Nuclear magnetic resonance studies of hemoproteins. VIII. Proton NMR studies of the binding of various nitrogenous ligands to ferric cytochrome c.

The structure of the heme environment of horse heart ferric cytochrome c was examined in the presence of various nitrogenous bases at several temperatures with the aid of hyperfine shifted proton NMR spectra at 220 MHz. The resonance positions and line widths of the signals for the peripheral methyl groups of the heme exhibited distinctive features of its low-spin state characteristic of each external ligand. In the imidazole complex of ferric cytochrome c, remarkable line sharpening of the heme-linked proton signals was encountered on raising the temperature. This may be related to the apoprotein perturbation on the binding of external ligand to the heme iron. These spectral peculiarities were discussed in relation to the electronic structure of the heme, the basicity of the external ligand and the van der Waals contact interaction between heme side chains and apoprotein.     

Genetic structure of <Emphasis Type="Italic">Cerasus jamasakura</Emphasis>, a Japanese flowering cherry,revealed by nuclear SSRs: implications for conservation

The genetic resources of a particular species of flowering cherry, Cerasus jamasakura, have high conservation priority because of its cultural, ecological and economic value in Japan. Therefore, the genetic structures of 12 natural populations of C. jamasakura were assessed using ten nuclear SSR loci. The population differentiation was relatively low (F _ST, 0.043), reflecting long-distance dispersal of seeds by animals and historical human activities. However, a neighbor-joining tree derived from the acquired data, spatial analysis of molecular variance and STRUCTURE analysis revealed that the populations could be divided into two groups: one located on Kyusyu Island and one on Honshu Island. Genetic diversity parameters such as allelic richness and gene diversity were significantly lower in the Kyushu group than the Honshu group. Furthermore, STRUCTURE analysis revealed that the two lineages were admixed in the western part of Honshu Island. Thus, although the phylogeographical structure of the species and hybridization dynamics among related species need to be evaluated in detail using several marker systems, the Kyusyu Island and Honshu Island populations should be considered as different conservation units, and the islands should be regarded as distinct seed transfer zones for C. jamasakura, especially when rapid assessments are required.     

高频度微卫星不稳定性大肠癌"修饰型"微卫星变化与MLH1及KRAS基因突变密切相关

本研究通过方法学的改良和观察方式的创新试图阐明这种现象的原因.微卫星非传统的检测方法仅能实现微卫星定性检测,我所在的研究组开发了自动片段分析双荧光标识技术,提高了微卫星检测的感度和重复性,并实现了微卫星片段变化长度的定量.小于6碱基的微卫星变化被定义为修饰型微卫星不稳定,大于8碱基的变化被定义为跳跃型微卫星不稳定,它们的电泳谱截然不同.前者表现为在非肿瘤来源微卫星位点基础上的增加或减少,后者表现为距离非肿瘤微卫星片段远隔部位的新波形的出现.通过研究我们发现,在DNA错配修复缺陷细胞系及基因敲除大鼠自发肿瘤样本,仅有修饰型微卫星不稳定性检出;在人类DNA错配修复缺陷细胞系连续80次传代也没有检出跳跃型变化.跳跃型变化不能通过简单重复序列不稳定基础上的增加或减少的累加而获得.在76例散发大肠癌,我们检测了微卫星不稳定性,KRAS基因突变,并对高频度微卫星不稳定性病例的两个主要DNA错配修复基因MSH2和MLHl进行了全长测序.我们发现,在大肠癌,按频度的传统分类与按波形变化的分类有高度的一致性,高频度微卫星不稳定性病例均检测到跳跃型表现,低频度微卫星不稳定性都表现为修饰型变化.在12例高频度微卫星不稳定病例,有三例检出了跳跃型和修饰型同时存在微卫星不稳定的特殊表型,这3例均检出KRAS的突变,更有趣的是该3例病例也同时检出了DNA错配修复基因MLH1的变异.而在其他9例高频度微卫星不稳定病例,KRAS突变及MLH1、MSH2交变未检出.通过对突变谱的分析我们还发现,修饰型微卫星不稳定与KTAS基因12号密码子的转换型突变高度相关,而微卫星稳定的病例检出的KRAS基因12号密码子突变多为颠换型突变.修饰型微卫星不稳定表型检出的高频度转换突变可由DNA错配修复缺陷的分子背景解释.通过本研究,我们认为以波形为基础的微卫星不稳定新分型可能是解决目前微卫星研究领域矛盾的一个选项.一直公认为高频度微卫星不稳定性是"真正"的DNA错配修复缺陷表型,我们的研究提示实际上高频度微卫星的可能是多元的.修饰型微卫星不稳定与DNA错配修复缺陷直接关联,而跳跃型微卫星不稳定的原因尚未阐明.在高频度为微型不稳定中,携带修饰型变化的病例可以通过DNA错配修复系统缺陷来解释其病因.     

Regulation of gonadotropin secretion and puberty onset by neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

Acquired Thermotolerance and Temperature-Induced Protein Accumulation in the Extremely Thermophilic Bacterium Rhodothermus obamensis

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95°C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.     

Effects of Amyloid β-Peptides and Gangliosides on Mouse Neural Stem Cells

The interaction of amyloid β-proteins (Aβs) with membrane lipids has been postulated as an early event in Aβ fibril formation in Alzheimer’s disease. We evaluated the effects of several putative bioactive Aβs and gangliosides on neural stem cells (NSCs) isolated from embryonic mouse brains or the subventricular zone of adult mouse brains. Incubation of the isolated NSCs with soluble Aβ1–40 alone did not cause any change in the number of NSCs, but soluble Aβ1–42 increased their number. Aggregated Aβ1–40 and Aβ1–42 increased the number of NSCs but soluble and aggregated Aβ25–35 decreased the number. Soluble Aβ1–40 and Aβ1–42 did not affect the number of apoptotic cells but aggregated Aβ1–40 and Aβ1–42 did. When NSCs were treated with a combination of GM1 or GD3 and soluble Aβ1–42, cell proliferation was enhanced, indicating that both GM1 and GD3 as well as Aβs are involved in promoting cell proliferation and survival of NSCs. These observations suggest the potential of beneficial effects of using gangliosides and Aβs for promoting NSC proliferation.     

Effect of Cycloheximide on Development of Mitochondria in Germinating Pea Cotyledons

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 µg protamine sulfate/ml, 0.15 m sucrose, and 10 m m MgSO₄ in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/µg DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.     

The yeast Tor signaling pathway is involved in G2/M transition via polo-kinase

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.     

Cathepsin E from rat neutrophils: its properties and possible relations to cathepsin D-like and cathepsin E-like acid proteinases

An extract of rat neutrophils was found to contain a high hemoglobin-hydrolyzing activity at pH 3.2, about 70% of which does not cross-react with anti-rat liver cathepsin D antibody. A neutrophil non-cathepsin D acid proteinase was successfully isolated from cathepsin D and characterized in comparison with the properties of rat liver cathepsin D. The neutrophil enzyme differed from cathepsin D in chromatographic and electrophoretic behaviors as well as immunological cross-reactivity, and its molecular weight was estimated to be 98,000 by gel filtration on Toyopearl HW 55. These findings strongly suggest that the neutrophil enzyme could be classified as cathepsin E. The enzyme, now designated rat cathepsin E, had an optimal pH at 3.0-3.2, preferred hemoglobin to albumin as substrate, and was markedly resistant to urea denaturation. Rat cathepsins D and E cleaved the insulin B-chain at six and eight sites, respectively; five sites were common for both enzymes. Possible relations among cathepsin E and cathepsin D-like or E-like acid proteinases reported so far were discussed.