Effect of link protein concentration on articular cartilage proteoglycan aggregation

Previous work has shown that alterations in proteoglycan aggregates are among the first changes detected with aging, disuse, and degeneration of articular cartilage, yet the cause or causes of these alterations remain unknown. To determine if differences in link protein concentration can explain alterations in the assembly, size, and stability of articular cartilage proteoglycan aggregates, we isolated proteoglycan monomer (aggrecan) and link protein from adult bovine articular cartilage and then assembled proteoglycan aggregates from aggrecan and 0.8% hyaluronan relative to aggrecan weight, in the presence of 0, 2, 4, 6, 8, 10, 15, and 20% concentrations of link protein relative to aggrecan weight. We determined the amount, sedimentation coefficient, and stability of the aggregates by analytical ultracentrifugation and measured their dimensions by electron microscopy with use of the monolayer technique. Increased aggregate size, as determined by ultracentrifugation, was directly correlated with an increased number of aggrecans per aggregate and with increased hyaluronan length, as determined by electron microscopy. The concentration of link protein significantly influenced aggregation: concentrations of 6–8% produced maximum aggregation, aggregate stability, and uniformity of aggrecan spacing; concentrations greater than 10% led to the formation of superaggregates (aggregates with sedimentation velocities greater than 100 S that may result from linking two or more hyaluronan filaments) but decreased aggregate stability; and concentrations of less than 4% link protein significantly decreased aggregation, the size and stability of aggregates, and the regularity of aggrecan spacing. The latter observations suggest that a decline in the concentration of link protein could decrease the organization and stability of the articular cartilage matrix.     

Biotransformation of caffeine, paraxanthine, theobromine and theophylline by cDNA-expressed human CYP1A2 and CYP2E1.

Six human cytochrome P450s expressed in HepG2 cells using vaccinia virus cDNA-directed expression, were used to study the biotransformation of caffeine and its metabolites. CYP1A2 alone was responsible for caffeine 3-demethylation and paraxanthine 7-demethylation; in addition, 1A2 catalysed virtually all reactions related to caffeine and its metabolites. The metabolic profile of caffeine biotransformation by CYP1A2 averaged 81.5% for paraxanthine, 10.8% for theobromine and 5.4% for theophylline formation. It remained quite uniform when caffeine concentrations were varied. The most striking finding was that CYP2E1 (the ethanol-inducible form) had major influences upon caffeine metabolism: in particular, it catalysed the formation of theophylline and theobromine from caffeine. Thus, the in vivo metabolite profiling of caffeine may reveal CYP2E1 activities in addition to the previously documented activities of CYP1A2, polymorphic N-acetyltransferase and xanthine oxidase.     

去纤酶对高血脂兔血小板功能、纤溶系统、血液流变学的影响

观察了食饵性高血脂对新西兰兔血小板功能、纤溶系统和血液流变学的影响以及去纤酶的调节作用,并动态观察了指标的变化.结果发现,高血脂能引起兔的血小板数增加,粘附力增强,血液粘滞性增加,红细胞流动变慢,呈现高凝和低纤溶状态,且主动脉形成明显粥样斑块.去纤酶在降低血脂的同时,可明显降低血小板的粘附力,降低血液粘滞性,呈现低凝和高纤溶状态,其主动脉粥样斑块亦明显减轻和缩小.提示去纤酶既能降低血脂又能降低血粘稠度,促进纤溶,起到抗凝及防止血栓形成,抑制动脉硬化的形成和发展.     

Enhancement of fibronectin synthesis and fibrillogenesis by BMP-4 in cultured rat osteoblast.

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin (Fn), which regulates the adhesion, differentiation, and function of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. In this study, the effect of BMP-4 on Fn fibrillogenesis in cultured rat osteoblasts was examined. BMP-4 enhanced Fn synthesis and extracellular Fn assembly in primary cultured osteoblasts. In addition, the extracellular assembly of Fn from exogenously applied soluble human Fn was also increased by BMP-4. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. The synthesis of both alpha5 and beta1 integrins was upregulated by BMP-4. Immunocytochemistry showed that the clustering of alpha5 and beta1 integrins was also increased by BMP-4. BMP-4 increased fibril formation of Fn and the adhesion of osteoblasts onto Fn matrix, which was inhibited by disintegrin triflavin and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. Phosphorylation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) was increased by BMP-4. Enhancement of extracellular Fn fibrillogenesis and the mRNA expression of beta1 integrin by BMP-4 were inhibited by ERK kinase (MEK) inhibitor PD98059. These results suggest that the enhancement of extracellular Fn fibrillogenesis by BMP-4 in cultured osteoblasts is related to the increase of the synthesis of Fn and clustering of alpha5 and beta1 integrins. ERK is involved in the signaling pathway of BMP-4 in regulating Fn fibrillogenesis in osteoblasts.     

人精子穿卵改良试验对不同生育状态男子生育力测定的价值

目的　本研究用一种经改良的人精子穿卵试验 (SPA)处理人精子 ,以探讨一种更简便、快速、经济 ,适合一般实验室要求的方法。方法　将精子体外获能液用磷酸氢二钠把pH值调理至 8.2± 0 .2 ;用普通隔水式恒温培养箱进行培养 ;对 75例不同生育状态男子生育力进行测定 (其中 2 5例有生育力男子 ;2 5例“原因不明”不育男子 ;及 2 5例输精管吻合术后 )。结果　 3种不同生育状态男子的SPA值分别是 (4 7.12± 9.92 ) % ;(2 1.73± 9.91) % ,及 (2 8.0 4± 9.91) %。前两者在统计学上有显著差异 ,(P<0 .0 1) ;后两者在统计学上无明显差异 (P >0 .0 5 )。结果表明经用改良的精子获能液并在普通恒温培养箱培养后精子的活率和活力都有较大的提高 ,这有利于精子的长期获能及提高精子的穿透能力。结论　本研究提示改良后的人精子穿卵试验处理精子确实更简便、快速、经济 ,适合一般实验室要求 ,便于推广。     

Lack of spontaneous activity of cutaneous spinal dorsal horn neurons in awake, drug-free, spinally transected cats

Extracellular single-unit activity was recorded from neurons with cutaneous input in the dorsal horn of the spinal cord (L4-L6) of awake, drug-free cats before (for several weeks) and after (day 1 through day 7) cord transection (T12). The spontaneous activity of the neurons was minimal or nonexistent in both recording conditions. The lack of spontaneous activity following spinal cord transection contrasts sharply with activity recorded in acute spinal-cord transected preparations in which high rates of spontaneous activity have been reported. This discrepancy may reflect an important difference between the chronic, awake, drug-free and acute preparations.