Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Does boron play only a structural role in the growing tissues of higher plants?

In species in which boron (B) mobility is limited, B deficiency only occurs in growing plant organs. As a consequence of the highly localized patterns of plant growth and the general immobility of B it has been extremely difficult to determine the primary function of B in plants. In species in which B is phloem mobile, the removal of B from the growth medium results in the depletion of B present in mature leaves. Thus, it is possible to develop mature leaves with increasingly severe levels of B depletion, thereby overcoming the complications of experiments based on growing tissues. Utilizing this approach we demonstrate here that B depletion of mature plum (Prunus salicina) leaves did not result in any discernible change in leaf appearance, membrane integrity or photosynthetic capacity even though B concentrations were reduced to 6-8 µg/g dwt, which is less than 30% of the reported tissue B requirement. Boron depletion, however, results in a severe disruption of plant growth and metabolism in young growing tissues. This experimental evidence and theoretical considerations suggest that the primary and possibly sole function of B, is as a structural component of growing tissues.     

Lysosomal tartrate sensitive acid phosphatase deficiency in cells which contain lysosomal "high uptake forms"

The catalytic and immunological properties of acid phosphatases (EC 3.1.3.2.) in different tissues were studied. It was demonstrated that high uptake forms of lysosomal enzymes like beta-galactosidase isolated from human platelets and bovine testis are mature enzymes, which have not lost their mannose-6phosphate marker. The results presented indicate that this phenomenon is related to a low activity or the complete absence of the lysosomal tartrate sensitive acid phosphatase activity in the tissues concerned.     

Phase I study of combination recombinant interleukin-2 and interferon gamma in patients with advanced malignancies

A phase I trial of interleukin-2 and interferon gamma combination treatment in patients with advanced malignancies was performed based on preclinical in vitro and in vivo data which demonstrated synergistic antitumor effect. The toxicities, immune parameters, and tumor responses are described. The clinical and biologic maximal tolerated doses were extrapolated from these data.     

染色体辐射效应的微机检测 Ⅱ断片率 微核率与细胞畸变率间的相关性检测

本文继前文后,按照设计的线性回归程序在“IBM—PC/XT”微型计算机上,进一步检测了断片率、微核率与细胞畸变率之间的相关性,肯定了微核测定法,断片测定法可以替代染色体畸变分析法。     

广东汉族健康人载脂蛋白AⅠ-CⅢl-AⅣ基因簇DNA多态性及其单倍型分布

本文研究了中国广东汉族健康人群apoAI-CⅢ-AIV基因簇DNA限制性内切酶PstI、SstI和EcoRI片段长度多态性。其中等位基因P_1,P_2,S_1,S_2,R_1和R_2的频率分别为0.98,0.02,0.96,0.04,0.90和0.10。经卡方检验符合Hardy-Weinbery氏遗传平衡,与其他种族比较,本文结果显示中国广东汉族人P_2等位基因频率低于日本人、亚洲印第安人和高加索人,S_2等位基因频率低于日本人、菲律宾人、沙特阿拉伯人和亚洲印第安人,而与高加索人相近,R_2等位基因频率稍高于高加索人。不同种族间apoAI-CⅢ-AIV基因簇DNA多态频率无疑存在差异,这种差异可能是由于遗传漂变和自然选择单独或联合作用所致。对P_1、P_2,S_1、S_2和R_1、R_2构成的单倍型和连锁平衡程度进行了分析,结果显示这些单倍型处于连锁不平衡状态。