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Biomedical subjects

R D Barber

Publications and source records attributed to R D Barber.

16 recordsLinked to original sources
PubMed2001

Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease.

Animals↗
PubMed2000

The role of histidines in the acetate kinase from Methanosarcina thermophila.

The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.

Acetate Kinase↗
PubMed2000

Isolation and in vitro differentiation of conditionally immortalized murine olfactory receptor neurons.

Two major challenges exist in our understanding of the olfactory system. One concerns the enormous combinatorial code underlying odorant discrimination by odorant receptors. The other relates to neurogenesis and neuronal development in the olfactory epithelium. To address these issues, continuous cell cultures containing olfactory receptor neurons (ORNs) were obtained from olfactory epithelia of H-2K(b)-tsA58 transgenic mice. ORNs were detected and characterized by immunocytochemistry, RT-PCR, and Western blot for the markers Galpha(olf), adenylyl cyclase III, the olfactory cyclic nucleotide-gated channel subunits, and olfactory marker protein. In culture, epidermal growth factor and nerve growth factor stimulated proliferation, and brain-derived neurotrophic factor and neurotrophin-3 induced cellular maturation. Clonal cell lines were isolated by fluorescence-activated cell sorting with anti-neural cell adhesion molecule antibodies, and of 144 single cells plated, 39 clones were expanded, propagated, and stored in liquid nitrogen. All attempts at recovery of clonal lines from frozen stocks have been successful. The most thoroughly characterized clone, 3NA12, expressed ORN markers and responded to stimulation by single odorants. Each odorant activated approximately 1% of cells in a clonal line, and this suggests that many different odorant receptors may be expressed by these clonal cells. Therefore, these cell lines and the method by which they have been obtained represent a significant advance in the generation of olfactory cell cultures and provide a system to investigate odorant coding and olfactory neurogenesis.

Animals↗
PubMed2000

Reconstructing smell.

Odorant signal transduction and neurogenesis are fundamental properties of the olfactory epithelium. Many preparations have been used to elucidate some of the mechanisms underlying these properties. In this article, we briefly review these research areas and describe some of the techniques used to obtain the data. We focus specifically on the cell-culture paradigm and the data obtained from various immortal cell lines in their attempts to reconstruct the olfactory epithelium in vitro.

Animals↗
PubMed1998

Pathways for transcriptional activation of a glutathione-dependent formaldehyde dehydrogenase gene.

The widespread occurrence of glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) suggests that this enzyme serves a conserved function in preventing the cytogenetic and potentially lethal interaction of formaldehyde with nucleic acids, proteins and other cell constituents. Despite this potential role of GSH-FDH, little is known about how its expression is regulated. Here, we identify metabolic and genetic signals that activate transcription of a GSH-FDH gene (adhI) in the bacterium Rhodobacter sphaeroides. Activity of the adhI promoter is increased by both exogenous formaldehyde and metabolic sources of this toxin. Elevated adhI promoter activity in DeltaGSH-FDH mutants implicates formaldehyde or the glutathione adduct that serves as a GSH-FDH substrate, S-hydroxymethylglutathione, as a transcriptional effector. From studying adhI expression in different host mutants, we find that the photosynthetic response regulator PrrA and the trans-acting spd-7 mutation increase function of this promoter. The behavior of a nested set of adhI::lacZ fusions indicates that activation by formaldehyde, PrrA and spd-7 requires only sequences 55 bp upstream of the start of transcription. A working model is presented to explain how GSH-FDH expression responds to formaldehyde and global signals generated from the reduced pyridine nucleotide produced by the activity of this enzyme.

Aldehyde Oxidoreductases↗
PubMed1998

Function of a glutathione-dependent formaldehyde dehydrogenase in Rhodobacter sphaeroides formaldehyde oxidation and assimilation.

