Membrane expression of platelet calpain

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.     

Parathyroid hormone resistance and B cell lymphopenia in propionic acidemia

The mechanisms of hypocalcemia, recurrent infections and hypogammaglobulinemia associated with metabolic decompensation of propionic acidemia due to propionyl-CoA carboxylase deficiency have not been defined. A 7-week-old infant with this disorder presented with severe hypocalcemia and B cell lymphopenia during an episode of metabolic acidosis and hyperammonemia. Hypocalcemia (1.1 mmoll ¹) was associated with elevated serum intact parathyroid hormone (122 ng 1 ¹), hyperphosphatemia, hypophosphaturia and hypercalcuria, indicating parathyroid hormone resistance. B cell lymphopenia (20 cells μl^-1) was associated with transient neutropenia, anemia and subsequent hypogammaglobulinemia (IgG < 294mgdl^-1, IgM < 8mgdl^-1, IgA < 8mgdl ¹), while T cells were normal. Parathyroid hormone resistance and B cell lymphopenia resolved following treatment with hemodialysis, diet and carnitine. These complications may be due to interference with parathyroid hormone renal tubular action and B cell maturation/proliferation by accumulated organic acids.     

Infant growth and aorta total lipid fatty acids

Abnormal fetal and infant growth have increasingly been correlated with adult onset cardiovascular disease. To date, there is little known about the lipid fatty acid profiles in infant cardiovascular tissue. Therefore, we analysed total lipid fatty acids from thoracic and abdominal aorta intima and media from 24 normally grown sudden infant death syndrome cases. Aorta from small for gestational age (n = 2), failure to thrive from birth (n = 3), and premature (n = 1) infants were also examined. Dihomo-gamma-linolenic acid (C20:3n-6) and oleic acid (C18:1n-9) concentrations were significantly lower in the thoracic than in the abdominal aorta. Similar dietary related differences were found in the subgroup (n = 15) of infants fed on formula milks. Both abdominal and thoracic intimal arachidonic (C20:4n-6) to dihomo-gamma-linolenic acid ratios were greater in the infants with retarded growth after birth than in their normally grown counterparts. Growth restriction in infancy might disrupt the normal accretion of vascular endothelial polyunsaturated fatty acids.     

肱骨干骨折后桡神经麻痹的系统评价

目的采用循证医学研究方法,系统评价肱骨干骨折后桡神经麻痹(radialnervepalsy,RNP)的不同处理方法,为临床治疗决策提供证据基础。方法采用PubMed、Datastar及CochraneDatabase等互联网公共搜索引擎作为检索工具,检索过去40年中发表的有关肱骨干骨折后桡神经麻痹的文献,再对入选文献的参考文献行手工检索,制定数据抽取问表,对入选文献进行数据抽取、汇总、归纳和荟萃分析。结果共检索到391篇原始文献,其中324篇得自电子检索,67篇得自手工检索。有35篇符合最终的入选标准,总计随访患者1045例。其中21篇文献共记录了发生于4517例肱骨干骨折中的532例桡神经麻痹,肱骨干骨折后桡神经麻痹的发生率为11.8%。肱骨干中1/5和中远1/5部位的桡神经麻痹发生率明显高于其他部位(P<0.05)。不同骨折类型中,横形和螺旋形骨折较斜形和粉碎性骨折更易于并发桡神经麻痹(P<0.0001)。肱骨干骨折后桡神经麻痹总的恢复率为88.1%,早期保守治疗的病例自发性恢复率为70.7%。观察等待组和早期手术探查组之间神经恢复的最终结果没有明显差别。结论对肱骨干骨折后桡神经麻痹采用早期保守治疗不会影响神经的最终恢复结果,可以避免许多不必要的手术。     

Comparison of techniques for HIV-1 RNA detection and quantitation in cervicovaginal secretions

PRINCIPLES: HIV-1 in female genital secretions has been measured using swabs, Sno Strips (Akorn, Inc., Buffalo Grove, IL), and cervicovaginal lavage (CVL), but little is known regarding the comparability of these collection techniques. METHODS: We compared HIV-1 RNA detection and quantity in specimens obtained from HIV-1-seropositive women in Kenya using three sample collection techniques and three storage techniques and evaluated reproducibility in samples collected 5 days apart. Specimens were stored in no medium, freezing medium, or TRI Reagent (Molecular Research Center, Cincinnati, OH) for 2 to 15 months. RESULTS: HIV-1 RNA assays were conducted on 640 specimens from 20 antiretroviral naive women. Storage in TRI Reagent significantly enhanced detection of genital HIV-1 and yielded significantly higher mean log10 RNA levels than specimens collected in either no or freezing medium. The prevalence of HIV-1 RNA detection in TRI Reagent ranged from 50% to 80% depending on collection method and was highest in cervical swabs. Mean log10 HIV-1 RNA levels were 3.1 log10 copies/cervical swab, 2.6 log10 copies/cervical Sno Strip, 2.5 log10 copies/vaginal swab, 2.4 log10 copies/vaginal Sno Strip, 2.9 log10 copies/ml for cervicovaginal lavage (CVL) cell pellet, and 2.1 log10 copies/ml in CVL supernatant. Comparing specimens from days 1 and 6, there was significant concordance of HIV-1 RNA detection and correlation of HIV-1 RNA levels for cervical swabs, vaginal swabs, vaginal Sno Strips, and CVL cell pellets (kappa, 0.5-0.9; r, 0.5-0.9), but not for cervical Sno Strips or CVL supernatants. CONCLUSIONS: Cervical or vaginal swab, vaginal Sno Strip, and CVL collection led to reproducible measurement of genital HIV-1 RNA, despite storage for several months and international transport. Collection using swabs was simpler than Sno Strips or cervicovaginal lavage, and yielded the highest prevalence of HIV-1 RNA detection and reproducibility.