymosan, but not that induced by the proteolytic enzymes, was absent in C5-deficient mice. The mobilization by C activators in these mice could be restored by injection of C5-sufficient serum, suggesting a critical role for C5. The manner in which C5 was involved in the C activation-mediated stem cell mobilization was studied using a serum transfer system. C5-sufficient serum, activated in vitro by incubation with Lm and subsequently liberated from the bacteria, caused mobilization in both C5-sufficient and C5-deficient mice. C5-deficient serum was not able to do so. The resistance of the mobilizing principle to heat treatment (56 degrees C, 30 min) strongly suggests that it is identical with the C5 split product C5a, or an in vivo derivative of C5a. This conclusion was reinforced by the observation that a single injection of purified rat C5a into C5-deficient mice also induced mobilization of CFU-s.     

Response of the serotoninergic brain system to social stress of various duration in male mice C57BL/6J and CBA/Lac]

The effects of social stress caused by experience of defeats in mice during 3 or 10 consecutive days of intermale confrontations on serotonergic brain activity (5-HT, 5-HIAA levels and 5-HIAA/5-HT ratio) in some brain regions of CBA/Lac (CBA) and C57BL/6J (C57) inbred mice have been studied. It was revealed the significant changes in 5-HT methabolism in the brain regions of defeated mice (losers) of CBA strain after 3 intermale confrontations. However, after 10 days of social stress these changes (excluded amygdala) turned to the control measures testifying to the adaptive mechanisms of serotonergic system in CBA losers. In C57 strain, the three-day social stress produced the mild changes in the brain serotonergic activity both quantitatively as well as qualitatively. Nevertheless, losers subjected to ten-day intermale confrontations had more expressed changes in 5-HT, 5-HIAA levels of 5-HIAA/5-HT ratios in the brain regions studied. It seems that long lasting social stress induced the development of disbalance of the brain serotonergic activity in C57 losers: it was shown the hyperactivity in the hypothalamus and hypoactivity in the amygdala and nucl. accumbens. Apparently, this cause leads to the development of the pronounced anxiety shown earlier in this mouse strain.     

The C5 isozyme of serum cholinesterase and adult weight.

The relationship between the CHE2 locus of serum cholinesterase (BChE) and adult human weight was studied in a sample of 225 CHE2 C5+ individuals and 225 CHE2 C5- controls matched for sex, height, age and race. With respect to the intensity of the C5 band staining (scored 1-6), 113 individuals had faint C5 bands (scores 1-3) and 112 intense C5 bands (scores 4-6). The individuals with intense CHE2 C5+ phenotype showed a significantly lower mean adult weight (64.66 +/- 0.73 kg) when compared to their controls (70.59 +/- 0.97 kg) and a significant reduction in weight variance (59.81 and 105.18, respectively). Individuals with faint C5 bands, although showing a negative correlation between weight and C5 band intensity, did not differ from their controls in mean weight.     

The oxidation of proline by mitochondrial preparations

The oxidation by mitochondria of various rat tissues of proline, pyrroline-5-carboxylate (P5C) and a number of aldehydes has been studied and ADP/O ratios determined for liver mitochondria. High oxidative activity for proline and P5C was found only in the liver and kidney. During the oxidation by liver and kidney mitochondria of proline and P5C; glutamate, ammonia, aspartate and some ornithine accumulated, thus suggesting that proline may normally be converted to ornithine by mitochondria. The oxidation of P5C (glutamic acid semialdehyde) by a mitochondrial dehydrogenase may be the same enzyme that oxidizes succinic acid semi-aldehyde but different from that oxidizing acetaldehyde.     

Relative inefficiency of terminal complement activation

The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.     

Potentiation of the primary humoral immune response in vitro by C5a anaphylatoxin

The effect of the anaphylatoxin C5a on the primary humoral immune response to SRBC was studied in culture of spleen cells from C3H mice. The addition of human C5a to antigen-stimulated cultures resulted in a significant, dose-dependent augmentation of the primary PFC response to antigen. The specificity of this effect was affirmed by the ability of C5ades Arg, but not of the structurally analogous C3a anaphylatoxin, to act in a parallel fashion. Enhancement could be observed over a range of doses of antigen. Brief preincubation of macrophages, but not of lymphoid cells, with C5a was sufficient to cause subsequent enhancement of the primary humoral immune responses. The duration of preincubation required for this effect closely paralleled that for binding of C5a to peritoneal cells. Immunopotentiation by C5a appears to involve the function of C5a receptor-bearing, Ia- accessory cells as well as Ia+ antigen-presenting cells. Immunopotentiation could be observed when the addition of C5a was delayed for up to 24 hr after initiation of culture. Additionally, augmentation of tritiated thymidine uptake in mixed lymphocyte reactions was enhanced by the addition of C5a in a fashion parallel to that for the primary response to SRBC. These observations support a role for C5a as a modulator of cellular immunity in addition to its role in acute inflammation. Possible cellular mechanisms and implications for immunomodulation of immune responses are discussed.     

白骨壤模拟湿地系统中氮的分配、循环及其净化效应

在温室中建立红树林植物白骨壤模拟湿地系统 ,分别用正常、5倍和 10倍浓度的人工配置的城市污水每周定时定量对模拟系统污灌 2次 ,用海水作对照 ,持续 1年。研究营养污染物氮在系统中的分配、循环及被净化的效果。结果表明 ,随污水处理浓度的升高 ,被更换的潮汐水中的含氮量和土壤含氮量都有较大提高 ;植物体各部分含氮量的高低顺序排列为叶 >茎>根 ;各处理组植物中氮元素的周转期依次为 4 81年、5 81年、4 5 8年和 5 86年 ;模拟系统对污水中氮的净化效果显著。     

Sterol-phospholipid interaction in model membranes: role of C5-C6 double bond in cholesterol

Several double-bond isomers of cholesterol where the normal C5-C6 double bond (delta 5) has been moved to different positions in the ring skeleton, i.e., delta 1, delta 4, delta 7, delta 8(9), delta 8(14), and delta 14, have been synthesized and incorporated in phosphotidylcholine vesicles. In addition, dienes like delta 5,7, delta 7,14, and delta 8,14 have also been studied. Many of these cholesterol analogues are intermediates in the sterol biosynthesis in different organisms. The incorporation studied indicated that more than 90% of the sterol was present in the vesicles. The effect of these cholesterol analogues was studied by glucose permeability, electron spin resonance, and fluorescence polarization spectroscopy. These studies indicated that delta 14-cholesten-3 beta-ol was most effective in restricting glucose permeability or in increasing the order parameter but was still not as effective as cholesterol. This was followed by delta 8(14)- and delta 8(9)-cholesten-3 beta-ol. The delta 1, delta 4, and delta 7 analogues and the dienols were relatively less effective in condensing the membrane. These studies indicate that the double bond at C5-C6 in cholesterol is most effective for optimal sterol-phospholipid interaction and may have formed the basis of the migration of the double bond from rings C and D in sterols to C5-C6 during the evolution of cholesterol.