原位PCR和原位杂交检测蛋鸡J亚群禽白血病病毒

根据ALV_J原型株HPRS10 3株gp85基因的内部序列 ,和pol基因的 3′端设计一对引物H5 H7。从发生ML病死蛋用型鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取总RNA ,反转录为cDNA ,经PCR扩增得到长度为 5 4 5bp的ALV_JcDNA特异性探针。探针定位于 5 2 5 8～ 5 80 2bp。将病鸡的组织石蜡切片置HybaidExpress原位PCR仪平台上 ,以H5 H7为引物进行原位PCR扩增。应用地高辛标记的cDNA探针对原位PCR扩增后切片进行了原位杂交检测。结果在待检组织肿瘤组织、十二指肠、骨髓中出现明显的阳性信号。睾丸、肺、胰腺、大脑、输卵管、肾脏均检出散在的阳性信号。这是国内外首次从分子水平证明蛋鸡J亚群禽白血病。     

Broad-host-range cloning vectors derived from the W-plasmid Sa

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (M_r 5.6?7.2 × 10⁶), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,     

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis–Menten‐like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis–Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system‐level implications for bacterial phenotype.     

De novo MECP2 duplication derived from paternal germ line result in dysmorphism and developmental delay

Xq28 duplications encompassing the methyl CpG binding protein 2 (MECP2) in males exhibit a distinct phenotype, including developmental delay, facial dysmorphism, muscular hypotonia, intellectual disability, poor or absent speech, recurrent infections and early death. The vast majority of affected males inherit the MECP2 duplication from their usually asymptomatic carrier mothers. Only a few cases with Xq28 duplication originating from de novo unbalanced X/Y translocation have been reported and the paternal origin of the aberration has only been validated in three males in the related literature. Here we present a karyotypically normal male with features characteristic of the MECP2 duplication syndrome. The genome-wide SNP genotyping shows a de novo 2.26-Mb duplication from Xq28 to the terminus. The genotypes of the SNPs within the duplicated region indicated a paternal origin. Furthermore, the results of fluorescence in situ hybridization (FISH) indicated a novel Xq:Yp translocation, characterized as der(Y)t(Y;X)(p11.32;q28), which suggests an aberrant that occurred during spermatogenesis. The phenotype is compared to the previously reported cases with Xq28 duplication originated from an unbalanced X/Y translocation, and there was no specific part of the phenotype that could be contributed to the origin of parental imbalances. This report further highlights the capacity of high-molecular cytogenetic methods, such as SNP array and FISH, in the identification of submicroscopic rearrangement, structural configuration and parental origin of aberrant while in the evaluation of children with idiopathic developmental delay and intellectual disability.     

Comparative Development of Eimeria tenella from Sporozoites to Oocysts in Primary Kidney Cell Cultures from Gallinaceous Birds

Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.     

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE⁻/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE⁻/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.     

Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T₉–T₁₀) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.