Despite its reactivity with many biological molecules, formaldehyde can be commonly encountered by virtually all cells. The widespread existence of glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) in procaryotes and eucaryotes suggests this enzyme plays a central and universal role in biological formaldehyde oxidation. This work sought to determine the role of GSH-FDH in the facultative phototrophic bacterium Rhodobacter sphaeroides. Growth phenotypes of wild-type and mutant cells, changes in enzyme specific activities, and the pattern of 13C-labeled compounds detected by NMR spectroscopy cumulatively suggest that R. sphaeroides GSH-FDH can play a critical role in formaldehyde metabolism under both photosynthetic and aerobic respiratory conditions. In photosynthetic cells, the data indicate that GSH-FDH generates reducing power, in the form of NADH, and one-carbon skeletons that are oxidized to carbon dioxide for subsequent assimilation by the Calvin-Benson-Bassham cycle. For example, use of methanol as a sole photosynthetic carbon source increases the specific activities of GSH-FDH, an NAD-dependent formate dehydrogenase, and the key Calvin-Benson-Bassham cycle enzyme, ribulose-1,5-bisphosphate carboxylase. This role of GSH-FDH is also supported by the pattern of [13C]formaldehyde oxidation products that accumulate in photosynthetic cells and the inability of defined GSH-FDH or Calvin cycle mutants to use methanol as a sole carbon source. Our data also suggest that GSH-FDH acts in formaldehyde dissimilation when aerobic respiratory cultures cometabolize methanol and succinate.

Aerobiosis↗
PubMed1997

Potassium conductances and proliferation in conditionally immortalized renal glomerular mesangial cells from the H-2Kb-tsA58 transgenic mouse.

The effects of the temperature-sensitive, immortalizing Simian Virus 40 T antigen, tsA58, on whole-cell potassium conductances were assessed in renal glomerular mesangial cells from H-2Kb-tsA58 transgenic mice [1]. MTT cell viability assay data indicated that in permissive (33 degrees C, 50 U ml-1 gamma-interferon, IFN+) and non-permissive (37 degrees C, without gamma-interferon, IFN-) culture conditions the oncogene was active and inactive respectively. In IFN+ cells whole-cell currents were inhibited by 10 mM 4-aminopyridine, 1 mM ATP and glibenclamide (glyburide, IC50 = 0.4 microM) and stimulated by cromakalim (EC50 = 40 microM). Furthermore, increases in pipette free calcium activity stimulated the potassium conductance (EC50 = 0.5 microM). Apamin inhibited this conductance (IC50 = 9 nM). None of these effects were observed in IFN- cells. The potassium conductance in IFN- cells was activated by a hyposmotic shock and this was inhibited by Gd3. These data indicate that (1) conductances consistent with ATP-sensitive and small, calcium-activated potassium channels are found in IFN+ cells, (2) an osmotically-sensitive channel is found in IFN- cells and (3) channel expression is dependent upon the activation of tsA58.

4-Aminopyridine↗
PubMed1996

Insulin restores the calcium-activated chloride conductance in dedifferentiated renal glomerular mesangial cells from the H-2KB-TSA58 [corrected] transgenic mouse.

The effects of duration in culture were assessed in mesangial cells from the H-2Kb-tsA58 transgenic mouse using the whole-cell configuration of the patch clamp technique. The whole-cell potassium conductance remained constant in cells from passages 8-17 while the chloride conductance was found to decrease in cells after passage 15. This reduction of the chloride conductance, indicating cell dedifferentiation, was the result of complete loss of the calcium-activated component of the conductance and loss of part of the calcium-insensitive component. Insulin, which increases the calcium-sensitivity of the calcium-activated component of the chloride conductance in early passage cells, restored part of the calcium-sensitive component of the chloride conductance in dedifferentiated cells. Vasoactive agonists such as arginine vasopressin and angiotensin II stimulate a chloride-dependent depolarization in mesangial cells that initiates cell contraction. In view of the importance of the chloride flux it is possible that the loss of a significant magnitude of chloride conductance may underlie mesangial cell pathology to some degree. This is supported by data that show insulin can reverse many of the early renal changes observed in diabetic nephropathy and, as shown in this study, that insulin enhances the chloride conductance in dedifferentiated, cultured mesangial cells.

Animals↗
PubMed1996

Inhibition by P1075 and pinacidil of a calcium-independent chloride conductance in conditionally-immortal renal glomerular mesangial cells.

