Objective To observe the influence of low protein diet with α-keto acid on kidney sclerosis and renin-angiotensin system in renal ablation rats. Methods Chronic renal failure rat model was established by renal ablation in 30 male SD rats,then the animals were randomly assigned to the following diet groups:normal protein group (NPD:18%casein protein),low protein group (LPD:6%casein protein) and supplemented low protein group (LK:5%casein protein+1%α-keto acids).Ten male SD sham-operated rats received 18%casein protein as control.All the rats were killed at the end of the 12th week.Pathologic changes were assessed by PAS staining.Ang II in homogenate and plasma were measured by radioimmunoassay and ELISA respectively.Immunohistochemistry and Western blotting were used to detect the protein expression of TGF-β1,renin and AT1R.Real-time PCR was used to detect the gene expression of renin and ATla,the main subtype of AT1 receptor. Results Body weight,total protein and serum albumin had not significant difference among the four groups(all P>0.05).Serum creatinine and proteinuria of nephrectomized rats were significantly higher compared to the control group (all P<0.05).Proteinuria of the LK group was lower than that of NPD and LPD groups (all P<0.05).Pathological results indicated fibrosis indices were significantly improved after LPD and LK intervention.Expressions of renin,Ang II and AT1R in LK group were significantly lower than those in NPD group (all P<0.05). Conclusions Low protein diet with α-keto acids supplement therapy exhibits renal protective effects of reducing urine protein excretion and improving renal fibrosis,which might be related to the attenuation of local renin-angiotensin system in activity nephrectomized rats.

Objective To detect the mutation of CARD15 gene in a patient with sarcoidosis and tuberculosis.Methods Clinical data were collected from a 32-year-old male patient with early-onset sarcoidosis and tuberculosis.Peripheral blood was obtained from the patient,both of his parents,and 102 healthy controls.All the 12 exons of the coding regions as well as flanking intronic sequences of the CARD15 gene were amplified by PCR followed by direct sequencing.The resulted sequences were blasted against the reference sequences of CARD15 gene.Results Both clinical features and histopathological findings of the patient were consistent with sarcoidosis.Furthermore,the patient presented with flexion contractures of fingers and toes,as well as iridocyclitis.A heterozygous missense recurrent mutation c.1000C ＞ T (p.R334W) was detected in exon 4 of the CARD15 gene in the patient,but not in either of his parents or any of the 102 healthy controls.Conclusions A p.R334W mutation in the CARD15 is identified in the patient,which may be responsible for the clinical phenotype of earlyonset sarcoidosis.Gene analysis may be a useful method to clarify the etiology of early-onset sarcoidosis.

Objective To observe the ultrastructural features of recessive dystrophic epidermolysis bullosa inversa(RDEB-Ⅰ)and to detect the mutations of COL7A1 gene in a family with RDEB-Ⅰ.Methods A 24-year-old male patient complained of recurrent vesicles in the skin for 24 years.The lesions began as generalized pruritic vesicles and bullae soon after birth,with a predilection for areas subject to friction,and showed a trend to be worse in summer but mild in winter.No photosensitivity was observed.When he was 3 to 4 years old,the lesions were decreased in number,with the only involvement of the trunk and abdomen;thereafter,the lesions were improved year by year.The patient suffered from nephritis at the age of 5 years,which progressed into renal failure at the age of 15 years.He received renal transplantation and was given long-term oral tacrolimus and mycophenolate mofetil,which leaded to an improvement in the lesions.The family history was unremarkable,and the marriage between her parents was not consanguineous.Dermatological examination revealed large areas of irregularly-marginated,hypopigmented,atrophic scar on the waist,back and abdomen with onychodystrophy involving multiple nails.No vesicles were observed.Immunofluorescence antigen mapping and transmission electron microscopy were conducted to observe the expression of type Ⅶ collagen in and ultrastructure of cutaneous lesions from the patient.Venous blood samples were obtained from the patient as well as his parents and 3 sisters,and drill biopsy specimens were obtained from the margin of vesicular lesions and unaffected anterior tibial skin of the patient.DNA specimens were obtained from the blood samples of the family members and 150 unrelated healthy controls,and RNA was extracted from the biopsy samples of the patient.PCR and direct sequencing were carried out to detect mutations in COL7A1 gene,and reverse transcription-PCR was conducted to confirm the mutation at mRNA level.Results Skin cleavage was observed under lamina densa in the dermis,with a decrease in anchoring fibrils and expression of type Ⅶ collagen in the lesions of the patient.A heterozygous synonymous mutation c.C5499T which created a new splicing site and leaded to a premature terminal codon,as well as a heterozygous missense mutation c.C6205T(C-T transition at codon 2069:CGT to TGT)which leaded to the substitution of arginine by cysteine,were identified in the COL7A1 gene of the proband and all of his sisters,but not in any of the unrelated controls.The c.C5499T and c.C6205T mutations were inherited from the patient's father and mother respectively.Conclusion The compound heterozygous mutations c.C6205T and c.C5499T may be responsible for RDEB-Ⅰ in this patient.

