Search PubMed⌕ Search

Biomedical subjects

F Sherman

Publications and source records attributed to F Sherman.

At least 109 records · Page 6Linked to original sources
PubMed1990

Differential stability of two apo-isocytochromes c in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1-cytochrome c and iso-2-cytochrome c, encoded by the genes CYC1 and CYC7, respectively. The amino acid sequences of these two isozymes are approximately 80% identical. Cyc3- mutants lack both holocytochromes c, because of a deficiency of cytochrome c heme lyase, the enzyme catalyzing covalent attachment of the heme group to apocytochrome c. A deficiency of heme lyase also prevents import into mitochondria. Surprisingly, apo-iso-1-cytochrome c is absent in cyc3- strains, although apo-iso-2-cytochrome c is present at approximately the same level at which holo-iso-2-cytochrome c is found in CYC3+ strains. The lack of apo-iso-1-cytochrome c is not due to a deficiency of either transcription or translation, but to rapid degradation of the protein. Apocytochromes c encoded by composite cytochrome c genes composed of the central portion of iso-2-cytochrome c flanked by amino and carboxyl regions of iso-1-cytochrome c exhibit increased stability compared with apo-iso-1-cytochrome c. A region encompassing no more than four amino acid differences between iso-1- and iso-2-cytochromes c is sufficient to partially stabilize the protein. In contrast to what is observed in vivo with the apo forms, the holo forms of the composite isocytochromes c are even less stable to thermal denaturation than iso-1-cytochrome c or iso-2-cytochrome c. Either a small region of the sequence of apo-iso-1-cytochrome c is involved in degradation of the protein, or the corresponding region in apo-iso-2-cytochrome c is preventing degradation. The differential stability of the two isocytochromes c may be part of a regulatory process that increases the proportion of iso-2-cytochrome c under certain physiological conditions.

Alleles↗
PubMed1990

Isolation and characterization of omnipotent suppressors in the yeast Saccharomyces cerevisiae.

Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness. Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII). Suppressors belonging to the same locus had a wide range of different phenotypes. Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.

Alleles↗
PubMed1990

Chromosomal assignment of mutations by specific chromosome loss in the yeast Saccharomyces cerevisiae.

Yeast 2-microns plasmids were integrated near the centromere of a different chromosome in each of 16 cir0 mapping strains of Saccharomyces cerevisiae. The specific chromosomes containing the integrated 2-microns plasmid DNA were lost at a high frequency after crossing the cir0 strains to cir+ strains. A recessive mutation in a cir+ strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated 2-microns plasmid and the unmapped mutation on homologous chromosomes.

Centromere↗
PubMed1990

Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans.

At frequencies as high as 1.4%, the pathogenic yeast Candida albicans spontaneously gave rise to morphological mutants exhibiting more than 20 different types of abnormal colonies; approximately two-thirds of the mutants were stable, while the other one-third were unstable and produced mixtures of different colonial forms at very high rates. Abnormal electrophoretic karyotypes were observed for all of the 14 mutants that were examined, indicating that they were associated with different types of single and multiple gross chromosomal rearrangements. Because C. albicans is asexual and does not go through a meiotic cycle, we suggest that the high frequency of chromosomal rearrangements provides a means for genetic variation in this organism.

Candida albicans↗
PubMed1989

Expression and activity of a gene encoding rat cytochrome c in the yeast Saccharomyces cerevisiae.

A rat-processed pseudogene, which encodes normal rat cytochrome c, has been expressed in the yeast, Saccharomyces cerevisiae. The translated region of the chromosomal CYC1+ locus, which encodes yeast iso-1-cytochrome c, was replaced by the translated region of the gene encoding rat cytochrome c (CYC1-RAT), thus preserving the proper CYC1 transcription initiation and termination signals. Although the levels of transcription of the normal CYC1+ gene and the CYC1-RAT gene in yeast were equivalent, rat cytochrome c was produced at approx. 40% of the level of iso-1-cytochrome c. In addition, the specific activity in vivo was estimated to be approx. 60% that of the yeast iso-1-cytochrome c. N-terminal processing of indigenous rat cytochrome c, in which the N-terminal methionine residue is cleaved and the penultimate glycine residue is acetylated, also occurred in yeast. Methionine cleavage was complete, while acetylation proceeded to only 70% completion. Lys-72 was trimethylated to 66% completion in the rat cytochrome c produced in yeast. The near normal expression (40%) and specific activity (60%) in vivo indicates that the 40% difference in amino acid sequence is not critical for mitochondrial import, heme attachment and interactions with redox partners.

