Search PubMed⌕ Search

Biomedical subjects

B J Bowman

Publications and source records attributed to B J Bowman.

At least 19 recordsLinked to original sources
PubMed2001

Crosslinking studies in gelatin capsules treated with formaldehyde and in capsules exposed to elevated temperature and humidity.

Incomplete in vitro capsule shell dissolution and subsequent drug release problems have recently received attention. A modified USP dissolution method was used to follow capsule shell dissolution, and a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay was used to follow loss of epsilon-amino groups to study this shell dissolution problem postulated to be due to gelatin crosslinking. The dissolution problems were simulated using hard gelatin capsule (HGC) shells previously treated with formaldehyde to crosslink the gelatin. These methods were also used to study the effect of uncrosslinked HGC stored under stressed conditions (37 degrees C and 81% RH) with or without the presence of soft gelatin capsule shells (SGC). A 120 ppm formaldehyde treatment reduced gelatin shell dissolution to 8% within 45 min in water at 37 degrees C. A 200 ppm treatment reduced gelatin epsilon-amino groups to 83% of the original uncrosslinked value. The results also support earlier reports of non-amino group crosslinking by formaldehyde in gelatin. Under stressed conditions, HGC stored alone showed little change over 21 weeks. However, by 12 to 14 weeks, the HGC exposed to SGC showed a 23% decrease in shell dissolution and an 8% decrease in the number of epsilon-amino groups. These effects on the stressed HGC are ascribed to a volatile agent from SGC shells, most likely formaldehyde, that crosslinked nearby HGC shells. This report also includes a summary of the literature on agents that reduce gelatin and capsule shell dissolution and the possible mechanisms of this not-so-simple problem.

Aldehydes↗
PubMed2001

Identification of the gene at the pmg locus, encoding system II, the general amino acid transporter in Neurospora crassa.

Mutations at the pmg locus in Neurospora crassa cause a deficiency in a transport system for a broad range of amino acids. We have isolated a gene that encodes a protein with a high degree of sequence similarity to the GAP1 general amino acid permease in Saccharomyces cerevisiae. Our data indicate that this is the gene at the pmg locus. It encodes a 572-residue protein with a molecular mass of 62,649 Da. The predicted secondary structure has 12 membrane-spanning regions, a feature characteristic of a superfamily of permease proteins. Inactivation of the gene yielded a mutant strain with the same phenotype as the pmg- strain, and a cosmid containing a functional copy of the gene rescued the pmg- strain. Although the pmg- strain has previously been assayed in a genetic background that contains mutations in genes for two other amino acid transport systems, we have found conditions in which the pmg- strain has an identifiable phenotype in an otherwise wild-type genetic background.

Amino Acid Sequence↗
PubMed2001

Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-atpases.

A series of naturally occurring compounds reported recently by multiple laboratories defines a new small-molecule class sharing a unique benzolactone enamide core structure and diverse biological actions, including inhibition of growth of tumor cells and oncogene-transformed cell lines. Here we show that representative members of this class, including salicylihalamide A, lobatamides A-F, and oximidines I and II inhibit mammalian vacuolar-type (H+)-ATPases (V-ATPases) with unprecedented selectivity. Data derived from the NCI 60-cell antitumor screen critically predicted the V-ATPase molecular target, while specific biochemical assays provided confirmation and further illumination. The compounds potently blocked representative V-ATPases from human kidney, liver, and osteoclastic giant-cell tumor of bone but were essentially inactive against V-ATPases of Neurospora crassa and Saccharomyces cerevisiae and other membrane ATPases. Essential regulation of pH in cytoplasmic, intraorganellar, and local extracellular spaces is provided by V-ATPases, which are ubiquitously distributed among eukaryotic cells and tissues. The most potent and selective V-ATPase inhibitors heretofore known were the bafilomycins and concanamycins, which do not discriminate between mammalian and nonmammalian V-ATPases. Numerous physiological processes are mediated by V-ATPases, and aberrant V-ATPase functions are implicated in many different human diseases. Previous efforts to develop therapeutic pharmacological modulators of V-ATPases have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, availability of the unique benzolactone enamide inhibitor class may enable further elucidation of functional and architectural features of mammalian versus nonmammalian V-ATPase isoforms and provide new opportunities for targeting V-ATPase-mediated processes implicated in diverse pathophysiological phenomena, including cancer.

Animals↗
PubMed2000

Characterization and in vitro release of methotrexate from gelatin/methotrexate conjugates formed using different preparation variables.

