[Screening for carriers of pathogenic genes for methylmalonic acidemia and Wilson's disease in neonates in Qingdao].

Objective: To investigate the carrier frequency of pathogenic genes for methylmalonic acidemia and Wilson's disease in neonates in Qingdao. Methods: In this cross-sectional study, using computer random sampling, 5 020 neonates from the neonatal screening center in Qingdao area from June 2016 to December 2018 were selected, and 5 012 of them were included in the carrier screening study.DNA was extracted from dried blood stain specimens used in the screening of newborns. Multiplex PCR combined with next generation sequencing were used for gene detection of MMACHC gene, MUT gene and ATP7B gene. The carrying rate of hotspots of each gene were calculated, and binomial distribution method was used to calculate 95% confidence interval of pathogenic gene carrying rate. Results: A total of 5 012 neonates completed the screening for carriers of disease-causing genes, of which 5 006 neonates completed the screening of two diseases and the remaining 6 neonates completed the screening of Wilson disease only.For ATP7B gene, the carrier frequency of the 12 hot spot mutations was 1.46% (73/5 012),and the 95% confidence interval was 1.16%-1.83%. For MMACHC gene and MUT gene, carrier frequency of 18 hot spot mutations was 2.50% (125/5 006) , and the 95% confidence interval was 2.10%-2.97%, among which cblC type accounted for 87.2% and the MUT pathogenic gene accounted for 12.8%. Conclusion: The carrier frequency of methylmalonic acidemia and Wilson's disease are both high in the neonatal population in Qingdao.

[Expression of PAK1 in bladder cancer and its influence on invasion of bladder cancer cells].

Objective: To investigate the expression of p21-activated kinase 1 (PAK1) in bladder cancer and its biological influence on invasion ability of bladder cancer cells. Methods: A total of 54 paraffin-embedded bladder cancer tissue samples and 12 normal bladder tissue specimens were retrieved in Jinshan Hospital Affiliated to Fudan University between January 2009 and December 2012. The expression of PAK1 in these tissues was detected by immunohistochemical staining. The PAK1 mRNA and protein levels were measured in bladder cancer cell lines and human normal bladder epithelial cell line using real-time, fluorescence-based quantitative polymerase chain reaction (RT-PCR) and Western blot, respectively. A stable PAK1 gene silencing bladder cancer cell strain 5637 were successfully constructed. After treatment with PAK1 RNA interference(RNAi), the ability of migration and invasion of the 5637 cell was evaluated using a Transwell system. Results: The expression of PAK1 proteins was significantly higher in bladder cancer tissues than in normal bladder tissues (28/54 vs 1/12, P<0.05). The overexpression of PAK1 was positively correlated with high histological grade, lymph node metastasis, and tumor size (all P<0.05). The mRNA level and protein level of PAK1 was much higher in bladder cancer cell lines T24, 5637 than human normal urothelial cell line SV-HUC-1.PCR and Western blot showed satisfactory inhibitory effect of PAK1 short hairpin RNA (shRNA) on PAK1 expression in 5637 bladder cancer cells. The number of PAK1 RNAi-treated 5637 cells traversed the membrane was decreased compared with the control group in migration and invasion assays. Conclusions: Overexpression of PAK1 in bladder cancer tissues may be an important feature of bladder cancer and related with the metastasis and invasion of bladder cancer. The molecular mechanisms involved in regulation of PAK1 expression needs further research.

A two-step method for identification of the Chinese glutinous rice Suyunuo, based on ISSR-SCAR and allele-specific markers.

Suyunuo is a valuable glutinous rice variety cultivated mainly in the Lake Taihu area of China. Historically, Suyunuo was presented to emperors as a tribute, and, still today, enjoys a great reputation in China. This study aimed to develop a unique, specific molecular marker for the identification of Suyunuo rice. Polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) molecular markers was performed on Suyunuo and 11 other glutinous rice varieties that are mainly cultivated in the Yangtze River Delta region. A Suyunuo-specific band was detected in the PCR products generated from primer ISSR-807. A sequence characterized amplified region (SCAR) primer pair targeting a Suyunuo-specific band was subsequently designed. The SCAR primers amplified a target band in all individuals of Suyunuo and in four glutinous indica varieties, whereas no bands were found in the seven glutinous japonica varieties. Subsequently, sequences amplified by the SCAR primer pair were analyzed to facilitate the design of Suyunuo allele-specific primers. The allele-specific primer pair produced target bands in all individuals of Suyunuo rice but no bands in individuals of any of the other 11 rice varieties. This study provides a theoretical guideline for rice germplasm identification and innovation of other valuable rice landraces.

