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。     

Kinetic analysis of the activation of transducin by photoexcited rhodopsin. Influence of the lateral diffusion of transducin and competition of guanosine diphosphate and guanosine triphosphate for the nucleotide site.

The activation of transducin (T) by photoexcited rhodopsin (R*) is kinetically dissected within the framework of Michaelis-Menten enzymology, taking transducin as substrate of the enzyme R*. The light scattering "release" signal (Vuong, T.M., M. Chabre, and L. Stryer, 1984, Nature (Lond.). 311:659-661) was used to monitor the kinetics of transducin activation at 20 degrees C. In addition, the influence of nonuniform distributions of R* on these activation kinetics is also explored. Sinusoidal patterns of R* were created with interference fringes from two crossed laser beams. Two characteristic times were extracted from the Michaelis-Menten analysis: t(form), the diffusion-related time needed to form the enzyme-substrate R*-transducin is 0.25 +/- 0.1 ms, and T(cat), the time taken by R* to perform the chemistry of catalysis on transducin is 1.2 +/- 0.2 ms, in the absence of added guanosine diphosphate (GDP) and at saturating levels of guanosine triphosphate (GTP). With t(form) being but 20% of the total activation time t(form) + t(cat), transducin activation by R* is not limited by lateral diffusion. This is further borne out by the observation that uniform and sinusoidal patterns of R* elicited release signals of indistinguishable kinetics. When (GDP) = (GTP) = 500 microM, t(cat) is lengthened twofold. As the in vivo GDP and GTP levels are comparable, the exchange of nucleotides may well be the rate-limiting process.     

Alcohol and aldehyde dehydrogenase genotypes and alcoholism in Chinese men.

The liver enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which are responsible for the oxidative metabolism of ethanol, are polymorphic in humans. An allele encoding an inactive form of the mitochondrial ALDH2 is known to reduce the likelihood of alcoholism in Japanese. We hypothesized that the polymorphisms of both ALDH and ADH modify the predisposition to development of alcoholism. Therefore, we determined the genotypes of the ADH2, ADH3, and ALDH2 loci of alcoholic and nonalcoholic Chinese men living in Taiwan, using leukocyte DNA amplified by the PCR and allele-specific oligonucleotides. The alcoholics had significantly lower frequencies of the ADH2*2, ADH3*1, and ALDH2*2 alleles than did the nonalcoholics, suggesting that genetic variation in both ADH and ALDH, by modulating the rate of metabolism of ethanol and acetaldehyde, influences drinking behavior and the risk of developing alcoholism.     

Adult-type rhabdomyoma of the palate. Cytologic presentation of two cases with histologic and immunologic study

The cytologic presentations of two adult-type rhabdomyomas of the soft palate are reported. Fine needle aspiration smears of the lesions contained globular tumor cells that had striations and intracytoplasmic rod-shaped inclusions. These inclusions, corresponding to hypertrophied Z bands, were pathognomonic of rhabdomyoma tissue. The cytologic diagnosis was confirmed by histologic and immunologic studies in both cases. The cytologic preparations fixed in alcohol or air dried did not show the "spiderweb cells" observed in the histologic sections of tissue fragments fixed in formaldehyde; this clearly illustrates that the morphology of the cells of this tumor depends primarily on the specimen preparation technique used. These cases suggest that aspiration cytology can establish the diagnosis of this benign tumor of striated muscle, for which total surgical removal is usually sufficient to effect a cure. The differential diagnosis is discussed in detail.     

Purification and characterization of two β-N-acetylhexosaminidases from the tobacco hornworm,Manduca sexta (L.) (Lepidoptera: Sphingidae)

β-N-Acetylhexosaminidases were detected in 10 insects including species of Lepidoptera, Coleoptera, Hemiptera, and Orthoptera. Two enzymes were purified from the tobacco hornworm, Manduca sexta (L.). EI was detected in larval and pharate pupal molting fluid, integument, and pupal hemolymph while EII was found in larval and pupal hemolymphs. They are acidic hydrolases with similar molecular weights (6.1 × 10⁴), molar extinction coefficients at 280 nm (1.9 × 10⁵ liters mol^?1 cm^?1), and pH optima (pH 6). They differ in the number of polypeptide chains per molecule (EI is a single chain and EII consists of two polypeptide chains), amino acid composition, extent of glycosylation (EII is probably a glycoprotein), isoelectric point (pI_EI = 5.9 and pI_EII ～- 5.1), tissue distribution, and reactivities toward nitrophenylated N-acetylglucosamine (k_cat,I = 328 s^?1 and k_cat,II = 103 s^?1) and N,N′-diacetylchitobiose (k_cat,I = 307 s^?1 and k_cat,II = 3 s^?1). These results suggest that EI is a chitinase and that EII may function as a hexosaminidase in vivo.     

Rotational dynamics of monoclonal anti-dansyl immunoglobulins

The rotational motions of monoclonal mouse anti-dansyl immunoglobulins were studied by nanosecond fluorescence emission anisotropic spectroscopy using a mode-locked argon-ion laser as the pulsed excitation source. Three homogeneous antibodies of the immunoglobulin Gl (IgGl) subclass containing different V regions were prepared. The fluorescence emission maxima of these antibodies (designated as DNS1, DNS2 and DNS3) are at 515, 480 and 500 nm, respectively. Their mean rotational correlation times, 〈φ〉, are 84, 109 and 96 ns, respectively. The binding of protein A or a monoclonal anti-allotype antibody to the F_c unit of DNS1 increased 〈φ〉 to 142 and 150 ns, respectively, whereas reduction of the disulfide bond between the heavy chains decreased 〈φ〉 to 48 ns. These nanosecond measurements show that the rotational motion of the F_ab arms in mouse IgGl is restricted.     

Metabolic and temporal studies on pancreatic exocrine cells in culture

Summary An approach is described whereby cells with definitive markers are followed from their source through dissociation and fractionation, then during long-term maintenance in vitro. Such sequential studies should enable investigators to define factors regulating proliferation and function of specific cells since ambiguity concerning identity is readily avoided.Pancreatic cells of guinea pigs were isolated by enzymic dissociation, and exocrine cells were enriched by centrifugation with solutions of serum albumin. Resulting populations consisting of up to 95% exocrine cells were then incubated with gyration to produce aggregates, and these were seeded to standard culture plates for further study. Colonial aggregates of exocrine epithelia develop in culture and can be maintained for 20–30 days. The cells exhibit changes with time that are qualitatively similar to those known to occur during serial cultivation of diploid fibroblastlike cells from human and other species. The uptake of tritiated thymidine decreases with maintenance time. Autoradiographic examination indicates that this is due to a reduction in the number of epithelial cells incorporating the isotope. Cell diameters increase from an average of 21 m at day 0 to 44 m by day 26, and a marked increase in heterogeneity of this parameter is also evident. Cellular DNA and protein accumulate during the same interval. Incorporation of tritiated leucine during 24-h exposures increases until about the 10th day in vitro and remains relatively constant for at least 2 weeks thereafter.The data are consistent with the hypothesis that exocrine pancreatic cells like other diploid cells in culture, progress to terminal differentiation under the culture conditions employed. The role of physical, nutritional, and humoral evironmental factors on this process will be the subject of future reports.Supported in part by National Cancer Institute Contracts NO1-CP-43231 and NO1-CP-65751