Structural and mechanistic studies on the HeLa and chicken liver proteins that catalyze glycinamide ribonucleotide synthesis and formylation and aminoimidazole ribonucleotide synthesis

Glycinamide ribonucleotide (GAR) transformylase from HeLa cells has been purified 200-fold to apparent homogeneity with a procedure using two affinity resins. The activities glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase were found to copurify with GAR transformylase. Glycinamide ribonucleotide synthetase and GAR transformylase were separable only after exposure to chymotrypsin. Antibodies raised to pure L1210 cell GAR transformylase were able to precipitate the glycinamide ribonucleotide transformylase and GAR synthetase activities from HeLa and L1210 cells both in their native and in their proteolytically shortened forms. The compound N-10-(bromoacetyl)-5,8-dideazafolate was found to inhibit formylation but to leave the ATP-requiring synthetase activities intact.     

Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge.

Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.     

Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template.

Antibody to the poliovirus genome-linked protein, VPg, specifically immunoprecipitated the product RNA synthesized in vitro by the poliovirus RNA polymerase and HeLa cell host factor when VPg-linked poliovirion RNA was used as a template. The largest product RNA that was immunoprecipitated was twice the size of the template RNA. The complete denaturation of the product RNA with CH3HgOH had no effect on the immunoprecipitation reaction. In contrast, CH3HgOH denaturation prevented the immunoprecipitation of the oligo(U)-primed product RNA. Immunoprecipitation of the product RNA synthesized in the host-factor-dependent reaction was prevented if VPg was removed from the template RNA by pretreatment with proteinase K or if an RNA template without VPg was used in the reaction. The results support our previous evidence that a covalent linkage exists between the labeled negative-strand product RNA and the VPg-linked template RNA and suggest that the purified polymerase and host factor initiated RNA synthesis in vitro in the absence of VPg or a VPg-precursor protein.     

Disulfides of the lutropin receptor

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.     

一例硬皮病人自发抗核仁抗体的免疫荧光及其与银染的比较研究

从硬皮病人血清中筛选出一例含有自发抗核仁抗体的血清,利用这个血清对核仁抗原的性质及其在细胞中随分裂周期不同产生的分布变化做了初步研究,并把结果与核仁嗜银蛋白做了比较。间接免疫荧光染色及细胞化学分析表明,这种核仁抗原的性质是蛋白质,其分布与嗜银蛋白相似,在间期,抗原呈颗粒状簇集在核仁中,而在分裂中期,抗原颗粒与染色体NORs部位接合,但有证据指出,这种抗原蛋白与核仁嗜银蛋白有所不同,同时还发现,经长时间秋水仙素处理诱导产生微核化的多核细胞中尽管微核的数目远多于细胞中NORs的数目,免疫荧光染色和银染都显示出每个微核中类核仁小体的存在。这说明(1)类核仁小体也是由核仁物质构成;(2)某些类核仁小体的产生可能与NORs无关。对这个现象的意义进行了讨论。     

Human thermoregulatory responses to cold air are altered by repeated cold water immersion

The effects of repeated cold water immersion on thermoregulatory responses to cold air were studied in seven males. A cold air stress test (CAST) was performed before and after completion of an acclimation program consisting of daily 90-min cold (18 degrees C) water immersion, repeated 5 times/wk for 5 consecutive wk. The CAST consisted of resting 30 min in a comfortable [24 degrees C, 30% relative humidity (rh)] environment followed by 90 min in cold (5 degrees C, 30% rh) air. Pre- and postacclimation, metabolism (M) increased (P less than 0.01) by 85% during the first 10 min of CAST and thereafter rose slowly. After acclimation, M was lower (P less than 0.02) at 10 min of CAST compared with before, but by 30 min M was the same. Therefore, shivering onset may have been delayed following acclimation. After acclimation, rectal temperature (Tre) was lower (P less than 0.01) before and during CAST, and the drop in Tre during CAST was greater (P less than 0.01) than before. Mean weighted skin temperature (Tsk) was lower (P less than 0.01) following acclimation than before, and acclimation resulted in a larger (P less than 0.02) Tre-to-Tsk gradient. Plasma norepinephrine increased during both CAST (P less than 0.002), but the increase was larger (P less than 0.004) following acclimation. These findings suggest that repeated cold water immersion stimulates development of true cold acclimation in humans as opposed to habituation. The cold acclimation produced appears to be of the insulative type.     

Kinetics of chitinase production. I. Chitin hydrolysis

As part of the development of a comprehensive mathematical model for chitinase production by Serratia marcescens QMB 1466 growing on chitin, the different mass transport and kinetic steps involved during chitin hydrolysis were studied. The experimental results for the hydrolysis of chitin by a crude preparation of chitinase show a system kinetically limited by the overall rate of chitin hydrolysis. This rate is linearly related to the concentration of enzyme adsorbed on the chitin particle. Adsorbed and bulk enzyme concentration were found to be related through a Langmuir type of isotherm.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

Surface changes in chimpanzee sperm during epididymal transit

Intact chimpanzee caput and cauda epididymal sperm, sperm cell lysates, and caput and cauda epididymal fluid were radiolabeled by enzymatic iodination with lactoperoxidase and Na125 I and were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Caput epididymal sperm showed nine labeled macromolecular components of 90, 64, 56, 48, 38, 31, 20, 18 and 16 Kd and cauda epididymal sperm showed eleven macromolecular components of 90, 64, 55, 47, 42, 33, 27, 18, 17, 15 and 11 Kd. Six of the components labeled on caput sperm (90, 64, 56, 48, 18 and 16 Kd) were detected in equal amounts of cauda sperm and two (38 and 20 Kd) were detected at greatly reduced labeling intensities. In the cauda epididymidis, four new components (33, 27, 17 and 11 Kd) became prominent features of the sperm surface. Analysis of labeled caput and cauda sperm cell lysates resolved components distinct from those detected on sperm surfaces. Electrophoresis of caput epididymal fluid showed five labeled components of 66, 56, 47, 41 and 37 Kd, while electrophoresis of cauda epididymal fluid showed eight labeled components of 92, 66, 56, 48, 31, 27, 24 and 11 Kd. Three components (66, 56 and 47 Kd) were present in both caput and cauda fluid, two (41 and 37 Kd) in caput fluid only, and five (92, 31, 27, 24 and 11 Kd) in cauda fluid only. Components of 37 Kd were labeled in caput fluid and on caput sperm but not on cauda sperm, whereas components of 27 Kd and 11 Kd were labeled in cauda fluid and on cauda sperm but not on caput sperm. These data show that chimpanzee sperm undergo extensive surface modifications during epididymal maturation and that some of these modifications may be related to exogenous proteins/glycoproteins in epididymal fluids.