Expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in brain tissue of diabetic rats with ischemia reperfusion

Objective To discuss the expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in the brain tissue of diabetic rats with ischemia reperfusion. Methods A total of 60 male Wistar rats were randomly divided into the normal group, sham group, diabetic cerebral infarction group and single cerebral infarction group according to the random number table, with 15 rats in each group. The high sucrose diet and intraperitoneal injection of streptozotocin were performed for the modeling of diabetic rats, while the thread-occlusion method was employed to build the model of cerebral ischemia reperfusion. The immunohistochemical staining was performed to detect the expression of angiostatin, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in the brain tissue. Results The expression of angiostatin after the reperfusion in the brain tissue of rats in the single cerebral infarction group and diabetic cerebral infarction group was increased 6 h after the reperfusion, reached to the peak on 1 d and then decreased gradually. The expression of angiostatin in the diabetic cerebral infarction group 6 h, 1 d, 3 d and 7 d after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05). VEGF began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak at 6 h and then decreased gradually. The expression of VEGF in the diabetic cerebral infarction group at each time point after the reperfusion was significantly lower than that in the single cerebral infarction group (P < 0.05). MMP-9 began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak on 1 d and then decreased gradually. The expression of MMP-9 in the diabetic cerebral infarction group at each time point after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05). Conclusions The high glucose environment in which the diabetic cerebral infarction is occurred is to induce the formation of MMP-9 at first and then activate and increase the expression of angiostatin. Afterwards, the expression of VEGF is inhibited, resulting in the poor angiogenesis after cerebral infarction, which thus makes the injury of brain tissue after cerebral infarction even worse than the non-diabetes mellitus.

Expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in brain tissue of diabetic rats with ischemia reperfusion

OBJECTIVE@#To discuss the expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in the brain tissue of diabetic rats with ischemia reperfusion.@*METHODS@#A total of 60 male Wistar rats were randomly divided into the normal group, sham group, diabetic cerebral infarction group and single cerebral infarction group according to the random number table, with 15 rats in each group. The high sucrose diet and intraperitoneal injection of streptozotocin were performed for the modeling of diabetic rats, while the thread-occlusion method was employed to build the model of cerebral ischemia reperfusion. The immunohistochemical staining was performed to detect the expression of angiostatin, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in the brain tissue.@*RESULTS@#The expression of angiostatin after the reperfusion in the brain tissue of rats in the single cerebral infarction group and diabetic cerebral infarction group was increased 6 h after the reperfusion, reached to the peak on 1 d and then decreased gradually. The expression of angiostatin in the diabetic cerebral infarction group 6 h, 1 d, 3 d and 7 d after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05). VEGF began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak at 6 h and then decreased gradually. The expression of VEGF in the diabetic cerebral infarction group at each time point after the reperfusion was significantly lower than that in the single cerebral infarction group (P < 0.05). MMP-9 began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak on 1 d and then decreased gradually. The expression of MMP-9 in the diabetic cerebral infarction group at each time point after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05).@*CONCLUSIONS@#The high glucose environment in which the diabetic cerebral infarction is occurred is to induce the formation of MMP-9 at first and then activate and increase the expression of angiostatin. Afterwards, the expression of VEGF is inhibited, resulting in the poor angiogenesis after cerebral infarction, which thus makes the injury of brain tissue after cerebral infarction even worse than the non-diabetes mellitus.

Tanshinone IIA Prevents Rat Basilar Artery Smooth Muscle Cells Proliferation by Inactivation of PDK1 During the Development of Hypertension.

Basilar vascular smooth muscle cells (BASMCs) hyperplasia is a prominent feature of cerebrovascular remodeling and stroke during the development of hypertension. Tanshinone IIA (Tan) has been reported to exhibit a protective effect against the pathological features of hypertension. Previous studies have shown that phosphoinostitide-3 kinase (PI3K)/3'-phosphoinostitide dependent kinase (PDK1)/AKT pathway is involved in the regulation of proliferation of various cell types. Therefore, there may be a crosstalk between Tan antihypertension processes and PI3K/PDK1/AKT proliferative effect in BASMCs. To test this hypothesis, we used a 2-kidney, 2-clip hypertension model to examine the effect of Tan on PI3K/PDK1/AKT pathway by cellular, molecular, and biochemical approaches. Our results revealed that the abundance of PDK1 in plasma was paralleled with an increase in blood pressure and the cross-sectional area of basilar artery in hypertensive rats. Tan decreased blood pressure and hypertension-induced PDK1 phosphorylation but produced no effect on the phosphorylation of PI3K. Moreover, Tan attenuated endothelin 1 induced the activation of PDK1/AKT pathway in rat BASMCs. Tan could inhibit cell cycle transition by regulating the expression of cyclin D1 and p27, in turn, prevent proliferation of BASMCs. Our study provides a novel mechanism by which Tan prevents cerebrovascular cell proliferation during hypertension, and thus Tan may be a potential therapeutic agent for cerebrovascular remodeling and stroke.

Febrile seizures are associated with mutation of seizure-related (SEZ) 6, a brain-specific gene.

