Evidence for the involvement of unsaturated fatty acids in initiating chromosome replication in Escherichia coli

The analogue 3-decynoyl-N-acetylcysteamine inhibits the synthesis of unsaturated fatty acids in Escherichia coli, resulting in the accumulation of saturated fatty acids in the membrane (Kass, 1968).In the presence of this analogue, DNA, RNA and protein synthesis continue at a linear rate for approximately two doubling times, and then cease. On the other hand, the analogue will inhibit the formation of new replication forks (premature initiation), which normally arise as a result of thymine starvation.Unlike other temperature-sensitive DNA mutants, mutants that are defective in initiating DNA replication (dnaA or dnaC) are unable to replicate DNA at a permissive temperature if they terminate replication at 42 °C in the presence of 3-decynoyl-N-acetylcysteamine.When replication is terminated at 42 °C, cultures of dnaA or dnaC mutants normally will reinitiate replication upon lowering the temperature to 30 °C. For each mutant this reinitiation is characterized by a particular temperature sensitivity. Such mutants become more temperature sensitive if the temperature is lowered in the presence of 3-decynoyl-N-acetylcysteamine. All the effects of this analogue can be reversed by the addition of unsaturated fatty acids.These results are interpreted using a model in which replication is initiated at a particular lipid site on the membrane. In the absence of unsaturated fatty acids functional lipid sites are not made. Functional sites, however, can be used again provided they are not inactivated by interaction with an inactive dnaA or dnaC product.     

海南国产沉香的化学成分研究

从国产沉香(Aquilaria sinensis)的95%乙醇提取物中分离得到6个化合物,经波谱数据分析,分别鉴定为3,3'-(3-hydroxypropane-1,2-diyl)diphenol(1)、guaiacy lacetone(2)、6-羟基-2-(4'-羟基-2-苯乙基)色酮(3)、6-羟基-2-(2-羟基-2-苯乙基)色酮(4)、6-羟基-2-(2-苯乙基)色酮(5)和5α,6β,7α,8β-四羟基-2-(4'-甲氧基-2-苯乙基)-5,6,7,8-四氢色酮(6)。其中化合物1为新化合物,化合物3为首次从国产沉香中分离得到。     

Strain differences in the induction of soluble and microsomal enzymes by phenobarbital and 3-methylcholanthrene

The ability of phenobarbital and 3-methylcholanthrene (3MC) to induce liver microsomal and soluble enzymes was compared in Sprague-Dawley and Long-Evans rats. 3MC increased the V for the aniline hydroxylase and stimulated the formation of the hemoprotein P₄₄₈ to a similar extent in the 2 strains of rats. On the other hand phenobarbital increased the V for the microsomal enzyme aniline hydroxylase and aminopyrine demethylase and enhanced the activity of the soluble enzyme aldehyde dehydrogenase only in Sprague-Dawley rats. It induced a more marked increase of cytochrome P₄₅₀ in the Sprague-Dawley than in the Long-Evans strain.     

Conditional allelic replacement applied to genes encoding the histone variant H3.3 in the mouse

Post‐translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase‐mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild‐type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre‐mediated replacement of the wild‐type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild‐type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post‐translational modifications in development and disease. genesis, 51:142?146, 2013. © 2013 Wiley Periodicals, Inc.     

Histone 3 modifications and blood pressure in the Beijing Truck Driver Air Pollution Study

Context: Histone modifications regulate gene expression; dysregulation has been linked with cardiovascular diseases. Associations between histone modification levels and blood pressure in humans are unclear.

Objective: We examine the relationship between global histone concentrations and various markers of blood pressure.

Materials and methods: Using the Beijing Truck Driver Air Pollution Study, we investigated global peripheral white blood cell histone modifications (H3K9ac, H3K9me3, H3K27me3, and H3K36me3) associations with pre- and post-work measurements of systolic (SBP) and diastolic (DBP) blood pressure, mean arterial pressure (MAP), and pulse pressure (PP) using multivariable mixed-effect models.