1. Depolarization of mesangial cells has been shown to occur following an outward movement of chloride ions from the cell. We have shown previously that mesangial cells from the H-2Kb-tsA58 transgenic mouse possess a significant whole-cell chloride conductance and consequently are a suitable preparation for the study of potential chloride channel inhibitors. 2. The effects on the whole-cell chloride conductance of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and the potassium channel openers, (KCOs) P1075 and pinacidil were investigated in mesangial cells from the H-2Kb-tsA58 transgenic mouse cultured in permissive conditions (at 33 degrees C in the presence of 50 u ml-1 murine gamma-interferon). 3. In symmetrical solutions of 140 mM tetramethylammonium chloride (TMAC1) the whole-cell chloride conductance was 1.08 +/- 0.05 nS (n = 63) and this could be reversibly inhibited by 5 x 10(-5) M NPPB. 4. Both P1075 and pinacidil inhibited the whole-cell chloride conductance. This inhibition was not reversible after drug washout and was demonstrated only when drugs were applied to the extracellular surface of the cells. Very low concentrations of the drugs were found to reduce the chloride conductance after 16 h incubation but under no circumstances studied was the conductance totally inhibited, leaving a mean residual current of 0.33 +/- 0.03 nS (n = 12). 5. The effects of different peptide calcium concentrations on the magnitude of the residual current in the presence of the drugs were investigated. The residual current was reduced with 10(-8) M calcium in the pipette and increased with 10(-3) M pipette calcium. Therefore, these data suggest that P1075 and pinacidil selectively inhibit a calcium-independent chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse.

Animals↗
PubMed1996

Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides.

Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes. During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c2 family of electron transport proteins (isocytochrome c2). Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH. Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of approximately40-kDa subunits, analogous to other GSH-FDH enzymes. These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R. sphaeroides extracts. In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c2 and AdhI activity. While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R. sphaeroides. Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes.

Alcohol Dehydrogenase↗
PubMed1995

A characterization of the chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse.

The chloride conductance of conditionally immortalized mesangial cells isolated from the H-2Kb-tsA58 transgenic mouse was studied in cells grown in permissive and non-permissive culture conditions. No differences were found in the magnitude of the chloride conductance in 140 mM tetramethyl ammonium chloride between cells grown in permissive and non-permissive culture conditions (1.08 +/- 0.05 nS and 1.02 +/- 0.05 nS). Selectivity sequences were defined as 1.8I- > 1.2Br- > lCl- and 1.3I- > 1.1Br- > lCl- respectively and both the anion channel inhibitors niflumic acid and ochratoxin A inhibited the chloride conductance in a dose-dependent manner. The chloride conductance was made up from calcium-dependent and calcium-independent components and in the presence of 1 microM free calcium in the pipette this could be increased by the presence of 100 U ml-1 insulin added to the incubation medium. This appeared to increase the sensitivity of the conductance to levels of intracellular calcium as no differences were observed in the presence of either 1 mM or 100 nM pipette calcium. These data indicate that there is a significant chloride conductance in mesangial cells from the H-2Kb-tsA58 transgenic mouse and minimal changes are observed with changes from permissive to non-permissive culture conditions.

Animals↗
PubMed1989

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector.

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.

Bacteroides↗
PubMedDate not supplied

Evidence for regular sporulation by Haplosporidium nelsoni (MSX) (Ascetospora; Haplosporidiidae) in spat of the American oyster, Crassostrea virginica.

The spore stage of Haplosporidium nelsoni, the ascetosporan parasite causing multinucleated sphere unknown (MSX) disease in oysters, Crassostrea virginica, has been reported so rarely (less than 0.01% of infected oysters) that a second host has been postulated. However, recent intensive sampling of young (less than 1 year) oysters in Delaware Bay, U.S. suggests that spore formation occurs regularly in this group and that spores are produced in at least 75-85% of all infections reaching the advanced stage. Sporulation was seasonal, occurring over two to three weeks in late June/early July and again in late summer/early fall. Our data indicate that sporulation by H. nelsoni in oysters is more common than previously suspected, occurring in a segment of the host population that may not have been sufficiently sampled in the past, and that a direct life cycle should be reconsidered.

Animals↗