Objective To study cutaneous ultrastructural changes and FERMT1 gene mutations in a patient with Kindler syndrome. Methods Clinical data were collected, and tissue samples obtained from the lesions of poikiloderma were observed by using transmission electron microscopy. Fifteen coding exons and their flanking sequences of the FERMT1 gene were amplified by PCR and DNA sequencing was followed.Results Reduplication of lamina densa was seen between the dermal-epidermal junctions of the lesional skin. The patient was found to be homozygous for a novel splice-site mutation (IVS9 + 1G ＞ A) in FERMT1 gene, and his parents were heterozygous for it. The mutation was undetected in fifty normal control individuals.Conclusions Transmission electron microscopy may serve as an ancillary examination for the diagnosis of Kindler syndrome. The IVS9+1G＞A mutation of FERMT1 gene may contribute to the clinical phenotype of Kindler syndrome in this patient.

Objective To explore the application value of 320-slice spiral CT myocardial perfusion technique in the evaluation of cardiac function in patients with coronary artery disease.Methods 24 patients with known reversible myocardial ischemia with coro-nary heart disease and 13 cases of normal were detected by 320-slice spiral CT myocardial perfusion scan in resting and after dobu-tamine-induced stress,measured the values of cardiac function indexes(ESV、EDV、SV、EF)in resting and after dobutamine-induced stress respectively,the results were analyzed by paired t test and independent sample t test,when the P value is less than 0.05,con-sidered statistically significant or correlation.Results The values of cardiac function indexes (ESV,EDV,SV)between the resting and after dobutamine-induced stress were significantly different except EF value in normal group.But only the EF value was signifi-cantly different in CHD,the rest values of cardiac function indexes(ESV,EDV,SV)no significant difference.Each corresponding value of cardiac function indexes in resting and after dobutamine-induced stress was significantly different between the CHD and the normal.Conclusion The dobutamine-induced stress myocardial perfusion with 320-slice spiral CT can judge the impaired degree of cardiac function in patients with myocardial ischemia,provide the basis for comprehensive evaluation of coronary artery disease and its prognosis.

Objective To assess mutations in the ALDH3A2 gene in two patients with Sj(o)gren-Larsson syndrome manifesting primarily as congenital ichthyosis,mental retardation and spastic paraplegia.Methods Two patients,a 2-year-old girl and a 1.5-year-old boy,with Sj(o)gren-Larsson syndrome were included in this study.None of their family members suffered from this disease.Peripheral blood samples were collected from the two patients,their family members (an elder brother and both parents),and 100 unrelated healthy controls.DNA was extracted from the blood samples,and subjected to PCR for the amplification of 10 encoding exons and their flanking sequences of the ALDH3A2 gene followed by DNA sequencing.Results A homozygous missense mutation c.325G ＞ A,which leads to the substitution of glycine by arginine at position 109,was detected in the ALDH3A2 gene of patient 1,whose parents and elder brother were heterozygous carriers of this mutation.The patient 2 carried compound heterozygous mutations,including c.1157A ＞ G (p.Asn386Ser) inherited from his father and c.1294A ＞ T (p.Arg432X) inherited from his mother.None of these mutations was detected in the unrelated healthy controls.Conclusion The homozygous mutation p.Gly109Arg and compound heterozygous mutations p.Asn386Ser and p.Arg432X present in these patients may be associated with clinical phenotypes of Sj(o)grenLarsson syndrome.