The purpose of this study was to evaluate effects of preparation variables on the composition of gelatin-methotrexate conjugates, and to evaluate their in vitro stability. Conjugation variables of pH, amount of conjugating agent 1-ethyl-3-(diaminopropyl)carbodiimide HCl (EDC), and methotrexate (MTX), with unfractionated gelatin were examined. Conjugate composition was determined spectrophotometrically. The molar ratios of MTX to gelatin in the conjugates ranged from 5.9 to 64.9. Molar ratios increased with molecular weight (MW) of gelatin in the conjugate, but the weight ratio was constant. This common conjugating procedure, however, produces by-product crosslinking and produces a mix of covalent MTX binding to carboxyl and amino groups of the gelatin. For release studies, gelatin was fractionated by size exclusion spectra (SEC) into MW of 21, 91, and 195 kDa prior to conjugation. MTX release from conjugates in dialysis cassettes at 25, 37, and 50 degrees C, in isotonic pH 7.4, buffer over 72 h was assayed by high performance liquid chromatography (HPLC). There was no effect of gelatin MW on MTX release. MTX release was approximately linear and attained 2.3, 7.2, and 13% by 72 h at 25, 37, and 50 degrees C, respectively, for the 91 kDa conjugates. First-order release rate constants were 0.23 x 10(-3), 0.95 x 10(-3), and 1.8 x 10(-3) x h(-1), respectively. The calculated activation energy for MTX release was 15.8 kcal/mol. Rate constants and the activation energy for MTX release are consistent with hydrolysis of a peptide bond. Non-degraded MTX species were found in the release medium at amounts similar to free MTX and were attributed to MTX polymers and MTX/gelatin fragments < 10 kDa.

Antimetabolites, Antineoplastic↗
PubMed2000

Disruption of vma-1, the gene encoding the catalytic subunit of the vacuolar H(+)-ATPase, causes severe morphological changes in Neurospora crassa.

By using the process of Repeat-induced Point mutation (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874), we inactivated vma-1, the gene encoding subunit A of the V-ATPase of Neurospora crassa. Two vma-1 mutant strains were characterized. One was mutated at multiple sites, did not make a protein product, and produced spores that only rarely germinated. The other had four point mutations, made a protein product, and produced viable spores. Neither strain had detectable V-ATPase activity. The vma-1 mutant strains did not grow in medium buffered to pH 7.0 or above or in medium supplemented with the cation Zn(2+). They were completely resistant to inhibition by concanamycin C, supporting our hypothesis that the V-ATPase is the in vivo target of this antibiotic. Inactivation of the vma-1 gene had a pronounced effect on morphology and development of the organism. In the mutants tip growth was inhibited, and multiple branching was induced. The vma-1 mutant strains could not differentiate conidia or perithecia. They could grow slowly as mycelia and could donate nuclei in a sexual cross. A mutation in the plasma membrane ATPase, which suppressed the sensitivity of wild type N. crassa to concanamycin, also proved effective in suppressing the sensitivity of a vma-1 null mutant to basic pH but did not correct the morphological defects.

Crosses, Genetic↗
PubMed2000

Hex-1, a gene unique to filamentous fungi, encodes the major protein of the Woronin body and functions as a plug for septal pores.

We have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or Woronin bodies, of Neurospora crassa. Analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. When grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. The HEX-1 protein was predicted to be 19,125 Da. Analysis of the N-terminus of the purified protein indicated that 16 residues are cleaved, yielding a protein of 17,377 Da. A polyclonal antibody raised to the HEX-1 protein recognized multiple forms of the protein, apparently dimers and tetramers that were resistant to solubilization by sodium dodecyl sulfate and reducing reagents. Treatment of the protein with phosphatase caused dissociation of these oligomers. Preparations enriched in Woronin bodies contained catalase activity, which was not detected in comparable fractions from the hex-1(-) mutant strain. These results support the hypothesis that the Woronin body is a specialized peroxisome that functions as a plug for septal pores.

Amino Acid Sequence↗
PubMed2000

Characterization and in vitro methotrexate release from methotrexate/gelatin conjugates of opposite conjugate bond polarity.

PURPOSE: Our laboratory has previously prepared gelatin/ methotrexate (MTX) conjugates containing mixed conjugation sites and by-product crosslinking, both of which may alter conjugate effectiveness. In this study, we prepared and evaluated gelatin/MTX conjugates having specific conjugate bond sites and minimal by-product crosslinking. METHODS: Opposite polarity conjugates were produced by coupling gelatin having blocked amino groups with MTX (G-MTX) and by coupling MTX having blocked amino groups with gelatin (M-GEL) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. Amino groups were blocked using citraconic anhydride and deblocked under acidic conditions. Gelatin and MTX contents were determined spectrophotometrically. The stability of each conjugate was determined by evaluating their in vitro release of MTX in isotonic buffer at pH 7.4 and 37 degrees C for 7 days. RESULTS: The G-MTX and M-GEL conjugates contained 21 and 1.2 mole MTX/mole gelatin and released 12 and 17% MTX by 7 days resulting in pseudo-first order release rate constants of 0.76x10(-3) and 1.0x10(-3) hr(-1), respectively. Alternate MTX species (< or =10%) were detected during the release study and were attributed to low molecular weight gelatin/MTX fragments and MTX polymers. CONCLUSIONS: Gelatin/MTX conjugates having opposite conjugate bond polarities and minimal by-product crosslinking have been produced and slowly released MTX by hydrolytic cleavage indicating good stability for future cell culture studies.

