Biomedical subjects

F Sherman

Publications and source records attributed to F Sherman.

At least 19 recordsLinked to original sources

PubMed1999

Water determination in drugs containing thiols.

A new rapid analytical method is applied for water determination in alpha-Mono-thioglycerol and Captopril tablets containing thiols, and therefore, not amenable for direct Karl Fischer titration. The method is based on the consecutive titration first of thiol by a novel reagent, and then of water by a conventional K. Fischer reagent in the same sample and cell with the electrometric 'dead-stop' location of the end point in both titrations. The new reagent consists of iodine, potassium iodide and sodium acetate in non-aqueous medium. Estimated repeatability and accuracy of both water and thiol determinations are satisfactory.

Captopril↗

PubMed1999

Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae.

N-terminal acetylation can occur cotranslationally on the initiator methionine residue or on the penultimate residue if the methionine is cleaved. We investigated the three N-terminal acetyltransferases (NATs), Ard1p/Nat1p, Nat3p and Mak3p. Ard1p and Mak3p are significantly related to each other by amino acid sequence, as is Nat3p, which was uncovered in this study using programming alignment procedures. Mutants deleted in any one of these NAT genes were viable, but some exhibited diminished mating efficiency and reduced growth at 37 degrees C, and on glycerol and NaCl-containing media. The three NATs had the following substrate specificities as determined in vivo by examining acetylation of 14 altered forms of iso-1-cytochrome c and 55 abundant normal proteins in each of the deleted strains: Ard1p/Nat1p, subclasses with Ser-, Ala-, Gly- and Thr-termini; Nat3p, Met-Glu- and Met-Asp- and a subclass of Met-Asn-termini; and Mak3p subclasses with Met-Ile- and Met-Leu-termini. In addition, a special subclass of substrates with Ser-Glu- Phe-, Ala-Glu-Phe- and Gly-Glu-Phe-termini required all three NATs for acetylation.

Acetylation↗

PubMed1999

Phenotypic reversal of the btn1 defects in yeast by chloroquine: a yeast model for Batten disease.

BTN1 of Saccharomyces cerevisiae encodes an ortholog of CLN3, the human Batten disease gene. We have reported previously that deletion of BTN1, btn1-Delta, resulted in a pH-dependent resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP). This phenotype was caused by btn1-Delta strains having an elevated ability to acidify growth medium through an elevated activity of the plasma membrane H(+)-ATPase, resulting from a decreased vacuolar pH during early growth. We have determined that growing btn1-Delta strains in the presence of chloroquine reverses the resistance to ANP, decreases the rate of medium acidification, decreases the activity of plasma membrane H(+)-ATPase, and elevates vacuolar pH. However, an additional effect of this phenotypic reversal is that activity of plasma membrane H(+)-ATPase is decreased further and vacuolar pH is increased further as btn1-Delta strains continue to grow. This phenotypic reversal of btn1-Delta can be considered for developing a therapy for Batten disease.

Chloroquine↗

PubMed1999

Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production.

The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)-sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a.a(3) both disappeared in the cyc1-Delta mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1-Delta mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1-Delta mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0.46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0.38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen-limiting conditions.

Antifungal Agents↗

PubMed1999

Studies of pH regulation by Btn1p, the yeast homolog of human Cln3p.

Although the gene responsible for Batten disease, CLN3, was positionally cloned in 1995, the function of Cln3p and the molecular basis of the disease still remain elusive. We previously reported that the yeast Saccharomyces cerevisiae contains a homolog to Cln3p, designated Btn1p, and that the human Cln3p complemented the pH-dependent resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1, 3-propanediol in btn1-Delta yeast mutants. We have determined that yeast lacking Btn1p have an elevated ability to acidify media during growth that correlates with an elevated plasma membrane ATPase activity. Btn1p may be involved in maintaining pH homeostasis of yeast cells.

Cell Membrane↗

PubMed1999

Investigation of Batten disease with the yeast Saccharomyces cerevisiae.

The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.

Cell Division↗

PubMed1999

Action of BTN1, the yeast orthologue of the gene mutated in Batten disease.

Neuronal ceroid-lipofuscinoses (NCL) are autosomal recessive disorders that form the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births, and with approximately 440,000 carriers in the United States. Disease progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills and premature death. The CLN3 gene, which is responsible for Batten disease, has been positionally cloned. The yeast gene, denoted BTN1, encodes a non-essential protein that is 39% identical and 59% similar to human CLN3. Strains lacking Btn1p, btn1-delta, are resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) in a pH-dependent manner. This phenotype was complemented by expression of human CLN3, demonstrating that yeast Btn1p and human CLN3 share the same function. Here, we report that btn1-delta yeast strains have an abnormally acidic vacuolar pH in the early phases of growth. Furthermore, DNA microarray analysis of BTN1 and btn1-delta strains revealed differential expression of two genes, with at least one, HSP30, involved in pH control. Because Btn1p is located in the vacuole, we suggest that Batten disease is caused by a defect in vacuolar (lysosomal) pH control. Our findings draw parallels between fundamental biological processes in yeast and previously observed characteristics of neurodegeneration in humans.

