OBJECTIVETo study mitochondrial DNA (mtDNA) A1555G mutation in seven families with nonsyndromic hearing loss (NSHL).

METHODSPolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real time-amplification refractory mutation system-quantitative PCR (ARMS-qPCR) were applied to detect mtDNA A1555G mutation in seven NSHL families. Related clinical data were also collected and analyzed.

RESULTSThe mtDNA A1555G mutation was detected in members from the maternal side, including heteroplasmy and homozygosis, others were negative for this mutation. The copy number of homoplasmic or heteroplasmic mutations of mtDNA A1555G correlated well with the degree of deafness (R = 0.341, P = 0.022 and R = 0.85, P = 0.015, respectively).

CONCLUSIONThe mutation rate of the mtDNA A1555G is high in the NSHL patients, the mutation type include heteroplasmy and homozygosis. There is significant correlation between the mtDNA A1555G copy number and the severity of hearing loss.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child, Preschool ; DNA, Mitochondrial ; genetics ; Female ; Gene Dosage ; Hearing Loss ; genetics ; pathology ; Humans ; Infant ; Male ; Middle Aged ; Pedigree ; Point Mutation ; Young Adult

BACKGROUNDThe capsule associated protein 10 gene (cap10) is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus (C.) neoformans. In this study, we aimed to construct an short hairpin RNA (shRNA) expression vector targeting C. neoformans cap10 gene expression and confirm its biologic relevance.

METHODSA pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method.

RESULTSThe expression of cap10 in C. neoformans cells transfected with ps4.1 neo-cap10 ((175,535.00 ± 47,004.00) copies/µl) was lower than that of cells transfected with the empty vector ((512,698.89 ± 32,318.02) copies/µl) and mock transfected cells ((562,931.66 ± 65,928.41) copies/µl). The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-cap10 (0.21 ± 0.02, (19.06 ± 1.66)%) than for the control experimental group (0.08 ± 0.02, (6.57 ± 1.23)%) and the blank experimental group ((0.07 ± 0.01), (5.89 ± 1.07)%) (P < 0.05).

CONCLUSIONSThe cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans cap10 gene is effective, and it can induce phagocytosis of C. neoformans.

Animals ; Cell Line ; Cryptococcus neoformans ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Phagocytosis ; Plasmids ; genetics ; Polymerase Chain Reaction ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Background and Objective:Cytokine-induced killer(CIK)cells have high anti-tumor activity for hepatocellular carcinoma(HCC).Whether CIK cell therapy can eradicate residual cancer cells and prevent or postpone tumor relapse after transcatheter arterial chemoembolization(TACE)should be testified.This study was to evaluate the efficacy of CIK cell therapy combined with TACE on HCC.Methods:A total of 146 consecutive patients with unresectable HCC were divided into combination group (72 patients trealted with CIK cell therapy combined with TACE) and TACE group(74 patients treated only with TACE).The progression-free survival(PFS)and overall survival(OS)were analyzed.Results:The 6-month,1-year,and 2-year PFS rales were 72.2%, 40.4%, 25.3%in combination group, and 34.8%,7.7%,2.6%in TACE group.The median time to progression was 11 months[95%confidence interval(CI),8-1 4 months]in combination group and 5 months(95% CI,4-7 months)in TACE group.The estimated 6-month, 1-year, and 2-year OS rates were 90.3%, 71.9%. 62.4%in combination group,and 74.6%,42.8%,18.8%in TACE group.The median OS was 31 months(95%CI,27-35 months)in combination group and 10 months(95% CI,7-13 months)in TACE group.The times of TACE,ECOG performance status, and CIK cell therapy were independent prognostic factors for PFS and OS.Conclusion:Adjuvant immunotherapy with CIK cells could greatly improve the efficacy of TACE on HCC,and plays an important role in prolonging the PFS and OS of HCC patients after TACE.

BACKGROUNDA high mortality rate of pancreatic cancer becomes a bottleneck for further treatment with long-term efficacy. It is urgent to find a new mean to predict the early onset of pancreatic cancer accurately. The authors hypothesized that genetic variants of cationic trypsinogen (PRSS1) gene could affect trypsin expression/function and result in abnormal activation of protease activated receptor-2 (PAR-2), then lead to pancreatic cancer. The aim of this study was to elaborate some novel mutations of PRSS1 gene in the patients with pancreatic cancer.

METHODSTotally 156 patients with pancreatic cancer and 220 unrelated individuals as controls were enrolled in this study. The mutations of PRSS1 gene were analyzed by direct sequencing. K-ras Mutation Detection Kit was used to find the general k-ras gene disorder in the pancreatic cancer tissue. Then the clinical data were collected and analyzed simultaneously.