Antimetabolites, Antineoplastic↗
PubMed2000

Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A.

Vacuolar ATPases (V-ATPases) are large complex enzymes that are structural and mechanistic relatives of F(1)F(o)-ATPases. They hydrolyze ATP and pump protons across membranes to hyperpolarize membranes and, often, to acidify cellular compartments. The proton gradients generated are used to drive the movement of various compounds across membranes. V-ATPases are found in membranes of archaebacteria and some eubacteria, in various components of the endomembrane system of all eukaryotes and in the plasma membranes of many specialized eukaryotic cells. They have been implicated in a wide variety of cellular processes and are associated with several diseases. Bafilomycin and concanamycin, specific inhibitors of V-ATPases, have been instrumental in implicating the V-ATPase in many of these roles. To understand further the mechanism of inhibition by these antibiotics and the physiological role of the enzyme in the cell, we have isolated mutants of the filamentous fungus Neurospora crassa that are resistant to concanamycin. Concanamycin has a dramatic effect on hyphal morphology at acid pH and is lethal at basic pH. In the resistant mutants, the cells can germinate and grow, although abnormally, in basic medium. Thus far, none of the mutants we have characterized is mutated in a gene encoding a subunit of the V-ATPase. Instead, the largest class of mutants is mutated in the gene encoding the plasma-membrane H(+)-ATPase. Mutations in at least four uncharacterized genes can also confer resistance. Inactivation of the V-ATPase by disruption of vma-1, which encodes the catalytic subunit (A) of the enzyme, causes a much more severe phenotype than inhibition by concanamycin. A strain lacking vma-1 is seriously impaired in rate of growth, differentiation and capacity to produce viable spores. It is also completely resistant to concanamycin, indicating that the inhibitory effects of concanamycin in vivo are due to inhibition of the V-ATPase. How the multiplicity of ATPases within a cell is regulated and how their activity is integrated with other metabolic reactions is poorly understood. Mutant analysis should help unravel this puzzle.

Anti-Bacterial Agents↗
PubMed1999

The structure of the vacuolar ATPase in Neurospora crassa.

The filamentous fungus Neurospora crassa contains many small vacuoles. These organelles contain high concentrations of polyphosphates and basic amino acids, such as arginine and ornithine. Because of their size and density, the vacuoles can be separated from other organelles in the cell. The ATP-driven proton pump in the vacuolar membrane is a typical V-type ATPase. We examined the size and structure of this enzyme using radiation inactivation and electron microscopy. The vacuolar ATPase is a large and complex enzyme, which appears to contain at least thirteen different types of subunits. We have characterized the genes that encode eleven of these subunits. In this review, we discuss the possible function and structure of these subunits.

Amino Acid Sequence↗
PubMed1997

Mutations of pma-1, the gene encoding the plasma membrane H+-ATPase of Neurospora crassa, suppress inhibition of growth by concanamycin A, a specific inhibitor of vacuolar ATPases.

Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibited growth of Neurospora crassa in medium adjusted to pH 7 or above. Mutant strains were selected for growth on medium containing 1.0 microM CCA. Sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Analysis of V-ATPase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity showed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while the seven mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer. Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated the V-ATPase. Instead, we hypothesize that the mutations conferred resistance because the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.

Anti-Bacterial Agents↗
PubMed1997

Cultural pathways toward Antonovsky's sense of coherence.

This study empirically validated Antonovsky's suggestion that differing cultural pathways exist in the development of a Sense of Coherence Differences were found in the ways in which essentially identical levels of a Sense of Coherence were developed in Native Americans and Anglo-Americans. Native Americans' families appear to emphasize moral and religious values while Anglo-American families appear to emphasize achievement and independence.

Achievement↗
PubMed1997

The intriguing evolution of the "b" and "G" subunits in F-type and V-type ATPases: isolation of the vma-10 gene from Neurospora crassa.

