封闭负压引流联合局部给氧治疗兔耳缺血性创面的实验研究

目的：观察封闭负压引流联合局部给氧促进兔耳缺血性创面愈合的效果。方法：28只大耳白兔,双耳背各造成直径2．5cm全层皮肤缺损,结扎中央血管神经束及后边缘动静脉,形成缺血创面。56个创面随机分为七组：A组（-50mmHg负压同时给氧,浓度为40％±5％,每日4小时）、B组（持续-50mmHg负压4小时继之局部给氧l小时）、c组（负压治疗4min,停止1min,每日4小时,之后给氧1小时）,D组（．50mrnHg负压治疗每日4小时）、E组（-125rnmHg负压治疗每日4小时）、F组（单纯给40％±5％氧l小时）和G组（空白对照）。在创面形成第0、1、3、5、7、10、14、18天拍照,测量创面面积,计算创面愈合率及创面愈合时间;在各时相点切取创面标本,组织学观察创面肉芽、上皮生长、水肿和炎细胞,Ki67免疫组化标记增殖细胞并计算增殖指数,TUNEL法计算凋亡指数。结果：负压给氧组在相同时相点创面愈合率高于单纯负压、氧疗或空白组（P〈0．05）,创面肉芽生长快,水肿和炎症轻,细胞增殖指数高于对照组（P〈0．05）,而细胞凋亡指数显著低于对照组（P〈0．05）。结论：负压联合局部给氧能显著促进兔耳缺血创面的愈合。     

Feedback control of mesolimbic somatodendritic dopamine release in rat brain

The objective of this study was to examine the role of dopamine (DA) receptors in the nucleus accumbens (ACB) in controlling feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats using ipsilateral dual-probe in vivo microdialysis. Perfusion of the ACB for 60 min with the DA uptake inhibitor GBR-12909 (10-1,000 microM) or nomifensine (10-1,000 microM) dose-dependently increased the extracellular levels of DA in ACB and concomitantly reduced the extracellular levels of DA in the VTA. Coperfusion of 100 microM nomifensine with either 100 microM SCH-23390 (SCH), a D1 antagonist, or 100 microM sulpiride (SUL), a D2 receptor antagonist, produced either an additive (for SCH) or a synergistic (for SUL) elevation in the extracellular levels of DA in the ACB, whereas the reduction in the extracellular levels of DA in the VTA produced by nomifensine alone was completely prevented by addition of either antagonist. Application of 100 microM SCH or SUL alone through the microdialysis probe in the ACB increased the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA remained unchanged. Overall, the results suggest that (a) increasing the synaptic levels of DA in the ACB activates a long-loop negative feedback pathway to the VTA involving both D1 and D2 postsynaptic receptors and (b) terminal DA release within the ACB is regulated directly by D2 autoreceptors and may be indirectly regulated by D1 receptors, possibly on interneurons and/or through postsynaptic inhibition of the negative feedback loop.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Basic unit of population observed in the distribution of the rice-stem borer,Chilo simplex,in a paddy field. Pattern of the spatial distribution of insect. 4th report

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Contributions from the Entomological Laboratory, Kyoto University, No. 231.     

Differential negative air ion effects on learning disabled and normal-achieving children

Forty normal-achieving and 33 learning disabled (LD) children were assigned randomly to either a negative ion or placebo test condition. On a dichotic listening task using consonant-vowel (CV) combinations, both groups showed an ioninduced increase in the normal right ear advantage (REA). However, the mechanisms for this effect were different for each group. The LDs showed the effect at the right ear/left hemisphere (enhancement). The normal achievers showed the effect at the left ear/right hemisphere (inhibition). The results are consistent with an activation-inhibition model of cerebral function and suggest a functional relationship between arousal, interhemispheric activation-inhibition, and learning disabilities. The LDs may have an interhemispheric dysfunction. Both groups showed superior right ear report and the normal achiever showed overall superiority. Normal achievers showed higher consonant intrusion scores, probably due to a greater cognitive capacity. Age was a significant covariate reflecting developmental capacity changes. Negative air ions are seen to be a tool with potential theoretical and remedial applications.     

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

O⁶-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O⁶-methylguanine and O⁴-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.     

Characterization of Protease-Activated Receptor (PAR) ligands: Parmodulins are reversible allosteric inhibitors of PAR1-driven calcium mobilization in endothelial cells

Several classes of ligands for Protease-Activated Receptors (PARs) have shown impressive anti-inflammatory and cytoprotective activities, including PAR2 antagonists and the PAR1-targeting parmodulins. In order to support medicinal chemistry studies with hundreds of compounds and to perform detailed mode-of-action studies, it became important to develop a reliable PAR assay that is operational with endothelial cells, which mediate the cytoprotective effects of interest. We report a detailed protocol for an intracellular calcium mobilization assay with adherent endothelial cells in multiwell plates that was used to study a number of known and new PAR1 and PAR2 ligands, including an alkynylated version of the PAR1 antagonist RWJ-58259 that is suitable for the preparation of tagged or conjugate compounds. Using the cell line EA.hy926, it was necessary to perform media exchanges with automated liquid handling equipment in order to obtain optimal and reproducible antagonist concentration-response curves. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation.     

Statistical test for detecting overdispersion in offspring number based on kinship information

In this paper, we develop a theory of a new statistic that tests overdispersion in offspring number on the basis of exactly known kinship relationships. The statistic utilizes the sample size and the number of kinship pairs found in a sample, specially the number of mother–offspring (MO) and maternal–half-sibling (MHS) pairs. Given a sufficiently large sample size, the statistic proposed in this paper approximately follows a standard-normal distribution under non-overdispersed conditions (Poisson’s variance). We found that (1) the value of the statistic (\(\ge 2\) or \(<2\)) reasonably indicates whether reproduction is overdispersed at the 5% significance level; (2) the power of the statistic is determined primarily by the balance between the degree of overdispersion and the sample size; (3) in many cases, if the number of kinship pairs can be approximated by a normal distribution, false-positive and false-negative situations can be avoided. The proposed method can detect moderate-weak levels of overdispersion that produce few MHS pairs in a sample because the effect of the population size (which determines the number of detected MHS pairs) is canceled by the detection of the number of MO pairs. Once the kinship determination procedure is established, this indirect measurement will be readily applicable to species even with weak overdispersion, expanding the available opportunities for understanding how overdispersion in offspring number affects ecological processes.