Rat ferritin-H: cDNA cloning, differential expression and localization during hepatocarcinogenesis

Elevated serum ferritin levels, especially of the H subunit, accompany many clinical malignancies. By using the subtraction-enhanced display technique, we have recently isolated several cDNA clones which are over- expressed in rat hepatocellular carcinoma induced by diethylnitrosamine. One 830-base-pair clone was 88% similar to human ferritin-H cDNA and encoded a 182 amino acid protein which is 97% homologous to human ferritin-H chain. Hepatic mRNA levels of ferritin-H were increased markedly at the early stage of diethylnitrosamine- induced hepatocarcinogenesis in the rat (6 weeks) and appeared more than 10-fold overexpressed as the tumour progressed. In contrast, hepatic ferritin-H mRNA remained constant during liver regeneration after a 70% partial hepatectomy. In situ hybridization showed that over- expression of ferritin-H was exclusively localized to preneoplastic foci, to tumour nodules and to tumour cells invading blood vessels. These findings suggest that ferritin-H is a highly conserved protein, its over-expression during tumour development is phenotypically correlated with tumour initiation and/or progression, and it is useful as an early marker for hepatocellular carcinoma.      

Genomic screening for beta-sarcoglycan gene mutations: missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E)

Autosomal recessive limb-girdle muscular dystrophies (LGMDs) are genetically heterogeneous. A subgroup of these disorders is caused by mutations in the dystrophin-associated sarcoglycan complex. Truncating mutations in the 43 kDa beta-sarcoglycan gene (LGMD 2E) were originally identified in a sporadic case of Duchenne-like muscular dystrophy, and a common missense mutation (T151R) was identified independently in Indiana Amish pedigrees with a milder form of LGMD. To facilitate mutational analysis of larger numbers of patients directly from genomic DNA, as opposed to reverse transcribed RNA from muscle biopsies, we have determined the genomic structure of the beta-sarcoglycan gene. The open reading frame of the beta-sarcoglycan coding region extends over six exons. Primers were designed for PCR amplification of single exons from genomic DNA and subsequent single strand conformation polymorphism (SSCP) analysis. We screened 15 patients from the Brazilian LGMD patient population, 13 of whom followed a severe course. Most of the patients had been assessed previously for deficiency of alpha- sarcoglycan immunofluorescence on muscle biopsy sections as a marker for disease of the sarcoglycan complex. Novel mutations in two familial and two sporadic cases of severe childhood-onset LGMD were identified. Only one of these patients carried a truncating mutation (homozygous 2 bp deletion, FS164TER), while the other three carried missense mutations (homozygous R91P, homozygous M100K, heterozygous recessive L108R; only one allele could be identified in this family). All three missense mutations occurred in exon 3, coding for the immediate extracellular domain. Complete absence for all three of the known sarcoglycans was noted by immunohistochemistry on muscle biopsy sections of the patients.      

中药制剂承载丸对甲基强的松龙预处理的成骨细胞L型钙通道电流的影响

目的:观察承载丸含药血清对甲基强的松龙预处理的MC3T3-E1成骨细胞L-钙通道电流的影响。方法:实验于2006-04/06在中国中医科学院望京医院药理实验室及吉林大学基础医学院生理教研室完成。①实验材料:MC3T3-E1(购于北京协和细胞中心);8个月龄SD大鼠10只,雌雄各半;中药制剂承载丸(由鹿角霜、肉苁蓉、续断、黄芪、当归、炙水蛭、杜仲、蛋衣、皂角刺、香附、乌药等21味中药组成);甲基强的松龙(批号H20040338,Pharmacia NV/SA生产)。②实验干预:成骨细胞(MC3T3-E1)传代培养。承载丸药粉60g,加水至200mL搅匀,取大鼠6只(雌雄各半),每只灌服2.5mL/d,灌服4d后动脉取血,制备含药血清。另取大鼠4只,同法灌服生理盐水,制成不含药血清。甲基强的松龙的浓度为1×10-5mol/L。③实验分组:将培养后的成骨细胞株分为4组:正常组(加入正常大鼠不含药血清)、模型组(加入正常大鼠不含药血清及甲基强的松龙)、承载丸低剂量组(加入甲基强的松龙及0.5mL承载丸含药血清)和承载丸高剂量组(加入甲基强的松龙及0.1mL承载丸含药血清)。④实验评估:24h后,用全细胞膜片钳技术记录每组10个细胞的L-钙通道电流,并测定其峰值电流。结果:①各组电流峰值测定结果:模型组峰值电流比正常组下降19.5%[(0.1839±0.0179),(0.2284±0.0209)nA,P<0.01];承载丸低剂量组和高剂量组比模型组分别升高37.4%和45.2%[(0.2526±0.0093),(0.2671±0.0120),(0.1839±0.0179)nA,P<0.01];承载丸高低剂量组相比差异有显著性意义(P<0.05)。②MG3T3E1细胞钙电流的特性分析:正常组,钙通道大约在-20mV时开放,最大峰值电流在 20～ 30mV,反转电位在 60～ 70mV。当在浴液中加入Co2 ,可阻断此电流的大部分电流成份,其峰值电流为(0.1050±0.0097)nA,而钙离子激活剂Bayk8644增加了此电流(0.3335±0.0323)nA。结论:①甲基强的松龙抑制成骨细胞L-钙通道电流。②承载丸含药血清可升高甲基强的松龙预处理的成骨细胞L-钙通道电流。     