[Effect of prostate peripheral zones stromal cells on the proliferation of prostate cells by overexpression of LMO2 gene].

OBJECTIVE: To explore the biological effect of prostate peripheral zones (PZs) stromal cells on the proliferation of prostate cells by overexpression of LMO2 gene. METHODS: Genes expressional distinction of different prostate stromal cells was screened by gene expression arrays. To validate the microarray data, real time-polymerase chain reaction (RT-PCR), Western blotting analysis were used to check the over expression of LMO2 in PZs cells.To compare the effect of stromal cells which overexpressed LMO2 gene on in vitro proliferation ability of BPH-1 and PC3 cell lines, cell proliferation was measured by CCK-8 and EdU assay. Cytokines chip was used to screen expression of cytokines in WPMY-1-LMO2 conditioned medium. The changes of BPH-1 and PC3 proliferation associated proteins were assessed by Western blotting. RESULTS: A total of 512 genes were identified as markedly differentially expressed in stromal cells originated from different zones. Among these genes, LMO2 gene was overexpression in peripheral zones stromal cells, and confirmed by RT-PCR and Western blotting. Expression level of LMO2 gene was significantly up-regulated in peripheral zones stromal cells compared with transitional zones stromal cells, increased by 3.36 folds on average (P<0.01). The proliferation of both PC3 and BPH-1 were found increased and STAT3 phosphorylation and CCND1 expression were increased after cultured in conditioned medium from stromal cells which stably expressed LMO2. Cytokines chip found increased FGF-9 and IL-11 expression in the medium supernatant reserved from LMO2-overexpressed stromal cell line. CONCLUSIONS: Distinct gene expression exists among prostate stromal cells originated from different zones. LMO2 overexpressed stromal cells can induce prostate epithelial cell growth via paracrine of FGF-9, IL-11 or other cytokines.

Synthesis of 5-substituted analogues of huperzine A.

Three 5-isosteres of huperzine A (2-4) were first synthesized. The key intermediate 10 was prepared by the reaction of acid 13 with LTA. The compounds 2 and 3 had 50% inhibition by 35 and 47 microM, respectively, which still retained antiacetylcholinesterase activity.

The determination of vasoactive substances during autonomic dysreflexia.

Urinary bladder percussion induced autonomic dysreflexia (AD) was observed in spinal cord injured patients with a complete neurological lesion, the upper level being above T5. To document the pathology and study the etiology of autonomic dysreflexia to further investigate its mechanism, this paper presents some clinical data on the determination of vasoactive substances such as norepinephrine (NE), epinephrine (E), renin (R), angiotensin II (AII) and atrial natriuretic polypeptide (ANP) before and during bladder percussion in 30 patients with a thoracolumbar or cervical spine and spinal cord injury. It is demonstrated that tapping the urinary bladder of such patients can cause AD. Changes of some of the vasoactive substances in the plasma were also observed, which might indicate that autonomic dysreflexia result from excitation of the sympathetic nervous system. Overactivity of the sympathetic nervous system was antagonized by excitation of the vegetative nerve system. There was no correlation between changes of blood pressure and adrenal function as well as the change of R-A II system; during autonomic dysreflexia, the inclement of ANP secretion played an important role in recovering homeostasis.

Cloning and expression of avian glutamine phosphoribosylpyrophosphate amidotransferase. Conservation of a bacterial propeptide sequence supports a role for posttranslational processing.

The cDNA for glutamine phosphoribosylpyrophosphate amidotransferase, the regulatory enzyme of de novo purine nucleotide biosynthesis, has been cloned for the first time from an animal. The derived amino acid sequence of the avian amidotransferase is homologous with amidotransferase sequences from bacteria and yeast. An 11-amino acid propeptide in Bacillus subtilis amidotransferase is conserved in the avian enzyme. Expression in Chinese hamster ovary (CHO) cells and Escherichia coli provides evidence for two post-translational maturation steps needed for synthesis of active enzyme: incorporation of an iron component and processing of the 11-amino acid propeptide. Functional complementation of a CHO amidotransferase mutant suggests that both maturation steps take place in CHO cells. In contrast, function in E. coli requires deletion of the sequence encoding the propeptide. Defective assembly of the iron component may restrict propeptide removal and activation of the avian amidotransferase in E. coli.