This investigation is to explore the feasibility of applying reverse docking method to the selectivity studies of protein kinase inhibitors. Firstly, a database that consists of 422 protein kinase structures was established through collecting the reported crystal structures or homology modeling. Then a reverse docking based method of protein kinase target screening was established, followed by the optimization of related parameters and scoring functions. Finally, seven typical selective kinase inhibitors were used to test the established method. The results show that the selective targets of these inhibitors have relatively high scoring function values (ranking in the first 35% of the tested kinase targets according to the scoring function values). This implies that the reverse docking method can be applied to the virtual screening of kinase targets and further to the selectivity studies of protein kinase inhibitors.
Alternative Splicing ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; methods ; Gene Targeting ; Models, Molecular ; Protein Binding ; Protein Kinase Inhibitors ; chemistry

Objective To observe the changes of renin-angiotensin system (RAS) in cultured mesangial cells by serum from 3/4 nephrectomized rats feeding with low protein diet and α-keto acid. Methods Thirty male SD rats received 3/4 nephrectomy (Nx) were placed on 18%normal protein diet (NPD,n=10),6% low protein diet(LPD,n=10) or 5% low protein plus 1%α-keto acid diet (LK,n=10) flor 12 weeks.Ten male SD sham-operated rats fed with 18% normal protein diet were used as control (sham group).In addition,mesangial cells were cultured in sera (10%) collected from above animals treated with or without losartan (0.02 mmol/L)for 48 hours.ELISA was applied to detect the level of Ang II,TGF-β1 and fibronectin (FN) in cell medium.Westem blotting was used to determine the protein level of ATI receptor (AT1R)and real-time PCR was used to detect the mRNA level of AT1R,TGF-β1 and FN. Results (1) Nutritional indices including body weight,total protein and albumin had no significant difference in each group. (2) Serum creatinine and 24 h pruteinuria were significantly inceased in nephrectomized groups compared to sham group(P<0.05,respectively).24 h proteinuria was greatly lower in LK group than that in NPD and LPD groups(P<0.05,respectively).(3)LK greatly decteased the level of Ang II[NPD(12.70±0.12)mg/g protein;sham(8.04±0.62)mg/g protein]in supernatant as well as the protein and mRNA expression of AT1R in cultured mesangial cells (P<0.05).(4)NPD serum directly induced higher secretion[FN:sham(20.58±0.46)g/g protein,NPD (39.84±0.06)g/g protein;TGF-β1:sham(10.12±O.56)mg/g protein,NPD(83.85±3.58)mg/g protein] and mRNA expression of FN and TGF-β1 compared with sham group (P<0.05).LPD decreased these increment (P<0.05) and LK showed stronger inhibitory effect (P<0.05). (5)Losartan application sharply reduced FN and TGF-β1 production both in supematant and in mRNA expression in NPD serum treated cells (P<0.05,respectively). Conclusion Low protein diet with α-keto acids supplement directly inhibits the RAS in mesangial cells which may contribute to its beneficial effect on the kidney.

Objective To investigate the distribution of blood vessels and the relationship of angiogenesis with hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in meningiomas.Methods Fifteen specimens of meningiomas,collected in our hospital during the resection and conformed by pathology from May 2010 to March 2011,were chosen; 4 different tissues from each specimen were taken,including the tail sign,base center,tumor' s center and tumor' s margin; three successive slices were made from each tissue part.Envision immunohistochemistry was employed to detect the expressions of CD34,VEGF and HIF-1α and their different distributions were compared.Results CD34 mainly disturbed in the cell membrane and cytoplasm,showing yellow granules in the dyeing; VEGF located at the cytoplasm of the tumor cells,while HIF-1α mainly at the nucleus.The expressions of CD34,VEGF and HIF-1α in the tail sign and tumor's margin were significantly higher than those in the MVD in the base center and tumor's center (P＜0.05).Positive correlations between expressions of VEGF and MVD,expressions of HIF-1α and MVD,as well as expressions of HIF-1α and VEGF were noted (r=0.960,P=0.040; r=0.964,P=0.036; r=0.998,P=0.002).Conclusion The angiogenesis in the periphery ofmeningiomas is much richer than that in the center; H1F-1α and VEGF may induce and promote the angiogenesis of meningiomas.

OBJECTIVETo explore the correlativity of meridians and acupoints with fascial system.

METHODSThe lines and beads-like structures seen by fasciaology and scanning connective tissue, were combined with traditional meridians and collaterals, and acupoints to investigate channels, collaterals and acupoints.

RESULTSThe high correlativity of the meridians and acupoints with the fascial system was found.

CONCLUSIONThe concept, functions, clinical application and mechanisms of meridians and acupoints can be preliminarily explained.