We have characterized the vma-10 gene which encodes the G subunit of the vacuolar ATPase in Neurospora crassa. The gene is somewhat unusual in filamentous fungi because it contains five introns, comprising 71% of the region between the translation start and stop codons. The 5' untranslated region of the gene contains several elements that have been identified in other genes that encode subunits of the vacuolar ATPase in N. crassa. A comparison of G subunits from N. crassa, S. cerevisiae, and animal cells showed that the N-terminal half of the polypeptide shows the highest degree of sequence conservation. Most striking is the observation that this region could form an alpha helix in which all of the conserved residues are clustered on one face. Subunit G appears to be homologous to the b subunit found in F-type ATPases. The major difference between the b and G subunits is the lack of a membrane-spanning region in the G subunit. We have also identified homologous subunits in the operons which encode V-type ATPases in a eubacterium, Enterrococcus hirae, and an archaebacterium, Methanococcus jannaschii. As in eukaryotic vacuolar ATPases the G subunit homologs lack a membrane-spanning region. Although the b and G subunits appear to be derived from a common ancestor, significant changes have evolved. In F-type and V-type ATPases these subunits can have zero, one, or two membrane-spanning regions and can also differ significantly in the number of copies per enzyme.

Amino Acid Sequence↗
PubMed1996

Characterization of a vacuolar protease in Neurospora crassa and the use of gene RIPing to generate protease-deficient strains.

We have isolated a gene from Neurospora crassa that appears to encode a pepstatin-sensitive protease found both in membranes and in soluble contents of vacuoles. The gene contains two introns and encodes a 396-residue protein with a molecular mass of 42,900 Da. Because of the similarity of the protein to proteinase A in Saccharomyces cerevisiae the gene has been named pep-4. Strains with mutations in the pep-4 gene were generated in vivo by the gene RIPing procedure described by Selker and Garrett (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874). The mutant strains were deficient in pepstatin-sensitive protease activity and did not appear to produce a major 42-kDa polypeptide in the vacuole. The mutant strains grew at the same rate as the wild type and had no other observable phenotype. When compared with inactivation of the PEP4 gene of S. cerevisiae, inactivation of the pep-4 gene in N. crassa produced a phenotype that was different in several ways. In N. crassa the mutant strains did not exhibit reduced sporulation or reduced viability after nitrogen starvation, and they had elevated levels of proteinase B and carboxypeptidase activities. The pep-4 gene appears to encode the N. crassa, homolog of proteinase A, but the maturation of vacuolar hydrolases appeared to be less dependent on this protease than has been observed in S. cerevisiae.

Amino Acid Sequence↗
PubMed1996

Isolation of the vma-6 gene encoding a 41 kDa subunit of the Neurospora crassa vacuolar ATPase, and an adjoining gene encoding a ribosome-associated protein.

The vma-6 gene, encoding a membrane-associated subunit of the vacuolar H+-ATPase from Neurospora crassa, was cloned and sequenced. The gene contains three small introns and encodes a protein of 41 005 Da. When compared with homologous polypeptides from other species, the N. crassa protein contains a unique glycine-rich region. Three conserved cysteine residues, previously unrecognized, have been identified. An unrelated gene encoding a protein of 31 701 Da was found 2.1 kb downstream of vma-6. The second appears to encode the N. crassa homolog of a ribosome-associated protein identified previously in several plant and mammalian cells, and was named rap-1.

Adenosine Triphosphatases↗
PubMed1996

Cross-cultural validation of Antonovsky's Sense Of Coherence Scale.

This study empirically validated Antonovsky's suggestion that a Sense of Coherence is important in managing stress and remaining both physically and psychologically healthy. In both Anglo-American and Native American cultures, Sense of Coherence Measures were found to correlate negatively with measures of physical and mental distress. More interestingly, this study supported Antonovsky's prediction that people from various cultures may attain a similar level of a Sense of Coherence, despite great socioeconomic differences.

Adaptation, Psychological↗
PubMed1996

The vacuolar ATPase of Neurospora crassa is indispensable: inactivation of the vma-1 gene by repeat-induced point mutation.

To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.

Base Sequence↗
PubMed1995

Regulation of the expression of three housekeeping genes encoding subunits of the Neurospora crassa vacuolar ATPase.

The vacuolar ATPase is a complex enzyme and is encoded by at least nine genes, which appear to be scattered throughout the genome. We have examined the vma-1 vma-2, and vma-3 genes, which encode subunits present in multiple copies within the Neurospora crassa vacuolar ATPase. We wished to see if the expression of these genes is coordinately regulated and if these genes contain similar promoter elements. A region was sequenced of approximately 1 kb located upstream of the protein coding region for each gene. Several sequence elements were found in similar positions in each of the three genes. Each of the genes had several strong transcription initiation sites, clustered within 13-60 bp and located 112-193 bp upstream of the translation start site. The size and abundance of the RNA transcripts was also determined: the amount of RNA transcribed from each gene was roughly proportional to the numbers of each subunit present in the enzyme. A series of plasmids was constructed containing parts of the putative promoter region fused to beta-galactosidase. Analysis of these plasmids indicated that the essential region of the vma promoters lies within 370 bp of the protein coding region. Overall, the vma genes appear to have similar characteristics to "housekeeping" genes described in other organisms.

Adenosine Triphosphatases↗