1,25-双羟胆钙化醇及其受体含量检测的实验研究

 建立了一种改良的血清1,25-双羟胆钙化醇(1,25-Dihydroxycholecalciferol,DHCC)超微量放射受体检测(RRA)技术。完成了灵敏度、精密度、准确度、稳定性及特异性等技术指标。报告了我国健康青年血清DHCC正常值;检测了先天性佝偻病、青春期佝偻病病人及患肾性骨病奶牛等血清DHCC水平。 根据配体与受体相互结合的定量关系,建立了DHCCR(DHCC受体)检测技术。在游离与结合配基分离方面,除建立与比较了DCC(葡聚糖包埋的活性炭)及HAP(羟基磷灰石)方法外,还首次将IEF(等电聚焦电泳)应用于DHCCR分离技术。对佝偻病鸡小肠粘膜上皮细胞受体含量进行了检测并比较了鸡小肠、输卵管壳腺及肝组织DHCCR含量。     

Phagocytic cell molecules that bind the collagen-like region of C1q. Involvement in the C1q-mediated enhancement of phagocytosis

C1q binds to and elicits cellular responses by several cell types, including monocytes, macrophages, neutrophils, B cells, and fibroblasts. The cell-binding domain is located within the collagen-like pepsin-resistant region of the C1q molecule (C1q tails). An affinity matrix of C1q tails coupled to Sepharose was used to select C1q-binding proteins from detergent extracts of surface-iodinated human monocytes, polymorphonuclear leukocytes, and the U937 cells. The major radiolabeled polypeptide eluted specifically from the ligand affinity column had an apparent molecular mass (Mr) of 126,000. Minor iodinated components eluted from Sepharose-tails migrated with Mr of 216,000 and 55,000. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions no change in the migration of any of these polypeptide bands was detected. None of these polypeptides reacted with antibodies directed against the integrins alpha 5 beta 1 (fibronectin receptor) or alpha v beta 3 (vitronectin receptor), LFA-1, or to several other cell adhesion molecules. The Mr 126,000 band was found to contain more than one polypeptide. Lectin binding properties, susceptibility to glycosidases and proteases, and immunoreactivity with the monoclonal antibody L-10, indicated that CD43 (sialophorin/leukosialin) is a component of this band. However, further data show that a monoclonal antibody, generated by immunization with the isolated Clq-binding fractions, recognizes a cell surface sialoglycoprotein distinct from CD43 and inhibits the C1q-mediated enhancement of phagocytosis in monocytes. These latter observations provide the first definitive connection between a specific phagocytic cell surface protein and a known C1q-mediated function. While these proteins contain sialic acid, binding assays and functional assays using neuraminidase-treated cells demonstrate that the functional interaction between C1q and the cell surface is not via sialic acid. The data taken together indicate either that the functional C1q receptor on phagocytic cells is a multi-subunit complex or that multiple proteins can interact with the fragment of C1q containing the cell-binding domain, at least one of which is involved in the C1q-mediated enhancement of phagocytosis.     

Location-dependent variations in the material properties of the anterior cruciate ligament.

Our recent anterior drawer studies in human cadaveric knees [Guan and Butler, Adv. Bioengng 17, 5 (1990); Guan et al., Trans. orthop. Res. Soc. 16, 589 (1991)] have suggested that anterior bundles of the anterior cruciate ligament (ACL) develop higher load-related material properties than posterior bundles. This was confirmed when we reevaluated the axial failure data for these bundle-bone specimens from an earlier study [Butler et al., J. Biomechanics 19, 425-432 (1986)]. The purpose of this study was to determine, in a larger data set, if anteromedial and anterolateral bundles of the anterior cruciate ligament exhibit significantly larger load-related material properties than the posterior ligament bundles. Seven ACL-bone units from seven donors (the three tissues from the original study plus four new ones) were subdivided into three subunits, preserving the bone insertions. The subunits were failed in tension at a constant strain rate (100% s-1) and four material properties were compared within and between donors. The anterior bundles developed significantly larger moduli, maximum stresses, and strain energy densities to maximum stress than the posterior subunits. Moduli for the anterior vs posterior subunits averaged 284 MPa vs 155 MPa, maximum stresses averaged 38 MPa vs 15 MPa, and strain energy densities averaged 2.7 N m cc-1 vs 1.1 N m cc-1, respectively. No significant differences were found, however, among strains to maximum stress or between any of the other properties for the two anterior subunits. These results are important to the design of ligament replacements and suggest new experiments designed to distinguish in vivo force levels in these ACL bands, a possible reason for the material differences.     

Idiotypic and antiidiotypic T and B lymphocytes in myasthenia gravis.