Amino Acid Sequence↗
PubMed1989

Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes.

We have identified and purified a novel cytokine, NK cell stimulatory factor (NKSF), from the cell-free supernatant fluid of the phorbol diester-induced EBV-transformed human B lymphoblastoid cell line RPMI 8866. NKSF activity is mostly associated to a 70-kD anionic glycoprotein. The purified 70-kD protein, isolated from an SDS-PAGE gel, yields upon reduction two small species of molecular masses of 40 and 35 kD, suggesting that this cytokine is a heterodimer. When added to human PBL, purified NKSF preparations induce IFN-gamma production and synergize with rIL-2 in this activity, augment the NK cell-mediated cytotoxicity of PBL preparations against both NK-sensitive and NK-resistant target cell lines, and enhance the mitogenic response of T cells to mitogenic lectins and phorbol diesters. The three activities remain associated through different purification steps resulting in a 9,200-fold purification, and purified NKSF mediates the three biological activities at concentrations in the range of 0.1-10 pM. These data strongly suggest that the same molecule mediates these three activities, although the presence of traces of contaminant peptides even in the most purified NKSF preparations does not allow us to exclude the possibility that distinct biologically active molecules have been co-purified. The absence of other known cytokines in the purified NKSF preparations, the unusual molecular conformation of NKSF, the high specific activity of the purified protein, and the spectrum of biological activities distinguish NKSF from other previously described cytokines.

Biological Factors↗
PubMed1989

Production of hematopoietic colony-stimulating factors by human natural killer cells.

We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.

Antigens, Differentiation↗
PubMed1989

Identification and characterization of genes and mutants for an N-terminal acetyltransferase from yeast.

A gene from Saccharomyces cerevisiae has been mapped, cloned, sequenced and shown to encode a catalytic subunit of an N-terminal acetyltransferase. Regions of this gene, NAT1, and the chloramphenicol acetyltransferase genes of bacteria have limited but significant homology. A nat1 null mutant is viable but exhibits a variety of phenotypes, including reduced acetyltransferase activity, derepression of a silent mating type locus (HML) and failure to enter G0. All these phenotypes are identical to those of a previously characterized mutant, ard1. NAT1 and ARD1 are distinct genes that encode proteins with no obvious similarity. Concomitant overexpression of both NAT1 and ARD1 in yeast causes a 20-fold increase in acetyltransferase activity in vitro, whereas overexpression of either NAT1 or ARD1 alone does not raise activity over basal levels. A functional iso-1-cytochrome c protein, which is N-terminally acetylated in a NAT1 strain, is not acetylated in an isogenic nat1 mutant. At least 20 other yeast proteins, including histone H2B, are not N-terminally acetylated in either nat1 or ard1 mutants. These results suggest that NAT1 and ARD1 proteins function together to catalyze the N-terminal acetylation of a subset of yeast proteins.

Acetyltransferases↗
PubMed1989

Deletions and replacements of omega loops in yeast iso-1-cytochrome c.

omega (omega)-loops are protein secondary structural elements having small distances between segment termini. It should be possible to delete or replace certain of these omega-loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae were investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different omega-loops were individually deleted, or in which one omega-loop was replaced with five different segments. Deletions encompassing amino acid positions 27-33 and 79-83 either prevented synthesis of the holoprotein, or produced highly labile iso-1-cytochromes c, whereas deletions encompassing positions 42-45 and 48-55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue positions 24-33) with same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso-1-cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.

Amino Acid Sequence↗
PubMed1989

Experimental ductus arteriosus: the relationships of atrial pressure, dilatation and flow with ANF secretion.