Results: H3K9ac was negatively associated with pre-work SBP and MAP; H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP; and H3K27me3 was negatively associated with pre-work SBP. Among office workers, H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP. Among truck drivers, H3K9ac and H3K27me were negatively associated with pre-work SBP, and H3K27me3 was positively associated with post-work PP.

Discussion and conclusion: Epigenome-wide H3K9ac, H3K9me3, and H3K27me3 were negatively associated with multiple pre-work blood pressure measures. These associations substantially changed during the day, suggesting an influence of daily activities. Blood-based histone modification biomarkers are potential candidates for studies requiring estimations of morning/pre-work blood pressure.     

Gender-related differences in the formation of skatole metabolites by specific CYP450 in porcine hepatic S9 fractions

Higher accumulation of skatole in the fat of male pigs compared with female pigs might be due to gender-related differences in the rate of skatole degradation. In the present study, skatole metabolites and cytochrome P450 (CYP450) isoforms involved in skatole metabolism were for the first time investigated in hepatic S9 fractions from six male and four female pigs (crossbred Landrace×Yorkshire dams and Duroc boar). Surprisingly, the rates of production of major skatole metabolites were similar in male and female pigs. The most abundant metabolite of skatole was 3-hydroxy-3-methyloxindole (HMOI) followed by 3-methyloxindole and indole-3-carbinol in both male and female S9 fractions. Concentrations of formed HMOI and 3-methyloxindole did not differ between the genders (P=0.124 for HMOI, and P=0.575 for 3-methyloxindole). Indole-3-carbinol formation was higher in S9 fractions from the females compared with male pigs (P=0.0001). Enzyme kinetic parameters were similar for both genders (P>0.05). In both male and female pigs, ellipticine, diallyl sulphide (DAS) and quercetin inhibited HMOI formation, confirming the involvement of CYP1A1 and CYP2E1. The formation of 3-methyloxindole was reduced in the presence of the CYP2E1 inhibitor DAS, and formation of indole-3-carbinol was reduced in the presence of CYP1A1 and CYP2A19 inhibitors. We found only minor differences in skatole metabolism between male and female pigs, particularly the involvement of CYP2C and CYP3A in indole-3-carbinol formation in female but not in male pigs. This is a very essential finding, suggesting the involvement of larger number of CYP450 isoforms in female pigs. On the other hand, indole-3-carbinol is a minor skatole metabolite, and the physiological significance of CYP2C and CYP3A involvement in its formation in female pigs, but not in male pigs, needs to be elucidated. Our results, however, should be interpreted with caution because of the low number of animals and possibility of breed and age effects on skatole metabolism.     

灰绿藜和碱蒿NHX基因 3'-UTR序列的差异性分析

采用3'末端快速扩增技术,分别从新疆野生盐生植物灰绿藜和碱蒿中克隆CgNHX和AaNHX cDNA 3'末端.测序结果表明克隆获得的片段为CgNHX和AaNHX基因3'末端,并且CgNHX和AaNHX3'-UTR的不同拷贝之间存在很大的差异性.序列同源性分析结果显示,AaNHX基因3'-UTR与CgNHX基因3'-UTR同源性高达52%,两者与SsNHX(碱蓬NHX)同源性分别达到60%和54%,表明NHX基因的加工方式可能相同,同时说明该基因在野生盐生植物中的功能是很重要的.     

