摘要:

AbstractThe genetic characterization of pathogenic isolates of Leishmania was attempted by analysis of the molecular properties of kinetoplast DNA (kDNA) minicircles. Unit minicircle size is not conserved during speciation of Leishmania since the minicircles of strains and clones of L t major are smaller (700 bp) than those found in certain strains of L mexicana ssp (820 bp), L donovani (850 bp) or L t tropica (900 bp). Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA of Leishmania with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of species, strains, and clones of Leishmania into schizodemes. Southern blot hybridization experiments reveal that at high stringency overall minicircle sequence homology is conserved among clones and strains of one species (L t major) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species‐specific diagnostic test for the identification of unknown Leishmania isolates. The properties of kDNA from an L t tropica strain LRC‐L32 (a “recidiva” organism) are so diverged from those of L t major strains as to support the classification [22,23] of L t tropica and L t major as separate species of Leishmania rather than subspecies of L

ISSN:0730-2312    

DOI:10.1002/jcb.240240202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractWe have previously demonstrated the modification and processing of Escherichia coli prolipoprotein (Braun's) in vitro (Tokunaga M, Tokunaga H. Wu HC: Proc Natl Acad Sci USA 79:2255, 1982). Using this in vitro assay of prolipoprotein signal peptidase and globomycin selection, we have isolated and partially characterized an E coli mutant which contained a higher level of prolipoprotein signal peptidase activity. In contrast, the procoat protein signal peptidase activity was not increased in this mutant as compared to the wild‐type strain. Furthermore, E coli strains containing cloned procoat protein signal peptidase gene were found to contain elevated levels of procoat protein signal peptidase, but normal levels of prolipoprotein signal peptidase. These two signal peptidase activities were also found to exhibit different stabilities during storage at 4°C. Thus biochemical, immunological, and genetic evidence clearly indicate that prolipoprotein signal peptidase is distinct from procoat protein signal peptidase in E co

ISSN:0730-2312    

DOI:10.1002/jcb.240240203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractProteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage‐site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues (trypsinlike), Leu‐Ala, Leu‐Ser, or Tyr‐Ala bonds (chymotrysinlike) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B‐like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B‐like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide “leftovers,” such as the proinsulin C‐peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relationships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects in converting proteases per se represent interesting areas for study in the search for disturbances in the production of neuroend

ISSN:0730-2312    

DOI:10.1002/jcb.240240204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractVery low density lipoprotein (VLDL) is the major vehicle in the plasma which carries triacylglycerol synthesized in the liver to peripheral tissues for utilization. Estrogen‐induced chick parenchymal liver cells (hepatocytes) synthesize and secrete large amounts of VLDL. These cells, in a primary monolayer culture system developed in this laboratory, have been employed to study the operative and regulatory aspects of VLDL synthesis, assembly, and secretion. Some 10 min are required for the translation of the principle VLDL protein constituent, apolipoprotein B, and 30–35 min are required for the two newly translated chick VLDL apolipoproteins, apolipoprotein B and apolipoprotein II, to be secreted. Apolipoprotein B is synthesized on membrane‐bound polysomes as a contiguous polypeptide chain of 350K molecular weight (MW) and is not assembled posttranslationally from smaller‐peptide precursors. Translocation of puromycin‐discharged apolipoprotein B nascent chains into the endoplasmic reticulum lumen and their subsequent secretion are independent of both ongoing protein synthesis and the attachment of the nascent peptides to ribosomes. Apolipoprotein B nascent chains discharged by puromycin assemble with glycerolipid (mainly triacylglycerol) and are secreted as immunoprecipitable VLDL. Core oligosaccharides are added to the apolipoprotein B nascent chain co‐translationally in at least two stages, at molecular weights of ∼ 120K and ∼ 280K. Inhibition ofN‐linked glycosylation of apolipoprotein B with tunicamycin affects neither the assembly of glycerolipids into VLDL nor the secretion of the VLDL particle, indicating thataglyco‐apolipoprotein B can serve as a functional component for VLDL assembly and secretion. Active synthesis of the VLDL apolipoproteins is required, however, for glycerolipid assembly into VLDL and secretion from the hepatocyte. The differential kinetics with which newly synthesized apolipoproteins and glycerolipids are secreted as VLDL and the timing of the effects of protein‐synthesis inhibitors on their secretion indicate that VLDL constituents are assembled sequentially in the intact liver cell. The bulk of the VLDL triacylglycerol and some VLDL phosphoglyceride is introduced early in the secretory pathway proximal, yet subsequent to apopeptide synthesis, while a significant fraction of VLDL phosphoglyceride associates with the resulting triacylglycerol‐rich lipid‐protein complexes just prior to their secretion as mature VLDL. Within the context of current models for VLDL structure, the late assembly of phosphoglyceride into VLDL is taken to represent a surface maturation of

ISSN:0730-2312    

DOI:10.1002/jcb.240240205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractPreincubation of35S‐methionine‐labeled chloroplast extracts with ATP at 0°C potentiates the subsequent assembly of labeled large subunits into RuBPCase. This is correlated with the dissociation of newly synthesized large subunits from the 29S large subunit binding protein complex. These released large subunits then assemble into RuBPCase in a second, nucleotide‐stimulated reaction. The data demonstrate that the 29S complex can play an active role in the assembly of Ru

ISSN:0730-2312    

DOI:10.1002/jcb.240240206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractCultivation of Spirodela oligorrhiza (Kurtz) Hegelm on a sublethal dose of atrazine results in a higher linolenic to linoleic acid ratio in the thylakoid membrane lipids, less starch, more osmiophilic globules, and a reduced stroma lamellar system. Also, the grana become randomly oriented and contain more numerous and elongated lamellae. These alterations in the lipid composition and ultrastructure of the chloroplast resemble those previously observed in triazine‐resistant weed biotypes and in chloroplasts developed under low light. Thylakoid membranes from atrazine‐adapted plants revealed an additional high‐affinity binding constant for [14C]‐diuron but the number of diuron binding sites actually decreased by 20 times compared to controls. The 32,000‐dalton membrane protein of the chloroplast is synthesized actively, but its breakdown appears decreased compared to control plants. The adaptive reorganization of thylakoid components may be a compensatory mechanism for maintenance of a functional interaction of the proteins and lipids of the photosystem I

ISSN:0730-2312    

DOI:10.1002/jcb.240240207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractIn the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM‐9 human cultured lymphocytes. After a 24‐hr preincubation, phenformin induced a twofold increase in specific125I‐insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between125I‐insulin binding and energy metabolism in IM‐9 cells was investigated. The phenformin‐induced increase in insulin binding to IM‐9 cells was related to a time‐ and dose‐dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2‐deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM‐9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a l

ISSN:0730-2312    

DOI:10.1002/jcb.240240208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

摘要:

AbstractThe modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose‐linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose‐linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose‐linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose‐linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are con

ISSN:0730-2312    

DOI:10.1002/jcb.240240209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY

ISSN:0730-2312    

DOI:10.1002/jcb.240240201

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1984

数据来源: WILEY