The design of the study was to determine whether an increased blood flow as seen in shunt lesions could serve as a stimulus for the secretion of atrial natriuretic factor (ANF). Since atrial pressure, flow, and dilatation are closely related, an experimental ductus arteriosus model was utilized, in which acute changes of flow are assumed not to dilate the left atrium. In six dogs, a Dacron graft was constructed between the main pulmonary artery and the innominate artery. Constricting and releasing the tape around the graft adjusted the amount of "ductal" shunting. The total pulmonary flow and the shunt flow were measured by electromagnetic-flow transducers around the aortic root and around the graft. Plasma ANF concentration was measured from both cardiac atria. The size of the left atrium was determined from echocardiographic measurements made from a short-axis view. The total pulmonary flow varied between 1.2 and 5.8 1/min. The highest measured ANF was 396 pg/ml, and this was from the left atrium when the pressure was 18 mmHg, the highest left atrial pressure recorded. The highest right atrial pressure (5 mmHg) also correlated with the highest right-atrial level of ANF (366 pg/ml). The right atrial pressure had a significant correlation with plasma ANF concentration (R = 0.43, p less than 0.05). Pulmonary flow and plasma ANF concentration did not correlate; neither did left atrial size and ANF levels in 16 flow states where the size was measured. In the absence of atrial dilatation there was minimal stimulus for ANF secretion. A transient increase of left atrial pressure, without a concomitant significant atrial dilatation, did not serve as a significant stimulus for ANF secretion.

Animals↗
PubMed1989

Dramatic thermostabilization of yeast iso-1-cytochrome c by an asparagine----isoleucine replacement at position 57.

Two Saccharomyces cerevisiae yeast mutants, cyc1-73 and cyc1-190, contain nonfunctional and presumably unstable forms of iso-1-cytochrome c due to Gly-34----Ser and His-38----Pro replacements, respectively. Second-site reversions that produced Asn-57----Ile replacements at least partially restored function, presumably by alleviating the instability of these two altered iso-1-cytochromes c. Introduction of the Ile-57 replacement by site-directed mutagenesis in an otherwise normal protein resulted in a 17 degrees C increase in the transition temperature (Tm), corresponding to over a 2-fold increase in the free energy change (delta G degrees) for thermal unfolding.

Amino Acid Sequence↗
PubMed1989

Transcription terminates near the poly(A) site in the CYC1 gene of the yeast Saccharomyces cerevisiae.

A 38-base-pair region required for normal CYC1 mRNA 3' end formation in Saccharomyces cerevisiae was shown to be necessary for the termination of transcription in vivo by examining the stability of CEN3 plasmids. CEN3 plasmids were stably maintained during vegetative growth, unless a GAL1 transcript impinged on the CEN3 region. Transcription from the GAL1 promoter was terminated, and plasmid stability was restored by the insertion of a fragment containing the 38-base-pair region of CYC1. In contrast, a similar fragment lacking the 38-base-pair region had no such stabilizing effect. Furthermore, CYC1 mRNA transcription terminated in a region less than 100 nucleotides downstream from the normal poly(A) site, thus establishing that CYC1 mRNA 3' end formation does not involve overly extended precursors as are observed in higher eukaryotes.

Base Sequence↗
PubMed1988

Replacements of lysine 32 in yeast cytochrome c. Effects on the binding and reactivity with physiological partners.

Lysine 32 has been previously implicated by chemical modification and modeling studies as a key component of the domain which controls recognition and binding of cytochrome c to its physiological partners, e.g. cytochrome b2, cytochrome c peroxidase, and cytochrome oxidase. In order to quantitate the importance of this residue, we have investigated the role of Lys-32 in the reactivity of cytochrome c in redox reactions in vitro and in vivo with protein partners by using a series of altered forms of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae in which Lys-32 is replaced by Leu-32, Gln-32, Trp-32, and Tyr-32. Leu-32 and Gln-32 represent substitutions which change charge without seriously affecting the steric bulk of the side chain or the stability of the protein. For the Leu-32- and Gln-32-altered proteins, steady state kinetic studies with cytochrome c peroxidase, cytochrome b2, and cytochrome oxidase showed that neither of the steady state kinetic parameters, Km nor Vmax, were substantially modified by mutation. Studies of single turnover kinetics with a small molecule (ascorbate) or within bound complexes with either cytochrome b5 or cytochrome c peroxidase demonstrated that redox kinetics are only slightly affected by these substitutions. NMR experiments demonstrated that the Gln-32-altered protein can still bind strongly to a physiological partner, cytochrome c peroxidase. Growth in lactate medium demonstrated that the activity in vivo compared with the normal value was reduced to only 85% with the Gln-32- and Leu-32-altered proteins and to 65% with the Trp-32- and Tyr-32-altered proteins. These findings suggest that the evolutionary invariance of Lys-32 reflects only small quantitative changes in the binding and reactivity of cytochrome c.

Alleles↗
PubMed1988

Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32.

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.

Cytochrome c Group↗
PubMed1988

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c.

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.

Apoproteins↗