Gonadotropin regulation of testosterone production by primary cultured theca and granulosa cells of Atlantic croaker: I. Novel role of CaMKs and interactions between calcium- and adenylyl cyclase-dependent pathways

Theca and granulosa cells for in vitro primary culture were obtained by enzymatic digestion of mature ovarian tissue from Atlantic croaker (Micropogonias undulatus) and separation from the other cell types by Percoll density-gradient centrifugation. Histochemical staining and treatment with pregnenolone confirmed the presence in the cultured cells of enzymes involved in synthesizing the major sex steroids in croaker ovaries: testosterone, estradiol, and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Croaker theca and granulosa cells maintained their steroidogenic response to gonadotropin when cultured with serum-supplemented media and produced high levels of testosterone for up to 5 days, although estradiol production was low. Multiple signal transduction pathways mediating gonadotropin stimulation of androgen production were identified in Atlantic croaker ovarian theca and granulosa cells in primary co-culture. Inhibitors of voltage-sensitive calcium channels (VSCCs) and calmodulin decreased the steroidogenic response to gonadotropin, whereas activators of adenylyl cyclase and protein kinase A (PKA) increased testosterone production, indicating that both calcium and PKA-dependent signaling pathways are involved in the regulation of follicular steroid production. In addition, the first evidence in vertebrates for an involvement of calcium/calmodulin-dependent protein kinases (CaMKs) in gonadal steroidogenesis was obtained, since the stimulatory effects of gonadotropin on testosterone media accumulation were attenuated by specific inhibitors of CaMKs. Some interactions among the signaling pathways were observed as demonstrated by the positive effect of elevated intracellular calcium on adenylyl cyclase activity and the reduction of forskolin- and dbcAMP-induced testosterone production by inhibitors of VSCCs, calmodulin, and CaMKs.     

Iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation

Iron overload (IO) is poorly investigated in the congenital haemolytic anaemias (CHAs), a heterogeneous group of rare inherited diseases encompassing abnormalities of the erythrocyte membrane and metabolism, and defects of the erythropoiesis. In this study we systematically evaluated routine iron parameters and cardiac and hepatic magnetic resonance imaging, together with erythropoietin, hepcidin, non-transferrin bound iron (NTBI), and cytokine serum levels in patients with different CHAs. We found that 40% of patients had a liver iron concentration (LIC) >4 mg Fe/g dry weight. Hepatic IO was associated with ferritin levels (P = 0·0025), transferrin saturation (TfSat, P = 0·002) and NTBI (P = 0·003). Moreover, ferritin >500 μg/l plus TfSat >60% was demonstrated as the best combination able to identify increased LIC, and TfSat alteration as more important in cases with discordant values. Possible confounding factors, such as transfusions, hepatic disease, metabolic syndrome and hereditary haemochromatosis-associated mutations, had negligible effects on IO. Erythropoietin and hepcidin levels were increased in CHAs compared with controls, correlating with LIC and ferritin, respectively. Regarding cytokines, γ-interferon (IFN-γ) was increased, and both interleukin 6 and IFN-γ levels positively correlated with ferritin and hepcidin levels. Overall, these findings suggest the existence of a vicious cycle between chronic haemolysis, inflammatory response and IO in CHAs.     

球囊扩张硫酸钙骨水泥椎体增强与后外侧植骨融合结合短节段经伤椎椎弓根钉固定治疗胸腰椎爆裂性骨折疗效比较

目的分析比较球囊扩张硫酸钙骨水泥(Calcium sulfate cement,CSC)椎体增强与后外侧植骨融合结合短节段经伤椎椎弓根钉固定治疗胸腰椎爆裂性骨折中的临床效果。方法选取本院2012年1月~2014年12月经临床确诊为胸腰椎爆裂性骨折的60例患者,随机分成两组,各30例。椎体增强组患者采用球囊扩张CSC椎体增强结合短节段经伤椎椎弓根钉内固定;植骨融合组应用后外侧植骨融合结合短节段经伤椎椎弓根钉内固定。分别记录两组患者术前、术后1周及末次随访时视觉模拟评分(VAS)、Oswestry功能障碍指数(ODI)、伤椎中央高度比、矢状面Cobb角等指标,并对结果进行比较和统计学分析。结果与术前相比,术后两组患者的VAS得分和ODI分值均显著降低,术后疼痛情况明显改善(0.01);术后1年,椎体增强组的中央高度比均明显高于植骨融合组且术后1年椎体增强组的矢状面Cobb角远远小于植骨融合组,提示术后椎体增强组疗效优于植骨融合组,矫正丢失更少,差异均存统计学意义(0.01),所有患者均未出现内固定失败。结论球囊扩张CSC椎体增强与后外侧植骨融合均可有效治疗胸腰椎爆裂性骨折,但CSC椎体增强早期可更好的恢复伤椎的高度,增加伤椎的稳定性和椎体强度,减少矫正度丢失,保留脊柱的节段运动功能。     

Differential effects of cannabis extracts and pure plant cannabinoids on hippocampal neurones and glia

We have shown previously that the plant cannabinoid cannabidiol (CBD) elevates intracellular calcium levels in both cultured hippocampal neurones and glia. Here, we investigated whether the main psychotropic constituent of cannabis, Δ⁹-tetrahydrocannabinol (THC) alone or in combination with other cannabis constituents can cause similar responses, and whether THC affects the responses induced by CBD. Our experiments were performed with 1 μM pure THC (pTHC), with 1 μM pure CBD (pCBD), with a high-THC, low CBD cannabis extract (eTHC), with a high-CBD, low THC cannabis extract (eCBD), with a mixture of eTHC and eCBD (THC:CBD = 1:1) or with corresponding ‘mock extracts’ that contained only pTHC and pCBD mixed in the same proportion as in eTHC, eCBD or the 1:1 mixture of eTHC and eCBD.     

