[Analyses of coding sequence point mutation and polymorphism of TGFBI gene in Chinese patients with keratoconus].

OBJECTIVE: To investigate the point mutations and polymorphisms of transforming growth factor beta-induced gene (TGFBI) in Chinese patients with keratoconus and discuss the relationship between the feature of gene mutations and single nucleotide polymorphisms of TGFBI gene and keratoconus. METHODS: Polymerase chain reaction single strand conformation polymorphism and DNA direct sequencing were performed in 30 keratoconus cases and 30 healthy controls. All 17 exons of the TGFBI gene were analyzed for point mutations and single nucleotide polymorphisms. RESULTS: Totally two heterozygous nucleotide changes were identified in exon 12 of the TGFBI gene. The codon 535 is changed from GGA to TGA in 1 patient, leading to a substitution of glycine to a stop codon at the protein level (G535X). The codon 540 is changed from TTT to TTC in 2 patients and 1 control individual, resulting in a nonsense mutation (F54F), and is a single nucleotide polymorphism of the gene. CONCLUSION: Mutation and polymorphisms of the TGFBI gene were detected in Chinese patients with keratoconus in this study. The results suggest that TGFBI gene might play an important role in the pathogenesis of keratoconus.

Association analysis of COMT polymorphisms and schizophrenia in a Chinese Han population: a case-control study.

Schizophrenia is a common disease with complex mode of inheritance; great efforts have been made to identify the susceptible genes. Catechol-O-methyltransferase (COMT) gene has long been considered as a candidate gene mainly because of two reasons: First, it encodes a key dopamine catabolic enzyme. Second, it maps to the velocardiofacial syndrome (VCFS) region of chromosome 22q11, which is associated with schizophrenia predisposition. Numerous case-control and family-based studies have been conducted, majority of them focused on a functional Val/Met polymorphism (rs4680). Unfortunately, these studies have produced conflicting results. In a previous report, Shifman et al. found a three-marker haplotype (rs737865-rs4680-rs165599) that showed significant association with schizophrenia. In this study, we try to replicate their findings in Chinese Han population and failed to find any associations.

[Coding-sequence mutation and polymorphism analysis of EDNRB gene in patients with Hirschsprung's disease from Zhejiang region].

To determine the characters of EDNRB gene in patients with sporadic Hirschsprung's disease (HD) and discuss the relationship between mutation of EDNRB gene and HD, seventy five sporadic HD cases and forty normal cases as control were collected and DNA was extracted from peripheral white blood cells by standard method. All seven exons of EDNRB gene were analyzed by single strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP) and direct sequencing. In result, A G --> A transition in codon 277 was observed in exon 4 in six patients. This variation leads to a nonsense mutation (L277L) and is a previously described polymorphism, the rate of variation is 8% (6/75). A G --> A transition in codon185 was detected in exon 2 in two patients, this is a novel heterozygous mutation, leading to a missense mutation (V185M), and the rate of mutation is 2.7% (2/75). So we can conclude that mutation of EDNRB gene can be detected in Chinese patients with sporadic HD and EDNRB gene might play an important role in the pathogenesis of HD.

A mouse model of severe acute pancreatitis induced with caerulein and lipopolysaccharide.

AIM: To establish a non-traumatic, easy to induce and reproducible mouse model of severe acute pancreatitis (SAP) induced with caerulein and lipopolyasccharide (LPS). METHODS: Thirty-two healthy mature NIH female mice were selected and divided at random into four groups (each of 8 mice), i.e., the control group (NS group), the caerulein group (Cn group), the lipopolysaccharide group (LPS group), and the caerulein+LPS group (Cn+LPS group). Mice were injected intraperitoneally with caerulein only, or LPS only, and caerulein and LPS in combination. All the animals were then killed by neck dislocation three hours after the last intraperitoneal injection. The pancreas and exo-pancreatic organs were then carefully removed for microscopic examination. And the pancreatic acinus was further observed under transmission electron microscope (TEM). Pancreatic weight, serum amylase, serum nitric oxide (NO) concentration, superoxide dismutase (SOD) and malondialdehyde (MDA) concentration of the pancreas were assayed respectively. RESULTS: (1) NS animals displayed normal pancreatic structure both in the exocrine and endocrine. In the LPS group, the pancreas was slightly edematous, with the infiltration of a few inflammatory cells and the necrosis of the adjacent fat tissues. All the animals of the Cn group showed distinct signs of a mild edematous pancreatitis characterized by interstitial edema, infiltration of neutrophil and mononuclear cells, but without obvious parenchyma necrosis and hemorrhage. In contrast, the Cn+LPS group showed more diffuse focal areas of nonviable pancreatic and hemorrhage as well as systemic organ dysfunction. According to Schmidt's criteria, the pancreatic histologic score showed that there existed significant difference in the Cn+LPS group in the interstitial edema, inflammatory infiltration, parenchyma necrosis and parenchyma homorrhage in comparison with those of the Cn group, LPS group and NS group (P<0.01 or P<0.05). (2) The ultrasturcture of acinar cells was seriously damaged in the Cn+LPS group. Chromatin margination of nuclei was present, the number and volume of vacuoles greatly increased. Zymogen granules (ZGs) were greatly decreased in number and endoplasmic reticulum exhibited whorls. The swollen mitochondria appeared, the crista of which was decreased in number or disappeared. (3) Pancreatic weight and serum amylase levels in the Cn +LPS was significantly higher than those of the NS group and the LPS group respectively (P<0.01 or P<0.05). However, the pancreatic wet weight and serum amylase concentration showed no significant difference between the Cn+LPS group and the Cn group. (4) NO concentration in the Cn+LPS group was significantly higher than that of NS group, LPS group and Cn group(P<0.05 or P<0.01). (5) The SOD and MDA concentration of the pancreas in the Cn+LPS group were significantly higher than those of NS, LPS and Cn groups (P<0.05 or P<0.01). CONCLUSION: The mouse model of severe acute pancreatitis could be induced with caerulein and LPS, which could be non-traumatic and easy to induce, reproducible with the same pathological characteristics as those of SAP in human, and could be used in the research on the mechanism of human SAP.