江苏省不明原因轻型呼吸道传染病暴发的病因学研究

自2004年4月18日起至6月末,江苏省东台市发生以发热、咽部充血、扁桃体肿大为临床表现的原因不明的疫情暴发,发病人群以中小学生、幼儿为主,地域以许河镇、新街镇以及临近乡镇为主。疫情涉及东台市9个镇,合计报告709例,分布在75个学校,240个班级。暴发的特点为传染性强,短期内出现大量的患者,发病有班级聚集性特点,未发现成人感染。临床表现以发烧（100％）、咽部充血（91．40％）、扁桃体肿大（60．22％）、咽痛（50．00％）为主;X线检查可发现肺纹理增强（90．3％）。用Hep-2细胞从患儿20份咽拭子标本中分离到12株病毒,经PCR扩增腺病毒L3部分基因及其PCR产物的序列测定和分析,证实这12株病毒为腺病毒3型;对28份咽拭子标本直接进行DNA提取和PCR扩增,有17份标本PCR扩增L3部分基因呈阳性,经序列测定和分析,也为腺病毒3型。这29个阳性标本L3基因的1446个核苷酸的序列与GenBank上所发表的腺病毒3型序列同源性高达99,5％。双份血IgG检测：对江苏省采集10份患儿急性期和恢复期双份血清,以从患儿咽拭子分离到的腺病毒3型作为抗原,进行病毒抗体测定,6份患儿的双份血清腺病毒总IgG滴度呈4倍以上增高。患儿急性期血IgA检测：对暴发疫情34例患儿急性期血清（发病时间为1～3天）进行腺病毒IgA抗体测定,结果有9份标本为阳性,1份标本为可疑阳性,阳性率为26．47％。IgA阳性率低可能与血清采集时间过早有关,机体还未全部产生IgA。另外,还对江苏省送检的从患儿血培养基培养的细菌毒株进行了鉴定,对其16S rRNA基因进行了基因扩增及序列分析,序列测定和分析提示为A组M3型化脓性链球菌。实验研究结果并结合此次疫情的流行病学和患儿的临床表现,证实腺病毒3型是引起此次儿童不明原因轻型呼吸道传染病暴发的病原体,部分病例伴有原发或继发化脓性链球菌感染。     

易化葡萄糖载体研究进展

位于细胞膜上的易化葡葡糖载体(facilitated glucose transPorter，GLUTs)促进体内葡葡糖的跨膜转运，它对稳定细胞能量和维持生命活动起到至关重要作用。到目前为止已相继发现并鉴定了12种GLUTs，它们由不同基因编码，具有相似的蛋白质结构和生物功能，在基因表达和细胞内分布受多种因素调控，其中最重要的是胰岛素。胰岛素通过P13K信息通路和Cbl激活蛋白(Cbl actiVating Protn，CAP)-Cbl信息通路实现对GLUT4等的快速周转，从而改变葡葡糖进入细胞的量。深入了解GLUTs的生物学特性和调控机制具有重要的临床意义。     

New indices in morphometry of nuclear structure in the NIH 3T3 cell line

We analyzed the connection of changes in nucleus ploidy with changes in nucleolar apparatus of NIH 3T3 cells. The quantity of nucleoli does not depend on the quantity of nucleolar DNA, but instead depends on euploidy: the majority of euploid cells have 1-3 nucleoli. The quantity of DNA in the nucleolus is correlated with the quantity of nucleolar DNA, and does not depend on ploidy changes. The nucleolar area has a tendency to increase in line with an increase in their numbers in the nucleus. The relationship of the quantity of DNA in the nucleolus with that of the nucleus is stable. During the process of increase in the number of nucleoli in a nucleus, there is a corresponding decrease in the quantity of DNA in each nucleolus, and there is likewise no increase in the sum of nucleolar DNA. The ratio of sums of the nucleolar perimeters to nuclear perimeter is a significant factor, which increases linearly along with an increase in the number of nucleoli in a nucleus.     

Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

alpha-tocopherol increases the intracellular glutathione level in HaCaT keratinocytes

-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with -tocopherol for 24 h, the intracellular GSH was increased at every concentration of -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with -tocopherol at 50 μM for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of -tocopherol on the GSH level, BSO, which is a typical inhibitor of γ-glutamylcysteine synthetase ( γ-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of -tocopherol on the GSH level was observed. On the other hand, -tocopherol resulted in the up-regulation of γ-GCS-HS (heavy subunit) mRNA. In addition, water soluble -tocopherol derivatives ( -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of γ-GCS-HS mRNA.     