Cell Division↗

PubMed1999

Water determination in drugs containing ascorbic acid.

A new rapid analytical method was applied for water determination in tablets of vitamin C, Ce De Calcium Veterinary, and C-Tamin-500 containing ascorbic acid and therefore is not amenable for direct Karl Fischer (KF) titration. The method is based on the consecutive titration first of ascorbic acid by a novel reagent and then of water by a conventional KF reagent (KFR) in the same sample and cell with the electrometric "dead-stop" location of the end point in both titrations. The new reagent consists of iodine, potassium iodide, and sodium acetate in nonaqueous medium. Estimated repeatability and accuracy of both water and ascorbic acid determination are satisfactory.

Ascorbic Acid↗

PubMed1999

Appearance and properties of L-sorbose-utilizing mutants of Candida albicans obtained on a selective plate.

This is the first report that adaptive mutagenesis can arise by chromosomal nondisjunction, a phenomenon previously associated exclusively with DNA alterations. We previously uncovered a novel regulatory mechanism in Candida albicans in which the assimilation of an alternative sugar, l-sorbose, was determined by copy number of chromosome 5, such that monosomic strains utilized l-sorbose, whereas disomic strains did not. We present evidence that this formation of monosomy of chromosome 5, which is apparently a result of nondisjunction, appeared with increased frequencies after a selective condition was applied, i.e., by adaptive mutagenesis. The rate of formation of l-sorbose-utilizing mutants per viable cell per day ranged from 10(-6) at the initial time of detection to 10(-2) after 4 days of incubation on the selective plate.

Candida albicans↗

PubMed1999

Specific chromosome alterations in fluconazole-resistant mutants of Candida albicans.

The exposure of Candida albicans to fluconazole resulted in the nondisjunction of two specific chromosomes in 17 drug-resistant mutants, each obtained by an independent mutational event. The chromosomal changes occurred at high frequencies and were related to the duration of the drug exposure. The loss of one homologue of chromosome 4 occurred after incubation on a fluconazole medium for 7 days. A second change, the gain of one copy of chromosome 3, was observed after exposure for 35 or 40 days. We found that the mRNA levels of ERG11, CDR1, CDR2, and MDR1, the candidate fluconazole resistance genes, remained either the same or were diminished. The lack of overexpression of putative drug pumps or the drug target indicated that some other mechanism(s) may be operating. The fluconazole resistance phenotype, electrophoretic karyotypes, and transcript levels of mutants were stable after growth for 112 generations in the absence of fluconazole. This is the first report to demonstrate that resistance to fluconazole can be dependent on chromosomal nondisjunction. Furthermore, we suggest that a low-level resistance to fluconazole arising during the early stages of clinical treatment may occur by this mechanism. These results support our earlier hypothesis that changes in C. albicans chromosome number is a common means to control a resource of potentially beneficial genes that are related to important cellular functions.

Aneuploidy↗

PubMed1999

Toxicity of copper, cobalt, and nickel salts is dependent on histidine metabolism in the yeast Saccharomyces cerevisiae.

The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeast Saccharomyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium. Each of the his1-his7 mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His(+) strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His(-) strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H(+)-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni.

ATP-Binding Cassette Transporters↗

PubMed1998

Cyc2p is required for maintaining ionic stability and efficient cytochrome c import and mitochondrial function in Saccharomyces cerevisiae.

A previous study demonstrated that. Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein and that cyc2 mutants contained only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c. We report herein that deletion of the entire gene results in defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources. This defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a non-ionic compound. We suggest that Cyc2p serves to maintain the osmotic stability of mitochondria, and its defect is exacerbated by KCl.

Biological Transport↗

PubMed1998

Nip1p associates with 40 S ribosomes and the Prt1p subunit of eukaryotic initiation factor 3 and is required for efficient translation initiation.

Nip1p is an essential Saccharomyces cerevisiae protein that was identified in a screen for temperature conditional (ts) mutants exhibiting defects in nuclear transport. New results indicate that Nip1p has a primary role in translation initiation. Polysome profiles indicate that cells depleted of Nip1p and nip1-1 cells are defective in translation initiation, a conclusion that is supported by a reduced rate of protein synthesis in Nip1p-depleted cells. Nip1p cosediments with free 40 S ribosomal subunits and polysomal preinitiation complexes, but not with free or elongating 80 S ribosomes or 60 S subunits. Nip1p can be isolated in an about 670-kDa complex containing polyhistidine-tagged Prt1p, a subunit of translation initiation factor 3, by binding to Ni2+-NTA-agarose beads in a manner completely dependent on the tagged form of Prt1p. The nip1-1 ts growth defect was suppressed by the deletion of the ribosomal protein, RPL46. Also, nip1-1 mutant cells are hypersensitive to paromomycin. These results suggest that Nip1p is a subunit of eukaryotic initiation factor 3 required for efficient translation initiation.

