Designed polyanionic coiled-coil proteins: acceleration of heparin cofactor II inhibition of thrombin

Novel polyanionic proteins were designed to increase the rate of heparin cofactor II (HC) inhibition of α-thrombin, an essential protease in the coagulation cascade. Two α-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue dimer containing 14 Glu residues (E14C), plus two 31-residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two-chain α-helical coiled coils at pH 7 and 25 °C. The control peptides were only partially α-helical. E14C remained folded at 90 °C but E8C was half unfolded at 49 °C. Coiled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fold, respectively, the rate of HC inhibition of α-thrombin. None of these compounds accelerated antithrombin inhibition of α-thrombin, and neither control peptide accelerated HC inhibition of α-thrombin. Acceleration of the HC inhibition of α-thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary complex. The results suggest that antithrombotic polyanionic α-helical coiled-coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biologcal activity. © Munksgaard 1995.     

乳腺钼靶片计算机光密度测定的临床应用研究

目的：利用计算机对乳腺钼靶片上病灶中心及病灶周围影像光密度的测定，来探索良、恶性乳腺病变之间以及各种病变病灶中心与病灶周围影像的密度差异：探讨病理基础和临床意义。材料和方法：随机选取１００例临床可扪及乳腺＂肿块＂的女性病人，进行乳腺钼靶Ｘ线检查，将检出病变的１０２张钼靶片进行计算机处理后，对病灶中心及病灶周围组织影像的光密度进行测定。所有病例除炎性病变外均取得病理结果。结果：１．良性病变的病灶中心密度与病灶周围密度无显著差异（Ｐ＞０．５），良性肿块（指纤维腺瘤和囊肿）的病灶中心与病灶周围密度间仅提示一种可能存在显著差异的趋势（０．１＞Ｐ＞０．０５）；２．乳腺癌病灶中心与病灶周围密度之间存在非常显著的差异（Ｐ＜０．００１）；３．良、恶性肿瘤病灶中心密度之间存在较显著差异（Ｐ＜０．０５）。结论：本研究的结果有助于乳腺良、恶性病变的鉴别诊断和提高早期乳腺癌的检出率。     

Binding of cardiolipin to polystyrene beads: evidence for a lamellar phase orientation

Summary. The association of cardiolipin with polystyrene beads was studied using ³¹P-NMR and electron microscopy. In the presence and absence of fetal calf serum, cardiolipin appeared to bind to the polystyrene beads in lamellar phase as assessed by ³¹P-NMR imaging. Electron microscopic analysis revealed an even coating of phospholipid about the beads with extensive micelle binding. Cardiolipin-coated beads challenged with ACA-positive sera followed by immunogold indicated antibody bound to micelles associated with the bead. Studies conducted with ACA IgG purified from patient sera indicated that some ACA bound to CL beads in the absence of a source of ACA cofactor (i.e. gelatin-blocked beads), some ACA required β₂-GPI for binding (i.e. no binding in the presence of β₂-GPI-depleted plasma), whereas other ACA which showed negliglible binding with gelatin-blocked beads, showed enhanced binding in the presence of /?₂-GPI-depleted plasma. The data indicate that: (1) cardiolipin binds to polystyrene beads in lamellar phase, (2) ACA bind to phospholipid micelles bound directly to the polystyrene beads, and (3) ACA differ between individuals displaying varying phospholipid and phospholipid/cofactor substrate specificities.     

Molybdate repair of molybdopterin deficient mutants from Chlamydomonas reinhardtii

