Luo Xiao-ping

Methylmalonic acid in amniotic fluid and maternal urine as a marker for neural tube defects

SummaryTo evaluate the implication of methymalonic acid (MMA) in the early diagnosis of neural tube defects (NTD), a quantitative assay for MMA was established by using gas chromatographymass spectrometry with stable isotope of MMA as an internal standard. Amniotic fluid and maternal urine MMA concentration, maternal serum folate, red blood cell folate and vitamin B12 levels were measured in the middle term of NTD-affected and normal pregnancies. Amniotic fluid and maternal urine MMA concentrations in the middle term of NTD affected pregnancies (1.4±0.9 μmol/L, and 22.1±12.6 nmol/μmol creatinine) were significantly higher than that of normal pregnancies (1.0±0.4μmol/L, and 2.5±1.1 nmol/μmol creatinine). There was no significant differences between normal and NTD pregnancies for serum folate, red blood cell folate and vitamin B12 levels. The results suggested that MMAs in amniotic fluid and maternal urine are sensitive markers for early diagnosis of NTD. Vitamin B12 is an active cofactor involved in the remethylation of homocycteine and its deficiency is an independent risk factor for NTD. MMA is a specific and sensitive marker for intracellular vitamin B12 deficiency. This study suggests that it is necessary to monitor the vitamin B12 deficiency and advocates vitamin B12 supplementation with folate prevention program.

Expression of prothrombinase/fibroleukin gene fg12 in lung impairment in a murine severe acute respiratory syndrome model

To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) through trachea was established. Impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. MHV-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. As a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. In summary, the established murine SARS model could mimic the pathologic characteristics of lungs in patients with SARS. Besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of SARS associated lung damage.

Efficacy of subcutaneous administration of gonadotropin-releasing hormone agonist on idiopathic central precocious puberty.

In order to assess the feasibility of subcutaneous administration of Triptorelin with 6-week intervals for the suppression of pituitary-gonadal axis and changes of clinical signs in girls with idiopathic central precocious puberty (ICPP), 46 girls with ICPP were treated with GnRHa. Triptorelin (Decapeptyl, 3.75 mg) was administered subcutaneously (SC) at 6-weeks intervals or intramuscularly (IM) at 4-weeks intervals randomly for more than 12 months consecutively. During GnRHa therapy, clinical parameters and laboratory data, including height, weight, pubertal stage, bone age, uterine volume and ovarian size, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and estradiol (E2), were monitored and analyzed. It was found that both treatment regimes led to regression of precocious puberty and reversal of secondary sexual characteristics. Breast developments regressed. Uterine volume was decreased after treatment, but there was no statistically significant difference. Mean ovarian volume did not change significantly during treatment. The height velocity was decreased significantly from 6.3 ± 1.4 cm/year to 5.8 ± 1.2 cm/year in group SC and 6.7 ± 1.3 cm/year to 5.4 ± 1.0 cm/year in group IM, respectively. The rate of bone maturation was reduced significantly during treatment. The ratio of delta/BA/deltaCA was 1.2 ± 0.2 or 1.3 ± 0.3 at the onset of therapy and decreased significantly after the treatment to 0.7 ± 0.2 or 0.9 ± 0.1, respectively. The predicted adult height was increased significantly and progressively during therapy. The levels of serum LH, FSH and E2 returned to the prepubertal condition. No significant side effects of therapy were noted. The most common side effect during SC treatment was that a non-irritating, 1 cm in diameter mass was palpated at the site of subcutaneous injection in the abdominal wall of patients, which disappeared after 6–12 weeks. Two girls had minimal withdrawal vaginal bleeding episodes after the first injection. It was concluded that both IM and SC triptorelin administrations were clinically effective. They induce profound suppression of hypothalamic-pituitary-gonadal axis while stabilizing height velocity, slowing bone maturation and increasing predicted adult height. These results suggest that subcutaneous injection of triptorelin in 6-weeks intervals at a dosage of 3.75 mg be a safe and acceptable regimen for ICPP

Detection of HBV and HCV coinfection by TEM with Au nanoparticle gene probes

Gold(Au) nanoparticle HBV DNA or HCV cDNA gene probes were prepared and were used to detect HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection directly by transmission electron microscopy (TEM). PCR identifying HBV and HCV in serum of patients with HBV and HCV coinfection was established. Alkanethiol-modified oligonucleotide was bound with self-made Au nanoparticles to form nanoparticle HBV DNA or HCV cDNA gene probes through covalent binding of Au-S. HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfectionw as added to the detection system composed of nanoparticle HBV DNA and(or) HCV cDNA gene probes. The results showed that HBV DNA and HCV RNA could be specifically amplified by PCR. The zones of DNA amplification appeared in 431 bp and 323 bp respectively. When HBV DNA and HCV RNA extracted from positive serum of patients with HBV and HCV coinfection were added to the detection system, TEM displayed the nanoparticles self-assembled into large network aggregates. It was concluded that the detection of HBV and HCV coinfection by TEM was convenient and efficient with high specificity and sensitivity.

Targeted suppression of glutaryl-CoA dehydrogenase by lentivirus-mediated SHRNA and excessive intake of lysine induce the apoptosis of rat striatal neurons

Methods Four short hairpin RNA (shRNA) sequences targeting the GCDH gene (NM_001108896) were designed to construct four recombinant lentiviral vectors. The effectiveness of gene silencing in rat striatal neurons was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques. GCDH deficiency neurons (GCDH neurons), neurons transfected with negative control virus (NC neurons) and not intervention neurons (C neurons) were all incubated with lysine for 24h in concentrations of 0mmol/L, 5mmol/L, 10mmol/L respectively. The viability was measured with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT). The apoptosis of the neurons were detected by Hoechst33342 and PI. Tetramethylrhodamine methyl ester (TMRM) was used to determine the change of mitochondrial membrane potential. The expression of caspase-3, 8, 9, Bax and Bcl-2 were examined by RT-PCR and Western blotting. Results The efficiency of gene silencing of lentivirus-mediated shRNA4 was up to 60%, compared with the parental and control groups. The viability of C neurons, together with mitochondrial membrane potential and expression of caspase-3, 8, 9, Bax/Bcl-2 was not influenced by lysine, even when the concentration was 10mmol/L. The viability of NC neurons was significantly higher than GCDH neurons, when with 5mmol/L lysine interference. When without lysine, there is no difference between the two. Moreover, 5mmol/L lysine could induce GCDH neurons apoptosis, and 10mmmol/L lysine could induce NC neurons apoptosis. In these apoptotic neurons, the mitochondrial membrane potential decrased, the expressions of caspase-3, 8, 9, Bax all increased significantly and Bcl-2 expression decreased, compared to normal cells.

Protection of INS-1 cells from free fatty acid-induced apoptosis by inhibiting the glycogen synthase kinase-3

To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic β-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.