RESULTSThere were two patients who carried novel mutations which was IVS 3 + 157 G > C of PRSS1 gene in peripheral blood specimens and pancreatic cancer tissue. What's more, it was surprising to find a novel complicated mutation of exon 3 in PRSS1 gene (c.409 A > G and c.416 C > T) in another young patient. The complicated mutation made No. 135 and No. 137 amino acid transfer from Thr to Ala and Thr to Met respectively. No any mutation was found in the normal controls while no mutations of k-ras gene were detected in the three patients.

CONCLUSIONMutations of PRSS1 gene may be an important factor of pancreatic cancer.

Adult ; Asian Continental Ancestry Group ; Female ; Humans ; Male ; Mutation ; Pancreatic Neoplasms ; genetics ; Trypsin ; genetics

BACKGROUNDMutations in the cationic trypsinogen gene (PRSS1) have been detected in patients with hereditary pancreatitis (HP). This study investigated the prevalence of the R122H (c.365 G > A), A121T (c.361 G > A) and D162D (c.488 C > T) mutations or polymorphisms in the common, non-hereditary forms of chronic pancreatitis and in an HP family.

METHODSDNA was prepared from blood samples of 54 patients with chronic pancreatitis (35 alcoholic, 17 idiopathic and 2 hereditary) and 120 normal controls. The PRSS1 genes were amplified by polymerase chain reaction (PCR) and their products were analyzed by sequencing and related clinical data were also collected.

RESULTSA new polymorphism (c.488 C > T) of PRSS1 was found in 25 patients with chronic pancreatitis (including one affected member of the HP family) and six members of the normal controls. The C/T genotype was significantly increased in chronic pancreatitis (OR: 16.379, 95% CI: 5.7522 - 52.3663), the frequency of c.488 C > T change was in according with the Hardy-Weinberg equilibrium, but it doesn't affect the clinical phenotype. The commonly reported change of R122H (c.365 G > A) was not detected in any of the study subjects. c.361 G > A was found in 2 affected members and one unaffected carrier in an HP family. One of the affected members of an HP family had c.361 G > A mutation and polymorphism (c.488 C > T) in the PRSS1 gene at the same time. The patient's clinical values (C3, C4, CA19-9 and HbA1c) were higher than those of the other patients with chronic pancreatitis. The two patients with HP developed diabetes mellitus and their father died with pancreatic cancer.

CONCLUSIONA new polymorphism (c.488 C > T) in the PRSS1 gene is associated with chronic pancreatitis, but it did not affect the clinical phenotype while the A121T (c.361 G > A) mutation in the gene shows a significant correlation in the patients with HP.

Female ; Humans ; Male ; Mutation ; Pancreatitis ; genetics ; Pancreatitis, Chronic ; genetics ; Polymorphism, Genetic ; Trypsin ; Trypsinogen ; genetics

OBJECTIVETo explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).

METHODSVSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.

RESULTSThe resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).

CONCLUSIONTestosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Dinoprost ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism ; physiology

OBJECTIVETo determine the presence of membrane testosterone receptors in cultured vascular smooth muscle cells (VSMC), and investigate their relationship with classical intracellular androgen receptors (iAR).

METHODSVSMCs were cultured from the thoracic aorta of male Sprague-Dawley rats by the explant method. Subconfluent VSMCs were incubated with serum-free medium for 24 h to obtain quiescent non-dividing cells, and then treated with the indicated agents. The aliquots of VSMCs were labeled with testosterone-BSA-FITC (T-BSA-FITC) and analyzed by flow cytometry. Classical iARs in intact- and permeabilized-cells were detected with anti-iAR antibodies and FITC-labeled secondary antibodies by immunofluorescence, followed by flow cytometry analysis.

RESULTSIncubation of VSMCs with T-BSA-FITC obviously increased their relative fluorescence intensity at 10 sec as compared with the untreated controls (P < 0.01), and so did it at 10 min in comparison with the treatment with BSA-FITC alone or together with free testosterone (P < 0.01). Pretreatment with iAR antagonist flutamide exhibited no significant influence on the relative fluorescence intensity of VSMCs (P = 0.318). Traditional iARs were not detectable on the surface of intact VSMCs, although permeabilized cells contained iARs.

CONCLUSIONVSMCs contain testosterone receptors in the plasma membrane, and these membrane receptors are not identical to classical iARs.

Animals ; Cells, Cultured ; Male ; Membrane Proteins ; metabolism ; Muscle, Smooth, Vascular ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Androgen ; metabolism ; Testosterone ; metabolism

Purpose:In order to investigate the effects of repair of arteriovenous fistula on heart function soas to widen the range of the indication of allogenetic transplantation of kidney. Methods :Repair of arteriovenousfistula was performed in 8 patients who have received long term hemodialysis and then complicated with repeatedattack of heart failure 5 months before renal transplantation. Results:There are 7 patients had their cardiac cham-bers returned to normal, and 1 returned to normal upper limit. Renal transplantation was successfully performedin 6 patients, but death occurred in 1 case resulting from liver function failure. Superacute rejection and sponta-neous rupture of the kidney occurred in 1 patient. Conclusions:It is considered that the repair of arteriovenous fis-tula before renal transplantation is beneficial to improvement of heart function in these patients so as to widen therange of operative indications and create favourable condition for renal transplantation and decrease complica-tions. Color doppler sonography is of great value in monitoring pre-and postoperative heart function.