Genetic factors contribute significantly to the etiology of febrile seizures (FS), the most common type of seizures in childhood. However, in most patients with FS, the causative gene is unknown. The purpose of this study was to explore the relationship between human brain-specific gene SEZ-6 and FS. Through amplification of genomic DNA by PCR and sequencing of the resulting products, we screened 75 subjects for mutations in the coding region (17 exons) of the SEZ-6 gene. Fifteen subjects were healthy individuals and 60 subjects had FS. Patients with FS could be divided into sub-groups based on seizure type (42 simple and 18 complex) and family history (41 had a positive family history). All patients have been followed to date to evaluate seizure recurrence and the development of epilepsy. No mutations were found in healthy controls, but 21 of the patients with FS had mutations in SEZ-6, and the most common type of mutation was a heterozygous, cytosine insertion (frame shift mutation) at position 1435 of the cDNA. The mutation incidence was significantly higher in patients with complex FS (vs. simple FS) and in patients with a positive family history. Sixteen of 42 patients with simple FS experienced seizure recurrence during the 1-5-year follow-up period. Fifteen of 18 patients with complex FS also experienced a recurrence during this period. Among these patients with recurrences, five patients with simple FS and six patients with complex FS have developed epilepsy. The mutation incidence among these epileptic patients is 72.7%. The human SEZ-6 gene is related to the occurrence and development of FS and may be a novel candidate gene for epilepsy. Screening for mutations in SEZ-6 may be valuable in predicting FS recurrence or the development of epilepsy.

[A pedigree with myotonic dystrophy: non-CTG, non-CCTG repeat expansion].

OBJECTIVE: Two genetic loci are associated with the myotonic dystrophy (DM) phenotype: DM1 DMPK on chromosome 19, and DM2 ZNF9 on chromosome 3. The aim of this study was to investigate the molecular genetics of a pedigree with DM. METHODS: In twenty-six individuals from a family with DM, the CTG repeats in DMPK and CCTG repeats in ZNF9were evaluated genetically, using Long Expand trade mark Template polymerase chain reaction (PCR), Southern blotting and genomic scanning. RESULTS: The numbers of CTG and CCTG repeat were all in normal range. There was no significant difference between the CTG repeat size in DMPK gene and that 4 years later from the same individual. The Lod score values with short tandem repeats STR markers chosen in 19q and 3q were all smaller than 1, which suggested that no STR marker was linked with this DM family. CONCLUSION: There might be some other mutant in this DM pedigree. Further study should be done to find the genetic basis of this pedigree.

Clinicopathological features of giant cell carcinoma of the pancreas.

BACKGROUND: Giant cell carcinoma of the pancreas (GCCP) as a tumor of high malignancy, large size, and inflammatory reaction occupies 2.1%-12.8% of all cases of pancreatic malignancies. This study was to analyze cases of GCCP collected in 8 years at our hospital in an attempt to describe some features of GCCP in Chinese people. METHODS: The clinicopathological features of 19 patients who had been pathologically diagnosed as having GCCP from 1021 patients with pancreatic malignancies collected by Pancreatic Disease Research Group (PDRG) of Changhai Hospital were retrospectively analyzed compared with those of 96 patients with common pancreatic carcinoma (PC) who were randomly selected from 1002 patients with pancreatic carcinoma. The differences of location, clinical symptoms, imagings, laboratory test, operation and the prognosis of these two groups were defined. RESULTS: Tumors in the head of the pancreas were found in 8 patients (42.1%), and those in the body or tail of the pancreas in 11 (57.9%). The initial symptom was abdominal pain in most patients (57.9%). Abdominal pain (73.7%), dyspepsia (63.2%), weight loss (36.8%) but jaundice were common at the time of diagnosis. The abnormal rates of routine laboratory tests in the GCCP group were lower than those in the common PC group. The assay of tumor markers between the groups of GCCP and common PC was approximately the same. The sensitivity and accuracy of ultrasonography, spiral computed tomography and magnetic resonance imaging were considerably high. Large carcinoma in stage IV was seen in 9 patients or 47.4% of the GCCP group, a rate higher than that in the common PC group. Osteoid formation was found microscopically in some patients, and poorly differentiated tumor cells were found in most patients. The 1-year survival rate was 17.6%, which was lower than that in the common PC group. CONCLUSION: The clinicopathological features of GCCP are different from those of common PC. Imaging tests can be used together with the assay of tumor markers to diagnose GCCP as early as possible and to improve the prognosis of GCCP patients.

[Compared D-amino acid oxidase expression in different Pichia pastoris host strains].

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.

[Enhancement of the production of SAM by overexpression of SAM synthetase in Pichia pastoris].

S-Adenosyl-L-methionine(SAM) is an important metabolic intermediate in the metabolic flux of sulphur. SAM is involved in three key metabolic pathways: transmethylation, transsulfuration and polyamine synthesis. As a potential therapeutic agent, SAM is being used as over the counter drug and nutrient supplement. An expression vector, harboring SAM synthetase 2 gene from S. cerevisiae and regulated by the glyceraldehyde-3-phosphate dehydrogenase gene promoter P(GAP), was transformed into GS115 strain of P. pastoris. Through zeocin resistance and expression screening, a recombinant strain was obtained that had high SAM yield and the fermentation conditions were optimized. The results showed that carbon source, nitrogen source, pH and dissolved oxygen had significant effects on the accumulation of SAM. The SAM production of the recombinant cells reached 2.49 g/L after fermentation for three days under the optimized conditions. The present studies show that fermentation of recombinant P. pastoris strain, expressing heterologous SAM synthetase gene, may be a promising approach for the production of SAM.