Biomedical subjects
Publications and source records attributed to D Gage.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
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An aerobic thermophile has been isolated from an alpha-pinene enrichment culture. The isolate, which was designated BR425, has been tentatively identified as Bacillus pallidus using 16S ribosomal RNA gene sequencing and organism morphology. Monophasic and biphasic incubations of BR425 cells with alpha-pinene, beta-pinene, and limonene yielded a number of oxidized monoterpene metabolites with carveol as a common metabolite. A pinene degradation pathway with carveol and carvone as central metabolic intermediates is suggested.
Several animal models have been developed for the mucopolysaccharidoses (MPSs), a group of lysosomal storage disorders caused by lysosomal hydrolase deficiencies that disrupt the catabolism of glycosaminoglycans (GAG). Among the MPS, the MPS-III (Sanfilippo) syndromes lacked an animal counterpart until recently. In this investigation of caprine MPS-IIID, the clinical, biochemical, morphological, and immunohistochemical studies revealed severe and mild phenotypes like those observed in human MPS III syndromes. Both forms of caprine MPS IIID result from a nonsense mutation and consequent deficiency of lysosomal N-acetylglucosamine 6-sulfatase (G6S) activity and are associated with tissue storage and urinary excretion of heparan sulfate (HS). Using special stains, immunohistochemistry, and electron microscopy, secondary lysosomes filled with GAG were identified in most tissues from affected goats. Primary neuronal accumulation of HS and the secondary storage of gangliosides were observed in the central nervous system (CNS) of these animals. In addition, morphological changes in the CNS such as neuritic expansions and other neuronal alterations that may have functional significance were also seen. The spectrum of lesions was greater in the severe form of caprine MPS IIID and included mild cartilaginous, bony, and corneal lesions. The more pronounced neurological deficits in the severe form were partly related to a greater extent of CNS dysmyelination. These findings demonstrate that caprine MPS IIID is a suitable animal model for the investigation of therapeutic strategies for MPS III syndromes.
1. The effects of ipsapirone, nefazodone, tiaspirone, BMS-20661, buspirone and gepirone on the hypothalamic-pituitary-adrenal (HPA) axis were studied. These drugs were selected because they have serontonin 1A (5-HT1A) receptor-binding capability and have the potential for therapeutic activity in the treatment of major affective or anxiety disorders or both. 2. Plasma corticosterone level was used as the end point for determining the effect of each drug on the HPA axis. Each drug increased the plasma corticosterone levels in a dose-dependent manner. The ED50 values were 0.8 mg/kg for BMS-20661, 3.5 mg/kg for gepirone, 3.9 mg/kg for buspirone, 5.3 mg/ kg for tiaspirone, 10.5 mg/kg for ipsapirone and 73.5 mg/kg for nefazodone. Ipsapirone and buspirone were more efficacious than the other four drugs. 3. The effect of a 10-mg/kg (35 mg/kg for nefazodone) test dose of each drug reached a peak between 30 min and 1 hr, and plasma corticosterone levels generally returned to control levels after 2 hr. 4. When the drugs were given 30 min before decapitation, in conjunction with a rotatory stress, BMS-20661 significantly inhibited the stress-induced rise, whereas ipsapirone and gepirone caused a significant increase in plasma corticosterone levels. However, when the drugs were given 2 hr before decapitation, nefazodone caused a significant decrease, whereas ipsapirone, BMS-20661 and gepirone produced significant increases in HPA axis activity. An 0800 hr dose of 0.1 mg/kg of dexamethasone suppressed the 1500 hr HPA activity by 73.1%. The 0.1-mg/kg dose of dexamethasone significantly reduced the drug-activated HPA axis activity of all of the drugs from their saline-control levels. The rank order, from least to greatest inhibitory effect, produced by this dexamethasone treatment on the drug-control levels was gepirone (-42.6%), tiaspirone (-48.9%), buspirone (-56.1%), nefazodone (-68.5%), insapirone (-70.0%), and BMS-20661 (-74.3%).
