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971.
The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-alpha-factor gene encoding an alpha-factor precursor with a single alpha-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR downward arrowEAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-alpha-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >10(6)-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp. 相似文献
972.
D Brambilla S Leung J Lew J Todd S Herman M Cronin DE Shapiro J Bremer C Hanson GV Hillyer GD McSherry RS Sperling RW Coombs PS Reichelderfer 《Canadian Metallurgical Quarterly》1998,36(1):311-314
Use of a common set of human immunodeficiency virus type 1 (HIV-1) RNA standards eliminated differences among absolute HIV-1 RNA copy number estimates made with three commercially available assays. The relative changes in the viral RNA levels determined by the commercial assays were similar and were unaffected by the use of a common set of standards. 相似文献
973.
JC Barefoot BL Heitmann MJ Helms RB Williams RS Surwit IC Siegler 《Canadian Metallurgical Quarterly》1998,22(7):688-694
OBJECTIVE: To investigate the relationship of symptoms of depression to weight changes in healthy individuals of normal weight across a follow-up of over 20 y. PARTICIPANTS AND DESIGN: College students (3885 men and 841 women) were administered a self-report depression measure in the mid-1960s. Their baseline body mass index (BMI) was calculated from their college medical records. Participants were contacted by mail in the late 1980s and asked to report their current height and weight as well as their smoking and exercise habits. Another measure of depressive symptoms was obtained from 3560 individuals at follow-up. Multiple regression models were used to relate changes in weight to depression scores while controlling for background (gender, baseline BMI and the gender by BMI interaction) and behavioral (exercise and smoking) predictors. RESULTS: The relationship between depressive symptoms and body weight change took the form of an interaction with baseline BMI (P < 0.001). Those with high baseline depression scores gained less weight than their nondepressed counterparts if they were initially lean, but more if they were initially heavy. This trend was especially strong in those with high depression scores at both baseline and follow-up. CONCLUSIONS: The findings support the hypothesis that depression exaggerates pre-existing weight change tendencies. This pattern would not have been detected by an examination of main effects alone, illustrating the need to move toward more complicated interactive models in the study of psychological factors and weight. 相似文献
974.
RS Chaves P Herrero I Ordiz M Angeles del Brio F Moreno 《Canadian Metallurgical Quarterly》1997,198(1-2):165-169
The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted. 相似文献
975.
The publication of the complete genome sequence of Helicobacter pylori and the computer analysis of the genes it contains represents an enormous advance in H. pylori research. Besides providing an overview of H. pylori biology, the genome sequence will aid future research and radically alter the research process. In particular, it will enable a 'top down' approach whereby genes are investigated because of their similarity to genes of known function in other organisms. Other advances in biotechnology will allow all H. pylori genes to be studied simultaneously, proteins to be identified quickly, and potential drug targets to be evaluated efficiently. Progress in H. pylori research should now be rapid, and with the research interest and resources focused on it, H. pylori could become the paradigm for post-genomic research. 相似文献
976.
SD Boulware WV Tamborlane NJ Rennert N Gesundheit RS Sherwin 《Canadian Metallurgical Quarterly》1994,93(3):1131-1139
The actions of recombinant human insulin-like growth factor-I (rhIGF-I) and insulin were compared in 21 healthy young (24 +/- 1 yr) and 14 healthy middle-aged (48 +/- 2 yr) subjects during 3-h paired euglycemic clamp studies using one of three doses (rhIGF-I 0.2, 0.4, and 0.8 micrograms/kg.min and insulin 0.2, 0.4, and 0.8 mU/kg.min, doses chosen to produce equivalent increases in glucose uptake). In younger subjects, rhIGF-I infusions suppressed insulin by 19-33%, C-peptide by 47-59% and glucagon by 33-47% (all, P < 0.02). The suppression of C-peptide was less pronounced with insulin than with rhIGF-I (P < 0.007). The metabolic responses to rhIGF-I and insulin were remarkably similar: not only did both hormones increase glucose uptake and oxidation in a nearly identical fashion, but they also produced similar suppression of glucose production, free fatty acid levels, and fat oxidation rates. In contrast, rhIGF-I had a more pronounced amino acid-lowering effect than did insulin (P < 0.004). In middle-aged subjects, basal IGF-I levels were 44% lower (P < 0.0001) whereas basal insulin and C-peptide were 20-25% higher than in younger subjects. Age did not alter the response to rhIGF-I. However, insulin-induced stimulation of glucose uptake was blunted in older subjects (P = 0.05). Our data suggest that absolute IGF-I and relative insulin deficiency contribute to adverse metabolic changes seen in middle age. 相似文献
977.