【Objective】Diabetes mellitus is a risk equivalent for coronary heart disease. This retrospective study was designed to investigate the risk factors of the progression of coronary lesions in patients with type 2 diabetes（T2DM）and Non- diabetes Mellitus（NDM）.【Methods】 526 patients with T2DM and 425 patients with NDM at the Third Affiliated Hospital of Sun Yat-sen University between March 2001 and January 2017 who underwent coronary imaging studies（coronary angiography or coronary CTA）twice during the same period were enrolled. The effects of cardiovascular risk factors on the progression of coronary lesions were analyzed in parallel in these two types of patients.【Results】Risk factors of the progression of coronary lesions in T2DM patients included smoking（OR = 1.836，95% CI：1.030~3.371，P = 0.04），Lp（a） ［OR = 1.001，95% CI：1.000~1.002，P = 0.004（baseline）；OR = 1.001，95% CI：1.000~1.002，P = 0.009（re-examined）］，HbA1c leve［l OR = 1.471，95% CI：1.030~2.100，P = 0.034（re-examined）］，uncontrolled LDL-C（OR = 1.882，95% CI：1.091~3.245，P = 0.023），TC［OR = 2.029，95% CI：1.028~4.008，P = 0.041（re-examined）］and low HDL-C ［OR = 0.017，95% CI：0.040~0.729，P = 0.017（re-examined）］. Comparative risk factors in NDM included BMI［OR =1.746，95%CI：2.462~2.712，P = 0.026（baseline）；OR = 0.001，95%CI：0~0.394，P = 0.025（re-examined）］，uncontrolled LDL-C（OR = 2.875，95%CI：1.669~4.952，P < 0.001）and low ApoA［OR = 0.282，95%CI：0.082~0.971，P = 0.045 （baseline）；OR = 0.117，95%CI：0.038~0.835，P = 0.029（re-examined）］. Lowest level of progression was found in the group with HbA1c<6.5%［0（0~3.4）points/year vs 0.3（0~3.0）points/year vs 1.0（0~5.1）points/year，P = 0.049. 0（-0.4~2.7）points/year vs 0.6（0~4.0）points/year vs 0.9（0~4.2）points/year，P=0.029］in T2DM patients.【Conclusion】Except for achievement of LDL- C goals，there might be some differences in risk factors for progression of coronary lesions between T2DM and NDM patients. Smoking，Lp（a），TC，HDL- C and control levels of HbA1c are independent predictors in T2DM as well as BMI and ApoA in NDM. Lowering HbA1c to less than 6.5% may delay progression of lesion.

OBJECTIVE: To observe the effect of ligustrazine on cell proliferation in subventricular zone (SVZ) in rat brain with focal cerebral ischemia reperfusion injury. METHODS: Male SD rats were randomly divided into a normal group,a sham operation group,a ligustrazine treatment group, and a control group. The ligustrazine treatment group and the control group were further divided into 5 subgroups: 1d, 3d, 7d, 14d, and 21d reperfusion after 2h middle cerebral artery occlusion (MCAO). The focal cerebral ischemia-reperfusion model was made by MCAO. S phase cells were labelled with BrdU. Immunohistochemistry method was conducted to detect the BrdU positive cells. The total number of BrdU positive cells in the SVZ was measured. The expression of neuro nitric oxide synthase (nNOS) was detected with Western blot method. RESULTS: There was a significant increase of BrdU positive cells in SVZ of ligustrazine treatment in the 1d and 3d group compared with that of the control group (P<0.01). The total number of BrdU positive cells reached a peak in 7d group and declined afterwards. Cells proliferated also in SVZ on the contralateral side, and peaked at 7d. The nNOS expression of ligustrazine administration after the focal cerebral ischemia-reperfusion decreased at 1d and 3d after the reperfusion compared with that of the control group (P<0.05), and increased at 7d, but with no significant difference compared with that of the control group. CONCLUSION Ligustrazine may promote the cell proliferation in SVZ of adult rats with ischemia-reperfusion injury by decreasing the nNOS expression.
Animals ; Blotting, Western ; Brain Ischemia ; physiopathology ; Cell Proliferation ; drug effects ; Cerebral Ventricles ; metabolism ; pathology ; Infarction, Middle Cerebral Artery ; physiopathology ; Male ; Nitric Oxide Synthase Type I ; metabolism ; Pyrazines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; physiopathology ; Time Factors