Compliance with growth hormone treatment--are they getting it?

A study was undertaken to investigate compliance in patients receiving growth hormone treatment. Two hundred patients completed a questionnaire designed to establish understanding about and compliance with treatment; 50% of our patients failed to comply with all aspects of their treatment. Failure to respond to treatment seems to be associated with poor compliance.     

Systemic to pulmonary arterial shunts in neonates

Background The major strategy for palliation of cyanotic lesions in neonates is the systemic to pulmonary arterial shunt. Methods Between May 1995, and December 2002, 48 consecutive neonates underwent systemic to pulmonary arterial shunts for cyanosis with reduced pulmonary blood flow. The mean age was 11.6 days (±SD 7.38) and the mean weight, 3.2kg (±SD 0.52). The babies were classified into three groups: Group I-Tetralogy-pulmonary Atresia (n=18), Group II-single Ventricle-Pulmonary atresia without (n=19) and with (n=5) isomerism, Group III-Pulmonary Atresia with Intact ventricular septum (n=6). Diagnosis was made by 2D echocardiography. Indication for cardiac catheterization was delineation of pulmonary anatomy/ductus laterality (n=4) or balloon atrial septostomy (n=4). The surgical procedure was a modified Blalock-Taussig shunt on the side of the situs. Post-operatively, no anti-coagulation or anti-platelet medication was employed. Results There was no mortality. Four cases required revision of the shunt in the immediate post-operative period for shunt thrombosis. The mean follow up was 17.54 months (±SD 8.36). In Group I, nine patients have undergone total correction with or without a conduit, while three required new arterial shunts for shunt/pulmonary artery stenosis. In Group II, nine patients have undergone bi-directional Glenn with atrial septectomy (n=2) and pulmonary artery plasty (n=4) and one patient underwent Fontan completion. In Group III, two patients underwent bi-directional Glenn and two had pulmonary valvotomy with/without right ventricular outflow tract widening. All the remaining babies are waiting for the second/final stage palliation or total correction. Conclusion Systemic to pulmonary arterial shunts in neonates is a gratifying and reasonably safe surgical procedure. Most babies become candidates for eventual univentricular/bi-ventricular repair.     

Therapeutic effectiveness and safety of outdated human red blood cells rejuvenated to improve oxygen transport function, frozen for about 1.5 years at 80 C, washed, and stored at 4 C for 24 hours prior to rapid infusion

After storage at 4 C for 20 to 28 days, red blood cells were biochemically modified to improve their oxygen transport function which had deteriorated during liquid storage. The solution used for rejuvenation contained pyruvate, inosine, glucose, phosphate, and adenine (PIGPA Solution B). The rejuvenated red blood cells were frozen with 40% W/V glycerol in a polyolefin plastic bag and were stored in the frozen state for about 1.5 years at −80 C. After thawing and washing the red blood cells were stored at 4 C in a sodium chloride- glucose-phosphate solution for 24 hours before transfusion. A pool of four to ten units was rapidly transfused to each of 14 elderly anemic recipients, 11 of whom had cardiopulmonary insufficiency. Recovery of the red blood cells after the freeze-thaw process was about 97 per cent, and after the freeze-thaw-wash process about 90 per cent. The 24− hour posttransfusion survival values were about 75 per cent, and the long-term survival values were about 85 days depending on the disease state of the recipient. The red blood cells had 1.5 times normal 2.3- DPG levels and a decreased affinity for oxygen at the time of transfusion and were able to delivery oxygen at high oxygen tension immediately after the rapid infusion of pools of from four to ten units through a 40-or 170-micron filter. Plasma hemoglobin levels were consistent with extravascular sequestration of nonviable red blood cells, and uric acid levels were not increased during the immediate 24− hour posttransfusion period.