To establish a model system for the study of ganglioside metabolism of the human brain tumor, medulloblastoma, we have chemically characterized the gangliosides of the Daoy cell line. These cells contain a high concentration of gangliosides (143 +/- 13 nmol LBSA/10(8) cells). The major species have been structurally confirmed to be GM2 (65.9%), GM3 (13.0%), and GD1a (10.3%). Isolation of individual gangliosides homogeneous in both carbohydrate and ceramide moieties by reversed-phase HPLC and analysis by negative-ion fast atom bombardment collisionally activated dissociation tandem mass spectrometry have allowed us to unequivocally characterize ceramide structures. In the case of GM2, 10 major ceramide subspecies were identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18:1-C23:1, d18:1-C24:1, and d18:1-C24:0. Taken together with previous studies, these findings in buman medulloblastoma cells support the view that high expression and marked heterogeneity of ceramide structure are general characteristics of tumor gangliosides, molecules which are shed by the tumor cells and which are biologically active in vivo.
The effects of tandospirone, enciprazine, gepirone, buspirone (5-HT1A agents) and carvotroline (5-HT2) on hypothalamic-pituitary-adrenocortical activity (HPA) activity were studied. These drugs increased the plasma corticosterone levels in a dose-dependent manner. Their ED50 values were 3.8, 31.8, 3.1, 3.4 and 7.0 mg/kg, respectively. Drug effects peaked between 30 min and 1 h, and plasma corticosterone levels returned to control levels after 2 h. When the drugs were given in conjunction with a rotatory stress, gepirone and enciprazine increased and carvotroline decreased plasma corticosterone levels. Dexamethasone (0.1 mg/kg) pretreatment reduced drug-activated HPA axis activity.
Leishmania resistant to arsenicals and antimonials extrude arsenite. Previous results of arsenite uptake into plasma membrane-enriched vesicles suggested that the transported species is a thiol adduct of arsenite. In this paper, we demonstrate that promastigotes of arsenite-resistant Leishmania tarentolae have increased levels of intracellular thiols. High-pressure liquid chromatography of the total thiols showed that a single peak of material was elevated almost 40-fold. The major species in this peak was identified by matrix-assisted laser desorption/ionization mass spectrometry as N1,N8-bis-(glutathionyl)spermidine (trypanothione). The trypanothione adduct of arsenite was effectively transported by the As-thiol pump. No difference in pump activity was observed in wild type and mutants. A model for drug resistance is proposed in which Sb(V)/As(V)-containing compounds, including the antileishmanial drug Pentostam, are reduced intracellularly to Sb(III)/As(III), conjugated to trypanothione, and extruded by the As-thiol pump. The rate-limiting step in resistance is proposed to be formation of the metalloid-thiol pump substrates, so that increased synthesis of trypanothione produces resistance. Increased synthesis of the substrate rather than an increase in the number of pump molecules is a novel mechanism for drug resistance.
Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, GD2. GD2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of GD2 in vivo were then determined in an established murine model. GD2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus GD2-treated mice; p < 0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2 x 10(6) cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as GD2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.
Muropeptide composition of peptidoglycan isolated from isogenic vancomycin-resistant and sensitive Enterococcus faecium strains was analyzed by reverse-phase high-performance liquid chromatography combined with amino acid and fast atom bombardment mass spectrometric analyses. Peptidoglycan of the sensitive and resistant strains was the same and was composed of tri- and tetrapeptides stem peptide subunits with or without aspartate or asparagine substitutions on the epsilon-amino group of the lysine residue. Thus, the synthesis of lactate-terminating peptidoglycan precursors in vancomycin-resistant E. faecium did not affect the chemical composition of peptidoglycan.