Summary The phenotypically wild strain I₃ of Chlamydomonas reinhardtii, carrying a cryptic mutation at the nit-6 locus, was distinguished from strains 21gr (cryptic mutant at nit-5) and 6145c (wild type) because of the ability of I₃ to grow on nitrate media containing 2mM tungstate.Molybdopterin-cofactor (MoCo) mutants 102 (double mutant at nit-5 and nit-6) and 104 (mutant at nit-4) grew on nitrate media supplemented with high concentrations of molybdate, although final cell densities were 40–60% lower and generation times 3.5 to six fold longer than for wild type. Under these conditions, nitrate reductase (NR) activity of the mutants, when measured either in situ or in vitro, was practically undetectable. The MoCo defective mutant 307 (nit-3) was not molybdate repairable. Although NR activity was not restored in vitro by molybdate in any of the MoCo^– mutant strains, their extracts had free NR-diaphorase subunits together with NR-subunits assembled into high molecular weight species.Our results indicate that: a) nit-4, nit-5 and nit-6 loci are responsible for molybdate processing in the cell; b) nit-3 may encode a component of the pterin moiety biosynthetic route; c) NR subunits can assemble in the presence of an inactive MoCo; d) high concentrations of molybdate can replace partially in vivo but not in vitro the function of nit-4 and the combined function(s) of the nit-5 and nit-6 gene products.     

The fertilizing ability of human epididymal sperm

Purpose: Membrane cofactor protein (MCP), CD46, whose primary function is to protect host cells from homologous complement, has been presumed to serve as a sperm adhesion molecule for oocytes. The purpose of this study was to clarify the relationship between the properties of MCP expressed on epididymal sperm and their fertilizing ability in a recently developed strategy for assisted reproduction. Methods: We collected ejaculated sperm from normal subjects and epididymal sperm from vasectomized subjects and patients with congenital absence of the vas deferens. Western blotting and cofactor activity assay were performed to investigated the structural and functional properties of MCP. Results: Epididymal spermatozoa which showed a reduced fertilizing ability tended to react poorly with antibodies against MCP and also showed low cofactor activity, indicating weak complement regulatory activity compared to that of ejaculated spermatozoa. Conclusions: MCP is sufficiently expressed in ejaculated sperm in men with a normally developed epididymis but is diminished in epididymal sperm from men with congenital or acquired obstruction of the vas deferens.     

乳腺钼靶摄影中钙化对乳腺疾病的诊断价值分析

目的：探讨乳腺摄影中各种钙化形式对乳腺疾病的诊断价值。方法：采用美国GE公司生产乳腺机800T进行轴位（CC位）、斜位（MLO位）摄影。结果：经手术或活检病理证实在乳腺钼靶摄影中所见钙化118倒，其中良性钙化72例，多表现为粗大、空泡、团块状。恶性钙化46例，多表现为细沙、针尖、小叉、小杆状。结论：钙化发生部位、钙化形态、数目、密度等对乳腺疾病正确诊断具有重要意义。     

血清肝素辅助因子II 活性与下肢动脉硬化闭塞症介入术后再狭窄相关

目的：观察血清肝素辅助因子II(heparin cofactor II,HC II)活性与下肢动脉硬化闭塞症介入术后再狭窄的 关系。方法：因股浅动脉闭塞性疾病而成功施行支架植入术的患者62例,根据患者血清HC II活性高低,将患者分为 两组：HC II活性≥100%组(n=40)及HC II活性<100%组(n=22)。收集患者相关临床资料,随访观察6个月,观察术后 支架内狭窄、闭塞情况。结果：两组患者基线资料比较差异无统计学意义(P>0.05)。随访至第6个月月末时,HC II活 性<100%组支架内再狭窄程度较HC II活性≥100%组严重(P<0.05)。HC II活性<100%组支架内再狭窄发生率较HC II活 性≥100%组高(P<0.05)。进一步对危险因素行多元回归分析,结果显示血清HC II活性升高是减少术后再狭窄发生的 独立因素(OR=0.982,P=0.048)。结论：血清HC II活性与下肢动脉硬化症介入术后再狭窄相关;HC II活性越低,术后 发生再狭窄的危险越大。     

钼靶X线检查在乳腺癌诊断中的应用价值

目的：探讨乳腺癌诊断中应用钼靶X线检查的价值,提高乳腺癌的早期诊断率。方法回顾性分析经手术病理确诊的66例乳腺癌患者的临床及钼靶X线检查影像资料。结果66例乳腺癌患者钼靶X线片大部分有较敏感的显影及直接征象和间接征象,均行乳腺癌根治术或改良根治术,钼靶诊断与术后病理诊断符合率为100％。结论钼靶X线检查可以大大提高乳腺癌的诊断准确率,对乳腺癌的早期明确诊断具有重要的意义,可作为早期乳腺癌诊断的首选方法。     