Mutation of RET gene in Chinese patients with Hirschsprung's disease.

AIM: To investigate the pathogenic mechanism of Hirschsprung's disease (HD) at the molecular level and to elucidate the relationship between RET oncogene and Chinese patients with HD. METHODS: Exon 13 of RET oncogene from 20 unrelated HD patients was analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The positive amplifying products were then sequenced. According to the results of SSCP and DNA sequence, SSCP was done as well for the samples from the family other members of some cases with mutated RET gene. RESULTS: SSCP analysis indicated that mobility abnormality existed in 4 unrelated HD patients. Direct DNA sequence analysis identified a missense mutation, T to G at the nucleotide 18 888 and a frameshift mutation at the nucleotide 18 926 insG. In a HD family, the sicked child and his father were the same heterozygous missense mutation (T to G at nucleotide 18 888). CONCLUSION: Among Chinese HD patients, RET gene mutations may exist in considerable proportion with different patterns. These new discoveries indicate that RET mutations may play an important role in the pathogenesis of unrelated HD in the Chinese population. PCR-SSCP combined with DNA sequence can be used as a tool in the genetic diagnosis of HD.

Regulating effect of Chinese herbal medicine on the peritoneal lymphatic stomata in enhancing ascites absorption of experimental hepatofibrotic mice.

AIM: To observe the regulatory effect of Chinese herbal medicine on peritoneal lymphatic stomata and its significance in treating ascites in liver fibrosis model mice. METHODS: Two Chinese herbal composite prescriptions were used separately to treat the carbon tetrachloride-induced mouse model of liver fibrosis. The histo-pathologic changes of the liver sections (HE and VG stainings) were observed. The peritoneal lymphatic stomata was detected by scanning electron microscopy and computer image processing. The changes of urinary volume and sodium ion concentration were measured. RESULTS: In the model group, lots of fibrous tissue formed in liver and extended into the hepatic lobules to separate them incompletely. In the treated and prevention groups, the histo-pathologic changes of liver was rather milder, only showed much less fibrous tissue proliferation in the hepatic lobules. The peritoneal lymphatic stomata enlarged with increased density in the experimental groups (diameter: PA, 3.07 +/- 0.69 microm; PB, 2.82 +/- 0.37 microm; TA, 3.25 +/- 0.82 microm and TB, 2.82 +/- 0.56 microm; density: PA, 7.11 +/- 1.90 stomata.1000 microm(-2); PB, 8.76 +/- 1.45 stomata.1000 microm(-2); TA, 6.55 +/- 1.44 stomata.1000 microm(-2)and TB, 8.76+/-1.79 stomata.1000 microm(-2)), as compared with the model group (diameter: 2.00+/-0.52 microm density: 4.45+/-1.05 stomata.1000 microm(-2)). After treatment, the urinary volume and sodium ion excretion increased in the experimental groups (PA, 231.28+/-41.09 mmol.L(-1); PB, 171.69 +/- 27.48 mmol.L(-1) and TA, 231.44 +/- 34.12 mmol.L(-1)), which were significantly different with those in the model group (129.33 +/- 36.75 mmol.L(-1)). CONCLUSION: Chinese herbal medicine has marked effects in alleviating liver fibrosis, regulating peritoneal lymphatic stomata, improving the drainage of ascites from peritoneal cavity and causing increase of urinary volume and sodium ion excretion to reduce the water and sodium retention, and thus have favorable therapeutic effect in treating ascites.

Study on the mechanism of regulation on the peritoneal lymphatic stomata with Chinese herbal medicine.

AIM: To study the mechanism of Chinese herbal medicine (CHM), the prescription consists of Radix Salviae Miltiorrhizae, Radix Codonopsitis Pilosulae, Rhizoma Atractylodis Alba and Rhizoma Alismatis, Leonurus Heterophyllus Sweet,etc on the regulation of the peritoneal lymphatic stomata and the ascites drainage. METHODS: The mouse model of live fibrosis was established with the application of intragastric installations of carbon tetrachloride once every three days; scanning electron microscope and computer image processing were used to detect the area and the distributive density of the peritoneal lymphatic stomata; and the concentrations and NO in the serum were measured and analyzed in the experiment. RESULTS: Two different doses of CHM could significantly increase the area of the peritoneal lymphatic stomata, promote its distributive density and enhance the drainage of urinary ion such as sodium, potassium and chlorine. Meanwhile, the NO concentration of two different doses of CHM groups was 133.52+/-23.57 micromol/L and 137.2+/-26.79 micromol/L respectively. In comparison with the control group and model groups (48.36+/-6.83 micromol/L and 35.22+/-8.94 micromol/L, P<0.01),there existed significantly marked difference, this made it clear that Chinese herbal medicine could induce high endogenous NO concentration. The effect of Chinese herbal medicine on the peritoneal lymphatic stomata and the drainage of urinary ion was altered by adding NO donor(sodium nitropurruside,SNP) or NO synthase (NOS) inhibitor (N(G)-monomethyl-L-arginine, L-NMMA) to the peritoneal cavity. CONCLUSION: There existed correlations between high NO concentration and enlargement of the peritoneal lymphatic stomata, which result in enhanced drainage of ascites. These data supported the hypothesis that Chinese herbal medicine could regulate the peritoneal lymphatic stomata by accelerating the synthesis and release of endogenous NO.