The muropeptide compositions of isogenic vancomycin-resistant and -susceptible Enterococcus faecalis strains were analyzed by reverse-phase high-performance liquid chromatography combined with amino acid analysis and fast atom bombardment mass spectrometry. Peptidoglycan of the susceptible strain contained pentapeptides as stem peptides, whereas peptidoglycan of the isogenic resistant strain was composed of muropeptides with tetrapeptide stem peptides. Despite the synthesis of lactate-terminating peptidoglycan precursors, no lactate-containing muropeptides were detected in peptidoglycan of the resistant strain. These findings indicate that either lactate-terminating precursors are not incorporated into peptidoglycan of the resistant strain or that the lactate residues are removed from peptidoglycan during synthesis.
A highly homogeneously methicillin-resistant Staphylococcus aureus strain was grown in the presence of various concentrations of exogenous glycine. Increasing concentrations of glycine in the medium resulted in a decrease in methicillin resistance and the appearance of a heterogeneous resistance phenotype. Parallel to the gradual changes in resistance was an alteration in the muropeptide composition of peptidoglycan. Increasing concentrations of glycine in the medium resulted in peptidoglycan in which muropeptides with a D-alanyl-D-alanine terminus were replaced with D-alanyl-glycine-terminating muropeptides. The disappearance of D-alanyl-D-alanine-terminating muropeptides in peptidoglycan and the concomitant decrease in resistance indicate a central role for D-alanyl-D-alanine-terminating precursors in methicillin resistance.
A comparative evaluation of various biomaterials for their resistance to bacterial colonization and encrustation in infected urine is an important area in urological biomaterials research. This article describes an in vitro dynamic perfusion system that allows four reactors containing 24 1-in. catheter samples (6 per reactor) to be simultaneously perfused at a constant flow rate by synthetic urine. A common urease-producing urinary pathogen, Proteus mirabilis, was maintained at a level of 10(6) colony-forming units/mL for 7 days in the dynamic perfusion reactors. The pH and bacterial population were monitored every 24 h and the percentage of encrustation on latex and hydrogel-coated commercial catheter materials gave reproducible results in three different runs, 15.2 +/- 3.65% and 13.8 +/- 2.58%, respectively. A major issue of inlet clogging due to ascending bacteria or ammonia has been rectified using a dismountable inlet assembly. An incubator coupled with a cooling system allowed accurate temperature maintenance of 37 degrees C in all four reactors. Results from scanning electron microscopy of some latex samples are also presented.
A post 30,000 x g particulate fraction was isolated from rat heart. This mixed membrane fraction is enriched in a carnitine palmitoyltransferase which is sensitive to both malonyl-CoA and etomoxiryl-CoA at concentrations that inhibit the malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo/CPT-I) of intact mitochondria. Tritiated etomoxiryl-CoA labels two proteins with the same molecular weight as the labeled proteins from rat heart mitochondria. Malonyl-CoA-sensitive carnitine palmitoyltransferase in the particulate fraction is stable to freeze-thawing, and the activity is not latent. These data show that the carnitine palmitoyltransferase associated with this particle is CPTo/CPT-I. Positive Western blots were obtained, with the particle using anti-CPTi/CPT-II at a molecular weight identical with the CPT1/CPT-II purified from rat heart mitochondria. Catalytic activity was purified to near homogeneity in approximately 40% yield. The purified protein has a molecular weight identical with CPTi/CPT-II, it cross-reacts with antibody against CPTi/CPT-II, it is not inhibited by malonyl-CoA or etomoxiryl-CoA, and mass spectral analyses of the tryptic peptides give the same molecular masses as CPTi/CPT-II, and, when mixed with equal amounts of CPTi/CPT-II, one uniform spot is found by two-dimensional electrophoresis. These data indicate that the catalytic subunit of CPTo/CPT-I is the same as CPTi/CPT-II. The average inhibition of the CPT of frozen-thawed particles is 71% by 50 nM etomoxiryl-CoA and 62% by 50 nM malonyl-CoA. The inhibitor sensitivity, but not the catalytic activity, is lost by solubilization in 1% Triton X-114; removal of Triton X-114 using Extracti-Gel D restores etomoxiryl-CoA and malonyl-CoA sensitivity (both 50 nM) of CPT to an average of 77 and 48%, respectively. Consistent with previous reports, these results show that CPTo/CPT-I is NOT inactivated by detergents, rather detergents both desensitize it to malonyl-CoA and alter its Vmax. These data show the assumption that CPTo/CPT-I is inactivated by detergents is untenable.