Diagnosis of inherited von Willebrand disease: comparison of two methodologies and analysis of the discrepancies

Diagnostics of von Willebrand disease (VWD) includes assessment of factor VIII (FVIII) coagulant activity, von Willebrand factor (VWF) antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), and more specific tests as multimeric and genetic analyses are necessary for the correct VWD classification. The ACL AcuStar? analyzer introduces chemiluminescence (CL) technology in detection of VWD with automated VWF:Ag and VWF:RCo assays. Compare VWF:Ag‐ELISA and VWF:RCo by aggregometry conventional assays with new CL VWF:Ag‐IL and VWF:RCo‐IL assays, investigate the ability to make accurate VWD diagnosis and concordance with multimeric and genetic analyses. 146 patients with congenital VWD (51 Type 1; 34 Type2A; 16 Type 2B; 31 Type 2M; 5 Type 2N; 9 Type 3) and 30 healthy normal subjects were included. A comparison was made between CL and conventional methods. Diagnostic evaluation included: VWF:RCo/VWF:Ag ratio, multimeric distribution (sodium dodecyl sulfate [SDS]‐agarose gel) of VWF and genetic analysis in 110 of 146 patients. CL and conventional methods revealed good correlation. Kappa test agreement diagnosis was >0.8. CL diagnostic sensitivity was 100% and specificity 97%. Multimeric and genetic analysis were of help in clarifying 13 discrepancies of diagnosis between methods, of which six discrepancies were explained by lack of conventional methods′ sensibility. CL methodology can detect VWD and discriminate between type 1, 3 and variant forms and offers an automated, faster, sensitive and less cumbersome method when compared to conventional assays, in particular VWF:RCo by aggregometry. In some cases, even with all phenotype and genetic analyses, discrepancies exist in the classification of VWD.     

Towards improved diagnosis of von Willebrand disease: Comparative evaluations of several automated von Willebrand factor antigen and activity assays

Introduction

von Willebrand disease (VWD) is reportedly the most common bleeding disorder and arises from deficiency and/or defects of von Willebrand factor (VWF). Laboratory diagnosis and typing has important management implications and requires a wide range of tests, including VWF activity and antigen, and involves differential identification of qualitative vs quantitative defects.

Methods

We have assessed several VWF antigen and activity assays (collagen binding [VWF:CB], ristocetin cofactor [VWF:RCo] and the new Siemens INNOVANCE assay [VWF:Ac], employing latex particles and gain of function recombinant glycoprotein Ib to facilitate VWF binding and agglutination without need for ristocetin) using different instrumentation, including the new Sysmex CS-5100, with a large sample test set (n = 600). We included retrospective plus prospective study designs, and also evaluated desmopressin responsiveness plus differential sensitivity to high molecular weight VWF.

Results

VWF:Ag and VWF:RCo results from different methods were respectively largely comparable, although some notable differences were evident, including one high false normal VWF:Ag value (105 U/dL) on a type 3 VWD sample, possibly due to heterophile antibody interference in the latex-based CS-5100 methodology. VWF:Ac was largely comparable to VWF:RCo, but VWF:CB showed discrepant findings to both VWF:RCo and VWF:Ac with some patients, most notably patients with type 2M VWD.

Conclusions

(a) VWF:Ag on different platforms are largely interchangeable, as are VWF:RCo on different platforms, except for occasional (some potentially important) differences, and manufacturer recommended methods may otherwise require some assay optimization; (b) VWF:RCo and VWF:Ac are largely interchangeable, except for occasional differences that may also relate to assay design (differing optimizations); (c) VWF:CB provides an additional activity to supplement VWF:RCo or VWF:Ac activity assays, and is not interchangeable with either.