椎体成形术并椎弓根钉内固定治疗胸腰椎骨折52例

目的探讨经伤椎椎弓根硫酸钙骨水泥椎体成形术并后路椎弓根钉内固定术治疗胸腰椎骨折的临床疗效、并发症及对患者生活质量的影响。方法选择52例胸腰椎骨折患者,采用经后路椎弓根钉内固定联合硫酸钙骨水泥椎体成形术治疗。并分别于手术前、术后1周、术后1年取钉时、取钉后1年统计椎体前、中、后缘的椎体高度、后凸畸形角（Cobb角）以及进行Oswestry功能障碍指数（Oswestry Disablity Index,ODI）分级评价。结果术后1周、取钉时和取钉后1年患者椎体高度均较术前明显增加,差异均有统计学意义（P〈0．05）;其Cobb角均较术前减小,差异均有统计学意义（P〈0．05）;术后各次随访结果之间比较,差异无统计学意义（P〉0．05）。患者术前ODI评价的优良率为5．77％,取内固定术后1年的ODI的优良率为98．08％,两者比较差异有统计学意义（P〈0．05）。结论经伤椎椎弓根骨水泥椎体成形术并后路椎弓根钉内固定治疗胸腰椎骨折有效避免了单纯椎弓根螺钉内固定的并发症,且长期疗效肯定,可有效提高患者的日常生活质量。     

肾上腺髓质素对豚鼠心室肌细胞L-型钙通道的作用及其信号转导机制

目的: 研究肾上腺髓质素( adrenomedullin, ADM)抑制豚鼠心室肌细胞L-型钙通道的信号转导机制。方法: 应用全细胞膜片钳技术,记录应用ADM(1-100 nmol·L^-1)前后L-型钙电流(I_Ca,L),以及分别记录应用ADM特异性受体拮抗剂ADM_22-52(100 nmol·L^-1)+ADM(100 nmol·L^-1)、蛋白激酶A (PKA) 特异性拮抗剂H-89(10 μmol·L^-1) + ADM(100 nmol·L^-1)、蛋白激酶C (PKC) 特异性拮抗剂PKC_19-36(10 μmol·L^-1)+ADM(100 nmol·L^-1)、PKC特异性激动剂PMA(1 μmol·L^-1)前后I_Ca,L。结果: ADM(1-100 nmol·L^-1)浓度依赖性地抑制豚鼠心室肌细胞I_Ca,L,并可被ADM_22-52(100 nmol·L^-1)完全阻断;H-89(10 μmol·L^-1)对ADM抑制I_Ca,L的作用无影响。PKC_19-36(10 μmol·L^-1)可完全阻断ADM 对I_Ca,L的抑制效应,且PMA(1 μmol·L^-1)可模拟ADM 对I_Ca,L的抑制效应。结论: ADM作用于特异性ADM受体可浓度依赖性地抑制豚鼠心室肌细胞I_Ca,L,此作用有可能与PKC激活相关。     

Comprehensive hereditary hemochromatosis genotyping

Hereditary hemochromatosis (HH) is an iron-overload disease common in populations of Northern European origin. Patients display increased iron absorption leading to excessive iron deposition and potential multiorgan failure. Using polymerase chain reaction sequence-specific primer (PCR-SSP) technology, we have developed an HH diagnosis assay capable of detecting 19 non-synonymous HFE mutations (including a previously unreported mutation, V295A) and several TFR2, SLC11A3 and H ferritin alleles implicated in HH. As part of the validation process, 159 UK renal donors were genotyped to determine HH allele frequencies in the UK population. The alleles nominally identified as HFE*01 (C282Y), HFE*02 (H63D) and HFE*03 (S65C) were found at frequencies of 0.085, 0.173 and 0.009, respectively. All other potential HH-associated alleles were absent, confirming their low prevalence in this population. This assay enables comprehensive routine HH genotyping, producing rapid, accurate and reproducible results at low cost.     

清开灵对大鼠谷氨酸神经毒性脑水肿时突触体游离钙的影响

目的 探讨清开灵注射液对谷氨酸(Glu)神经毒性脑水肿时突触体内游离钙(［Ca2+］i)的影响。方法 30只大鼠分成正常对照组、模型组、治疗组3组,右侧脑室注射Glu制作大鼠神经毒性脑水肿模型,4 h后处死大鼠测定其脑皮质含水量、钠、钾、钙和突触体［Ca2+］i含量。结果 模型组脑含水量(82.04±0.8)%,钠含量(244.4±29.3) mmol/kg,［Ca2+］i (327.9±33.2) nmol/L分别较正常对照组(79.93±0.59)%,(213.8±13.0) mmol/kg,(159.6±18.6) nmol/L显著升高(P<0.01,P<0.05,P<0.01),模型组钙(4.76±0.74) mmol/kg较正常对照组(5.21±0.81) mmol/kg显著降低(P<0.05)。治疗组脑含水量(80.13±0.72)%,钠含量(209.4±14.7) mmol/kg,［Ca2+］i (162.5±17.6) nmol/L较模型组显著降低(P均<0.01),钙(5.44±0.76 mmol/kg.干重)却增高(P<0.05)。结论 清开灵对大鼠谷氨酸神经毒性脑水肿有保护作用,其机制可能与拮抗突触体钙离子内流有关。