Expression of hMSH2 gene and mutant p53 in sporadic digestive tract tumors

ＴｈｅｈｕｍａｎＤＮＡｍｉｓｍａｔｃｈｒｅｐａｉｒｓｙｓｔｅｍ (ＭＭＲＳ)ｃｏｎｓｉｓｔｓｏｆａｓｅｒｉｅｓｏｆｅｎｚｙｍｅｓｔｈａｔｃａｎｃｏｒｒｅｃｔｍｉｓｔａｋｅｓｄｕｒｉｎｇＤＮＡｒｅｐｌｉｃａｔｉｏｎ Ｕｓｉｎｇｔｈｉｓｓｙｓｔｅｍ ,ｍｕｔａｔｉｏｎｓｃａｎｂｅａｖｏｉｄｅｄ ,ａｎｄＤＮＡｃａｎｂｅｋｅｐｔｗｈｏｌｅａｎｄｓｔａｂｌｅｄｕｒｉｎｇｒｅｐｌｉｃａｔｉｏｎ ＳｉｘｇｅｎｅｓｏｆＭＭＲｈａｖｅｂｅｅｎｄｅｆｉｎｅｄ :ｈＭＳＨ2 ,ｈＭＬＨ1,ｈＭＳＨ6,ｈＭＳＨ3,ｈＰＭＳ1ａｎｄｈＰＭ…     

Analysis of clinical,biochemical and viral factors associated with early relapse after lamivudine treatment for hepatitis B e antigen-negative chronic hepatitis B patients in Taiwan

Summary.  The efficacy of lamivudine for HBeAg-negative chronic hepatitis B (CHB) Chinese patients has not been fully investigated. The role of the Hepatitis B virus (HBV) genotype on the treatment effect of lamivudine is controversial. Thirty-two consecutive patients with HBeAg-negative CHB were enrolled. All patients were treated with lamivudine 100 mg once daily of 7–12 months duration. The mean total period of follow-up since entry for all patients was 24 ± 3.5 months. HBV genotypes were classified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and verified by sequencing. Precore (G1896A) and basic core promoter (BCP, A1762T & G1764A) mutations were determined by PCR and direct sequencing. Twenty-one (65.6%) patients were infected by genotype B and, 11 (34.4%) by genotype C. G1896A was predominant in genotype B infected patients (95.2% vs 63.6%, P  = 0.037). At the end of treatment, 31 (96.8%) and 14 (43.8%) patients achieved biochemical and virological responses, respectively. The biochemical and virological response rates were 40.6 and 0% at 12 months after treatment. Eighteen (56.3%) patients had biochemical relapse within 12 months after withdrawal of lamivudine. By multivariate analysis, the pretreatment serum level of HBV DNA ≥12 Meq/mL was the only factor associated with early biochemical relapse (Odds ratio = 9.333, 95% CI = 1.497 ∼ 58.197, P  = 0.017). In conclusion, the virological effect of lamivudine for HBeAg-negative CHB is transient. Most patients had biochemical relapse within 12 months after lamivudine treatment regardless of HBV genotype. A high pretreatment viral load is the determinant for early biochemical relapse.     

Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress

Several neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD) are associated with elevated brain iron accumulation relative to the amount of ferritin, the intracellular iron storage protein. The accumulation of more iron than can be adequately stored in ferritin creates an environment of oxidative stress. We developed a heavy chain (H) ferritin null mutant in an attempt to mimic the iron milieu of the brain in AD and PD. Animals homozygous for the mutation die in utero but the heterozygotes (+/-) are viable. We examined heterozygous and wild-type (wt) mice between 6 and 8 months of age. Macroscopically, the brains of +/- mice were well formed and did not differ from control brains. There was no evidence of histopathology in the brains of the heterozygous mice. Iron levels in the brain of the +/- and wild-type (+/+) mice were similar, but +/- mice had less than half the levels of H-ferritin. The other iron management proteins transferrin, transferrin receptor, light chain ferritin, Divalent Metal Transporter 1, ceruloplasmin, were increased in the +/- mice compared to +/+ mice. The relative amounts of these proteins in relation to the iron concentration are similar to that found in AD and PD. Thus, we hypothesized that the brains of the heterozygote mice should have an increase in indices of oxidative stress. In support of this hypothesis, there was a decrease in total superoxide dismutase (SOD) activity in the heterozygotes coupled with an increase in oxidatively modified proteins. In addition, apoptotic markers Bax and caspase-3 were detected in neurons of the +/- mice but not in the wt. Thus, we have developed a mouse model that mimics the protein profile for iron management seen in AD and PD that also shows evidence of oxidative stress. These results suggest that this mouse may be a model to determine the role of iron mismanagement in neurodegenerative disorders and for testing antioxidant therapeutic strategies.     

A hot spot for hotfoot mutations in the gene encoding the delta2 glutamate receptor

The orphan glutamate receptor delta2 is selectively expressed in Purkinje cells and plays a crucial role in cerebellar functions. Recently, ataxia in the hotfoot mouse ho4J was demonstrated to be caused by a deletion in the delta2 receptor gene (Grid2) removing the N-terminal 170 amino acids of the delta2 receptor. To understand how delta2 receptors function, we characterized mutations in eight additional spontaneously occurring hotfoot alleles of Grid2. The mouse Grid2 gene consists of 16 exons, spanning approximately 1.4 Mb. Genomic DNA analysis showed that seven hotfoot mutants had a deletion of one or more exons encoding the N-terminal domain of delta2 receptors. The exception is ho5J, which has a point mutation in exon 12. Deletions in ho7J, ho9J, ho11J and ho12J mice result in the in-frame deletion of between 40 and 95 amino acids. Expression of constructs containing these deletions in HEK293 cells resulted in protein retention in the endoplasmic reticulum or cis-Golgi without transport to the cell surface. Coimmunoprecipitation assays indicated that these deletions also reduce the intermolecular interaction between individual delta2 receptors. These results indicate that the deleted N-terminal regions are crucial for oligomerization of delta2 receptors and their subsequent transport to the cell surface of Purkinje cells. The relatively large size of the Grid2 gene may be one of the reasons why many spontaneous mutations occur in this gene. In addition, the frequent occurrence of in-frame deletions within the N-terminal domain in hotfoot mutants suggests the importance of this domain in the function of delta2 receptors.     