C Sreekantaiah M Ladanyi E Rodriguez RS Chaganti 《Canadian Metallurgical Quarterly》1994,144(6):1121-1134
In recent years, significant progress has been made in identifying characteristic chromosomal rearrangements associated with several solid tumor types, notably sarcomas, a relatively rare subset of human cancer. Most sarcomas analyzed have been found to be characterized by recurrent chromosome translocations that are specific to histological types. We have reviewed published reports of chromosomal aberrations in benign and malignant soft tissue tumors and found an incidence of specific translocations in these neoplasms that ranged from 20% to 93% within histological tumor types. Identification of recurrent chromosomal abnormalities in benign tumors has resulted in a reappraisal of the general concept that benign tumors have a normal (diploid) chromosome constitution. The variety of recurrent changes present in the different tumor types attests to the cytogenetic diversity inherent in these tumors. The chromosomal rearrangements in each of the tumor types were unique and did not correspond to cancer-associated aberrations known from other solid or hematopoietic malignancies. Cytogenetics thus provides an essential adjunct to diagnostic surgical pathology in the case of malignant soft tissue tumors, which often present substantial diagnostic challenges. In addition, it represents another approach to determine the histogenetic origin of some tumors and identifies sites of gene deregulation for molecular analysis. Indeed, recent molecular analyses of several sarcoma-associated translocations have identified novel genes and novel mechanisms of their dysregulation. 相似文献
978.
Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and NADPH to support phototransduction and NADPH-requiring processes in this organelle. Glycolysis in isolated bovine rod outer segments produces 44.0 +/- 6.4 nmol of ATP/min/mg of protein or 5.7 mM ATP/min. This rate of ATP production is more than sufficient to maintain the basal rate of cGMP synthesis (0.86 mM cGMP/min) in the dark requiring 1.7 mM ATP/min. Following photoexcitation, the 4.5-fold increase in the turnover of cGMP requires an ATP synthesis rate of up to 7.7 mM ATP/min (Ames, A., Walseth, T. F., Heyman, R. A., Barad, M., Graeff, R. M., and Goldberg, N. D. (1986) J. Biol. Chem. 261, 13034-13042). Under these conditions the rate of ATP production by glycolysis as measured in isolated rod outer segments is not sufficient for the regeneration of cGMP. Additional energy is most likely provided by the phosphocreatine shuttle which transports high energy phosphate groups in the form of creatine phosphate from the rod inner segment to the rod outer segment for conversion to ATP. The hexose monophosphate pathway in bovine rod outer segments can produce up to 39.8 +/- 2.2 nmol of NADPH/min/mg of protein. This rate of NADPH production is sufficient to support both the reduction of retinal to retinol (1.2 +/- 0.2 nmol of NADPH/min/mg of protein) following the photobleaching of rhodopsin and glutathione reduction (1.1 +/- 0.1 nmol of NADPH/min/mg of protein) for the protection of rod outer segments from oxidative damage. These studies provide insight into the contribution of anaerobic glycolysis and the hexose monophosphate pathway in providing energy and nucleotides for phototransduction and other outer segment processes. 相似文献
979.
KG MacCarthy JS Blake KL Johnson MV Dahl RS Kalish 《Canadian Metallurgical Quarterly》1994,3(2):66-71
Resistance to dermatophyte infections has been shown to be mediated in part by T lymphocytes. The dermatophyte antigens recognized by human T lymphocytes and their degree of cross-reactivity were analyzed. Dermatophyte-responsive T-cell lines were generated by in vitro sensitization to crude fungal extracts obtained from Trichophyton rubrum, Trichophyton tonsurans, Microsporum canis and Epidermophyton floccosum. Proliferation was measured by incorporation of 3H-thymidine. The human T-cell lines responded to fungal extracts derived from these various dermatophyte species, demonstrating the recognition of cross-reactive antigens by human T cells. However, the T cells were dermatophyte-specific as they did not respond to herpes antigen, nor did herpes-specific T cells derived from the same donors respond to dermatophyte antigens. The mannose-rich glycoprotein fraction (mannan) isolated from T. rubrum was able to induce proliferation of T-cell lines generated by stimulation with various fungal extracts. Furthermore, a T-cell line generated by stimulation with mannan derived from T. rubrum proliferated in response to extracts from various fungal species, indicating that a major cross-reactive dermatophyte T-cell antigen was present in the mannose-rich glycoprotein fraction. 相似文献
980.
LM Helms JP Felix RM Bugianesi ML Garcia S Stevens RJ Leonard HG Knaus R Koch SG Wanner GJ Kaczorowski RS Slaughter 《Canadian Metallurgical Quarterly》1997,36(12):3737-3744
Voltage-gated potassium (K(V)) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte K(V) channels. The binding of monoiodotyrosinyl margatoxin ([125I]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte K(V) channels. These data were compared with [125I]MgTX binding to heterotetrameric K(V) channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [125I]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [125I]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Na+. K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [125I]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl K(V) channel inhibitors was essentially the same for inhibition of [125I]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking 86Rb+ efflux from the CHO/K(V)1.3 cells, except that alpha-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [125I]MgTX binding, the antibody profiles, and the effects of the peptidyl K(V) inhibitors all indicate that the [125I]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain. 相似文献