Cell Division↗

PubMed1998

A yeast model for the study of Batten disease.

Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive. We previously cloned the Saccharomyces cerevisiae homologue to the human CLN3 gene, designated BTN1, which is not essential and whose product is 39% identical and 59% similar to Cln3p. We report that btn1-Delta deletion yeast strains are more resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (denoted ANP), a phenotype that is complemented in yeast by the human CLN3 gene. Furthermore, the severity of Batten disease in humans and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.

Chloramphenicol↗

PubMed1998

Monosomy of a specific chromosome determines L-sorbose utilization: a novel regulatory mechanism in Candida albicans.

We report the identification of the gene, SOU1, required for L-sorbose assimilation in Candida albicans. The level of the expression of SOU1 is determined by the copy number of chromosome III (also denoted chromosome 5), such that monosomic strains assimilate L-sorbose, whereas disomic strains do not, in spite of the fact that SOU1 is not on this chromosome. We suggest that C. albicans contains a resource of potentially beneficial genes that are activated by changes in chromosome number, and that this elaborate mechanism regulates the utilization of food supplies and possibly other important functions, thus representing a novel general means for regulating gene expression in microbes.

Alcohol Oxidoreductases↗

PubMed1998

Degradation of yeast cytochromes c dependent and independent on its physiological partners.

Altered iso-1- and iso-2-cytochromes c, with certain amino acid replacements, occur at diminished levels due to degradation in the yeast Saccharomyces cerevisiae. A subclass of the labile isocytochromes c are significantly protected from degradation by the presence of cytochromes a.a3 and c1, the physiological partners of cytochrome c. We have investigated the degradation that is dependent on physiological partners by examining the levels of iso-1-cytochrome c having all or most amino acid replacements at positions 6, 41, 52, and 78, in both rho+ strains and rho- strains, which lacks cytochrome a.a3. In addition, we have examined some of these replacements in strains also having the N52I replacement, which suppresses a variety of abnormal iso-1-cytochromes c, including those whose degradation is either dependent or independent on the physiological partners. Although some degree of preferential rho--dependent reductions was observed for iso-1-cytochromes c having replacements at each of the 6, 41, 52, and 78 sites, prominent effects of rho+/rho- ratios of approximately 100/0 to 30/0 were observed for iso-1-cytochromes c having replacements mainly at the 41, 52, and 78 sites, but not the G6 site. We suggest that prominent degradation dependent on physiological partners may be restricted to certain regions of the cytochrome c molecule. Furthermore, we suggest that the region of the largest confirmational difference between oxidized and reduced cytochrome c appears to be particularly protected by interactions with its physiological partners.

Amino Acid Sequence↗

PubMed1998

Maize contains a Lon protease gene that can partially complement a yeast pim1-deletion mutant.

We have identified a gene in maize that encodes a product belonging to the Lon protease family. In yeast and mammals, Lon-type proteases catalyze the ATP-dependent degradation of mitochondrial matrix proteins. The maize gene, which we have designated LON1, is predicted to encode a protein with a molecular mass of 97.7 kDa. Lon1p is more similar in sequence to bacterial Lon proteases than to the yeast and human mitochondrial Lon proteases. LON1 transcripts are present in shoots of 4-day-old etiolated maize seedlings, and transcript levels decrease when these seedlings are heat-shocked. LON1 transcripts are also present at comparable levels in leaves and roots of 2-week-old greenhouse-grown seedlings. In yeast, the mitochondrial Lon-type protease, Pim1p, has been implicated in mitochondrial protein turnover, the assembly of mitochondrial enzyme complexes, and mitochondrial DNA maintenance, and it is essential for respiratory function. We show that maize Lon1p can replace the Pim1p function in yeast for maintaining mitochondrial DNA integrity, but not in the assembly of cytochrome a x a3 complexes.

ATP-Dependent Proteases↗

PubMed1997

Differential ubiquitin-dependent degradation of the yeast apo-cytochrome c isozymes.

The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genes CYC1 and CYC7, respectively. The cytochromes c are synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome c heme lyase, which is encoded by CYC3. Apo-iso-2-cytochrome c but not apo-iso-1-cytochrome c was observed in cyc3(-) mutants. Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome c was due to its rapid degradation. We report herein that this degradation of apo-iso-1-cytochrome c is dependent on ubiquitination and on the action of the proteasome. Diminished degradation of apo-iso-1-cytochrome c was observed in pre2-2 and pre1-1 mutants having altered proteasome subunits; in ubc1, ubc4, and ubc5 strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin. In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c is ubiquitinated. Furthermore, the degradation of apo-iso-1-cytochrome c was diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation. We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochrome c is part of the regulatory process controlling the preferential expression of the iso-cytochromes c.

Amino Acid Sequence↗