Ataxia Jackson (<Emphasis Type="Italic">ax</Emphasis><Superscript><Emphasis Type="Italic">J</Emphasis></Superscript>): a genetic model for apoptotic neuronal cell death

Programmed cell death or apoptosis is an important process to form normal adult cytoarchitecture. But in vivo analysis of neuronal apoptosis has not been well advanced. Therefore, apoptotic cell death of a particular neuronal system or anatomical part in a mutant is an invaluable target to learn about a link between a gene and neuronal apoptosis. Ataxia (ax) is an autosomal recessive neurological mutant mouse. We recently investigated brains of homozygotes for ataxia Jackson (ax(J)), an allele of ax, using TUNEL method. A few TUNEL-positive cells were observed in the granular cell layer of the cerebellum, the dentate gyrus, and the olfactory bulb of phenotypically normal littermates (ax(J)/+ or +/+) aged at 23-38 days. In affected ax(J)/ax(J) mice, however, the number of TUNEL-positive cells was significantly increased in the cerebellum, particularly in the granular cell layer (p < 0.05). The ax(J) mouse will be an in vivo unique model for studies on the genetic basis of apoptotic neuronal cell death, and identification of the ax gene is desired to elucidate molecular basis of the apoptosis.     

Persistence of spinocerebellar afferent topography following hereditary Purkinje cell degeneration

Cerebellar Purkinje neurons play a significant role in the development and early maintenance of cerebellar afferent topography. Anterograde intra-axonal labeling experiments were designed to identify any role for Purkinje cells in maintaining spinocerebellar mossy fiber afferent topography in shaker mutant rats with adult onset Purkinje cell heredodegeneration. Following the death of Purkinje cells myelinated spinocerebellar axons persist and their terminal mossy fiber morphology remains normal in appearance and size. The relative percentage of labeled projections to each of the five anterior lobe lobules was comparable in mutant and normal rats. Finally, unfolded reconstructions of the anterior lobe illustrated that the organization of labeled terminals in clusters, patches and discontinuous parasagittally oriented stripes or transversely oriented bands were spatially distributed the same in both normal and mutant rats. These findings strongly infer that Purkinje cells are not necessary for the persistence and maintenance of spinocerebellar afferent pathways in adult animals.     

Immunohistochemical study on the distribution of MnSOD in the central nervous system of the transgenic mice expressing a human Cu/Zn SOD mutation

In the present study, we used the SOD1^G93A mutant transgenic mice as an animal model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of MnSOD in the central nervous system of transgenic mice at the age of 8, 13, and 18 weeks. In the spinal cord of wild-type SOD1 (wtSOD1) and SOD1^G93A transgenic mice, MnSOD-immunoreactive neurons were distributed mainly in the anterior horn, although they were also observed in the posterior horn. The staining intensity of MnSOD was significantly increased in the spinal cord of SOD1^G93A transgenic mice at presymptomatic and symptomatic stage. In the brainstem of symptomatic SOD1^G93A transgenic mice, significantly increased immunoreactivity for MnSOD was observed in abducens nucleus, facial nucleus, dorsal motor nucleus of vagus, hypoglossal nucleus, medullary and pontine reticular formation, superior and inferior olivary nucleus, and cochlear nucleus. The present study provides the first evidence that MnSOD immunoreactivity was increased in the central nervous system of SOD^G93A transgenic mice, suggesting that mitochondria may play an important role in the pathogenesis and progress of ALS. The mechanisms underlying the increased immunoreactivity for MnSOD, and the functional implications of these increases, require elucidation.     

The role of the c-kit receptor in the regenerative differentiation of rat Leydig cells

To determine the physiological role of the c-kit receptor, which is highly expressed in Leydig cells, the regenerative differentiation of Leydig cells was studied following transient degeneration induced by ethane dimethyl sulphonate (EDS) in c-kit-deficient mutant rats (Ws/Ws). EDS caused the destruction of Leydig cells; their functional recovery was evaluated by the weight change of the target organs of androgens, which occurred at the same rate in Ws/Ws and wild-type rats. These results indicate that the tyrosine kinase activity of the c-kit receptor does not play an essential role in the regenerative differentiation of Leydig cells.     

万古霉素产生菌的选育与发酵培养基优化

万古霉素产生菌东方拟无枝酸菌（Amycolatopsis orientalis）5－13经紫外线（30W，253.7nm，距离30cm，照射50s）诱变后，在含万古霉素1000μg/ml的浓度梯度平板上筛选万古霉素抗性变株，得到一株万古霉素抗性变株A.orientalis 6-21，其摇瓶效价较出发菌株提高23％，对万古霉素的抗性提高200％。传代试验表明该变株的高产性能遗传特性较稳定。用正交试验法对万古霉素发酵培养基进行了优化，与原发酵培养基相比，万古霉素的摇瓶发酵效价提高了14.3%。