Redox homeostasis is compromised in vivo by the metabolites accumulating in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency in rat cerebral cortex and liver

Abstract

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a disorder biochemically characterized by the predominant accumulation of 3-hydroxy-3-methylglutarate (HMG), 3-methylglutarate (MGA), 3-methylglutaconate and 3-hydroxyisovalerate in tissues and biological fluids of the affected patients. Neurological symptoms and hepatopathy are commonly found in HL deficiency, especially during metabolic crises. Since the mechanisms of tissue damage in this disorder are not well understood, in the present study we evaluated the ex vivo effects of acute administration of HMG and MGA on important parameters of oxidative stress in cerebral cortex and liver from young rats. In vivo administration of HMG and MGA provoked an increase of carbonyl and carboxy-methyl-lysine formation in cerebral cortex, but not in liver, indicating that these metabolites induce protein oxidative damage in the brain. We also verified that HMG and MGA significantly decreased glutathione concentrations in both cerebral cortex and liver, implying a reduction of antioxidant defenses. Furthermore, HMG and MGA increased 2’,7’-dichlorofluorescin oxidation, but did not alter nitrate and nitrite content in cerebral cortex and liver, indicating that HMG and MGA effects are mainly mediated by reactive oxygen species. HMG and MGA also increased the activities of superoxide dismutase and catalase in cerebral cortex and liver, whereas MGA decreased glutathione peroxidase activity in cerebral cortex. Our present data showing a disruption of redox homeostasis in cerebral cortex and liver caused by in vivo administration of HMG and MGA suggest that this pathomechanism may possibly contribute to the brain and liver abnormalities observed in HL-deficient patients.     

Large-volume sample stacking-capillary electrophoresis used for the determination of 3-nitrotyrosine in rat urine

Large-volume sample stacking using the electroosmotic flow (EOF) pump technique has been investigated for the quantification of 3-nitrotyrosine in urine of diabetic rats. The best separation conditions for these highly complex samples were obtained using capillary electrophoresis (CE) in the reversed polarity mode (i.e., injecting at the cathode and detecting at the anode) using cetyltrimethylammonium bromide (CTAB) in the running buffer. The optimum CE separation conditions were achieved using a phosphate buffer prepared with 0.15M phosphoric acid and 0.5 mM CTAB adjusted to pH 6.4 with sodium hydroxide. In such CE conditions, the limit of detection (LOD) was 1.77 microM for 3-nitrotyrosine with normal injection mode, meanwhile with the large-volume sample stacking technique a more than 20-fold improvement was observed (i.e., LOD = 0.08 microM was obtained) without noticeable loss of resolution. This value allowed the detection of 3-nitrotyrosine in urine from diabetic rats. To our knowledge, this work is one of the few applications showing the great possibilities of these stacking procedures to analyse biological samples by CE.     

Adrenalectomy of Rats Results in Hypomyelination of the Central Nervous System

The effect of adrenalectomy on CNS myelin accumulation was investigated to determine whether glucocorticoids play a role in regulating myelination. When 14-day-old rats were adrenalectomized and sacrificed 7-8 days later, the amount of bulk-isolated myelin in whole brain, as expressed per gram wet weight of brain or per milligram DNA-phosphate, was reduced to about 75% that of sham-operated controls. Both brain weight and DNA content were unchanged by adrenalectomy. Examination of individual brain regions also revealed decreased amounts of myelin in adrenalectomized animals. Brain glycerol 3-phosphate dehydrogenase specific activity was reduced in adrenalectomized animals to 40-60% that of controls, and serum corticosterone levels were less than 0.6% of control levels. The amount of cerebral myelin in animals adrenalectomized on day 21 and sacrificed 9 days later was not significantly reduced. This suggests a possible role of glucocorticoids during the early period of rapid myelination.