The prevalence of Id and anti-Id T and B cells as measured by their reactivities with two human mAb, one antiacetylcholine receptor mAb and one anti-Id mAb, was studied in 38 patients with myasthenia gravis and in 27 healthy individuals. Id and anti-Id T cells were estimated by enumerating the numbers of cells secreting IFN-gamma in response to 10 pg/ml of the human mAb. T cell stimulation, measured as numbers of IFN-gamma-secreting cells that exceeded the mean + 2 SD of controls, was induced by the Id mAb in 78.9% of the patients and in 7.4% of the controls, whereas the anti-Id mAb-stimulated T cells in 55.3% of the patients and in 3.7% of the controls. The mean value of the Id and anti-Id-reactive T cells in the patients was 18.3/10(5) and 10.1/10(5) PBMC, respectively. B cells secreting IgM antibodies binding to the human mAb were increased in patients with myasthenia gravis compared to healthy controls. Seventy-five percent of the patients and 12% of the controls had B cells secreting IgM antibodies binding to the Id mAb, although 89% of the patients and 16% of the controls had B cells secreting IgM antibodies binding to the anti-Id mAb. The mean value of B cells secreting IgM antibodies binding to Id or anti-Id mAb in the patients were 7.4 cells/10(6) and 5.5 cells/10(6) PBMC, respectively. We conclude that Id and anti-Id T and B cells are present in myasthenia gravis. These methods allow a quantitative estimation of T and B cells with defined specificities and thus a way of mapping the repertoire of lymphocytes.     

Evidence against the role of alpha 1-adrenoceptor reserve in buffering the inhibitory effect of nifedipine on the contractility of canine vascular smooth muscle

The relationship between the postsynaptic alpha 1-adrenoceptor reserve and the sensitivity of vasoconstriction induced by alpha-adrenoceptor agonists to the dihydropyridine Ca2+ entry blocker nifedipine was investigated in isolated muscle strips of dog mesenteric artery (DMA) and saphenous vein (DSV). The amplitudes of the contractile responses of DMA induced by phenylephrine were the same as those in DSV in the presence and in the absence of extracellular Ca2+. The use of 3 x 10(-9) M phenoxybenzamine to irreversibly block the alpha 1-adrenoceptors revealed a marked difference in the size of the alpha 1-adrenoceptor reserve between DMA (40%) and DSV (7%). In spite of a larger receptor reserve, the contractile responses induced by phenylephrine in DMA were more sensitive to nifedipine compared with those in DSV. These results suggest that the postsynaptic alpha 1-adrenoceptor reserve in vascular smooth muscle, at least in DMA and DSV, does not play an important role in buffering the inhibitory effect of nifedipine on the contractile response to a full agonist of alpha 1-adrenoceptors. Other factors, such as the difference in the membrane depolarizing effect, the ability to utilize intracellular Ca2+ for contraction, and the possible existence of alpha 1-adrenoceptor subtypes, may contribute to the different inhibitory effects of nifedipine on these blood vessels.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

A new metabolic pathway from n-dodecane to α, ω-dodecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-dodecanedioic acid via ,-dodecanediol from n-dodecane using a mutant S76 of Candida tropicalis was studied.It was found that resting cells of S76 produce ,-dodecanediol from n-dodecane. This intermediate was identified by different analytical methods. With n-dodecanol as substrate the quantitative changes in the concentrations of ,-dodecanediol as well as other intermediates, e.g. monoacid, -hydroxy acid and ,-dioic acid produced by resting cells of S76 for different periods of time were determined. With ,-dodecanediol as the sole carbon source, quantitative changes of -hydroxy acid and ,-dioic acid produced by S76 were also recorded.The results confirm the existence of a new metabolic pathway via ,-diol in the course of ,-dioic acid formation from n-alkane in the mutant S76 of C. tropicalis.     

A radioimmunoassay method for the rapid detection of Candida antibodies in experimental systemic candidiasis

Rabbits were employed as experimental models to evaluate a solid-phase radioimmunoassay (RIA) method for the diagnosis of systemic candidiasis. Ten rabbits were inoculated subcutaneously to mimic superficial candidiasis and were found to produce no antibodies to Candida as determined by both immunodiffusion and RIA procedures. However, 94 per cent of 18 rabbits systemically infected by intravenous injection of Candida cells were observed to produce antibody as assessed by the RIA technique. These data encourage further tests with human sera and the continued development of this RIA procedure as a useful tool in the early serodiagnosis of systemic candidiasis.     

哈尔滨城市和乡村青年居民肠道菌群多样性研究

【目的】分析居住于哈尔滨城市和乡村的青年居民肠道菌群多样性的异同。【方法】采用PCR和DGGE技术相结合的方法对生活于哈尔滨城市和乡村的青年志愿者肠道菌群多样性进行研究。基于DGGE指纹图谱,分别使用聚类和PCA分析对志愿者肠道微生物相似性进行分析,使用Shannon-Weine多样性指数(H′)、丰度(S)和均匀度(EH)对志愿者肠道微生物多样性进行分析,对图谱中具有代表性的共性和特异性条带进行胶回收和克隆测序以分析志愿者肠道微生物组成。基于PCR技术在种水平上对城乡志愿者肠道内乳杆菌属和双歧杆菌属多样性进行定性分析。【结果】相似性分析显示,城乡青年居民间肠道微生物群落结构存在分开趋势,相似性小于城市或乡村青年居民内部;多样性分析显示,城乡青年居民肠道微生物多样性差异不显著;测序结果表明,城乡居民肠道微生物组成在门水平上相同,但是在种属水平上存在差异。PCR定性分析显示Lactobacillus plantarum、L.casei和L.salivarius在哈尔滨城乡青年居民肠道内检出率接近100%,Bifidobacterium longum和B.breve的检测率约90%,在哈尔滨城乡青年居民肠道内普遍存在;乳杆菌属和双歧杆菌属各细菌种在城乡居民肠道中的检出频率差异不显著。【结论】哈尔滨城市和乡村青年居民肠道微生物多样性差异不显著。