The peptidoglycan of a Tn551 mutant of Staphylococcus aureus (RUSA208) selected for reduced methicillin resistance was analyzed by reversed-phase high pressure liquid chromatography and mass spectrometry. RUSA208 is a member of a cluster of Tn551 mutants located on fragment A of SmaI digests but is distinct from the femA and femB class of mutants. The peptidoglycan of RUSA208 contained normal parental muropeptides but in diminished amounts only. The major muropeptides of RUSA208 were new components eluting with somewhat longer retention times from the column. Amino acid analysis of these new muropeptides showed identical compositions to the corresponding peaks in the parental strain, but mass spectrometry revealed increased molecular weights by the following mass units: 1 (in monomers), 1 or 2 (in dimers), and 2 or 3 (in trimers). These observations suggest that in RUSA208 the mutational block may be in the amidation of the stem peptide glutamate residues, resulting in the replacement of isoglutamine with free glutamic acid in one or more of the cell wall stem peptides.
Ganglioside biosynthesis and subsequent shedding are a potential mechanism contributing to tumor cell escape from the host immune response. As a first step in identifying active molecular species, structural characterization and quantification of the purified individual cellular and shed gangliosides of YAC-1 murine lymphoma cells were undertaken. These studies uncovered three striking changes in ganglioside metabolism in cells passaged in vivo, compared with cells cultured in vitro. (i) Marked inhibition of GalNAcGM1b synthesis: GM1b was present in an equal proportion to its biosynthetic product GalNAcGM1b in vitro, but was present in a 6-fold higher concentration in vivo. (ii) Marked inhibition of NeuGc synthesis: NeuGc, present in vitro in an up to 7-fold higher concentration than its biosynthetic precursor NeuAc, was decreased in relative concentration in vivo (1:1). (iii) Selectivity of shedding: ganglioside shedding in vitro was generalized with respect to both carbohydrate structure and ceramide structure (mainly d18:1-C24:1 and d18:1-C16:0), while in vivo, there was selective shedding of gangliosides containing NeuGc and the shorter chain fatty acid. The reduced synthesis of NeuGc and of GalNAcGM1b in vivo, and the selective shedding of more polar ganglioside species, also in vivo, show that the extracellular environment can markedly affect cellular ganglioside metabolism.
It was shown that Tn551 inactivation of two chromosomal (so-called auxiliary) loci other than the mec gene result in a dramatic reduction of methicillin resistance and decreased cell wall turnover and autolytic capacity in a methicillin-resistant Staphylococcus aureus strain (de Jonge, B. L. M., de Lencastre, H., and Tomasz, A. (1990) J. Bacteriol. 173, 1105-1110). To understand the mechanistic basis of these phenomena we have examined the status of the autolytic enzymes and the muropeptide composition of peptidoglycan using reversed-phase high-performance liquid chromatography and mass spectral analyses. While no differences could be detected in the number of autolytic hydrolases, the mutants showed major changes in peptidoglycan composition. Nine prominent muropeptides of the parental strain each carrying a pentaglycyl substituent were missing from the cell wall of one group of mutants. The second mutant lacked four parental muropeptides which were composed of the unsubstituted disaccharide pentapeptide and its alanyl-tetraglycine derivative. The auxiliary genes are genetic determinants involved with the biosynthesis of peptidoglycan precursors, the presence of which in the cell wall may be needed for optimal cell wall turnover.