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Biomedical subjects

R Baker

Publications and source records attributed to R Baker.

At least 127 records · Page 7Linked to original sources
PubMed1999

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.

Animals↗
PubMed1999

Chemically synthesized ubiquitin extension proteins detect distinct catalytic capacities of deubiquitinating enzymes.

We have used solid-phase chemistry to synthesize proteins equivalent to a human ubiquitin precursor (ubiquitin-52-amino-acid ribosomal protein fusion; UBICEP52) and representative of isopeptide-linked ubiquitin-protein conjugates [ubiquitin-(epsilonN)-lysine]; these proteins were precisely cleaved by a purified recombinant Drosophila deubiquitinating enzyme (DUB), UCH-D. Along with the previously synthesized ubiquitin-(alphaN)-valine, these synthetic proteins were used as substrates to assess the catalytic capacities of a number of diverse DUBs expressed in Escherichia coli: human HAUSP; mouse Unp; and yeast Ubps 1p, 2p, 3p, 6p, 11p, and 15p and Yuh1p. Distinct specificities of these enzymes were detected; notably, in addition to UCH-D, isopeptidase activity [ubiquitin-(epsilonN)-lysine cleavage] was only associated with Yuh1p, Unp, Ubp1p, and Ubp2p. Additionally, human placental 26S proteasomes were only able to cleave UBICEP52 and ubiquitin-(epsilonN)-lysine, suggesting that 26S proteasome-associated DUBs are class II-like. This work demonstrates that the synthetic approach offers an alternative to recombinant methods for the production of small proteins in vitro.

Animals↗
PubMed1999

WIN 17317-3, a new high-affinity probe for voltage-gated sodium channels.

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.

Animals↗
PubMed1999

Incorporation of zidovudine into leukocyte DNA from HIV-1-positive adults and pregnant women, and cord blood from infants exposed in utero.

OBJECTIVE: The nucleoside analog 3'-azido-3'-deoxythymidine (ZDV) has widespread clinical use but also is carcinogenic in newborn mice exposed to the drug in utero and becomes incorporated into newborn mouse DNA. This pilot study was designed to determine ZDV incorporation into human blood cell DNA from adults and newborn infants. DESIGN: In this prospective cohort study, peripheral blood mononuclear cells (PBMC) were obtained from 28 non-pregnant adults and 12 pregnant women given ZDV therapy, six non-pregnant adults with no exposure to ZDV, and six non-pregnant adults who last received ZDV > or = 6 months previously. In addition, cord blood leukocytes were obtained from 22 infants of HIV-1-positive, ZDV-exposed women and from 12 infants unexposed to ZDV. There were 11 mother-infant pairs involving HIV-1 -positive women. METHODS: DNA was extracted from PBMC obtained from non-pregnant HIV-1-positive adults taking ZDV, pregnant HIV-1-positive women given ZDV during pregnancy, and from adults not taking ZDV. Cord blood leukocytes were examined from infants exposed to ZDV in utero and from unexposed controls. DNA samples were assayed for ZDV incorporation by anti-ZDV radioimmunoassay (RIA). RESULTS: The majority (76%) of samples from ZDV-exposed individuals, pregnant women (8 of 12), non-pregnant adults (24 of 28), or infants at delivery (15 of 22), had detectable ZDV-DNA levels. The range of positive values for ZDV-treated adults and infants was 25-544 and 22-452 molecules ZDV/10(6) nucleotides, respectively. Analysis of 11 mother-infant pairs showed variable ZDV-DNA incorporation in both, with no correlation by pair or by duration of drug treatment during pregnancy. Two of the 24 samples from individuals designated as controls were positive by anti-ZDV RIA. The 20-fold range for ZDV-DNA values in both adults and infants suggested large interindividual differences in ZDV phosphorylation. CONCLUSIONS: Incorporation of ZDV into DNA was detected in most of the samples from ZDV-exposed adults and infants. Therefore, the biologic significance of ZDV-DNA damage and potential subsequent events, such as mutagenicity, should be

Adult↗
PubMed1999

Otolith ocular reflex function of the tangential nucleus in teleost fish.

In teleost fish, the tangential nucleus can be identified as a compact, separate cell group lying ventral to the VIIIth nerve near the middle of the vestibular complex. Morphological analysis of larval and adult hindbrains utilizing biocytin and fluorescent tracers showed the tangential nucleus to be located entirely within rhombomeric segment 5 with all axons projecting into the contralateral MLF. Combined single-cell electrophysiology and morphology in alert goldfish found three classes of neurons whose physiological sensitivity could be readily correlated with rotational axes about either the anterior (45 degrees), posterior (135 degrees), or horizontal (vertical axis) semicircular canals. Tangential neurons could be distinguised from those in semicircular-canal specific subnuclei by an irregular, spontaneous background of 10-15 sp/s and sustained static sensitivity after +/- 4 degrees head displacements. Each axis-specific tangential subtype terminated appropriately onto oculomotor subnuclei responsible for either vertical, torsional, or horizontal eye movements and, in a few cases, axon collaterals descended in the MLF toward the spinal cord. We hypothesize, therefore, that the tangential nucleus consists of 3 axis-specific phenotypes that process gravitoinertial signals largely responsible for controlling oculomotor function, but that also in part, maintain body posture.

Animals↗
PubMed1999

Abducens internuclear and ascending tract of deiters inputs to medial rectus motoneurons in the cat oculomotor nucleus: synaptic organization.

Abducens internuclear and ascending tract of Deiters (ATD) inputs to medial rectus motoneurons in the oculomotor nucleus are important for conjugate horizontal movements. In the present study, the organization of these separate populations of neurons and their synaptic connections with medial rectus motoneurons in the cat oculomotor nucleus have been examined by light and electron microscopy by using retrograde and anterograde axonal tracers. Consistent with the patterns of retrograde horseradish peroxidase labeling, the abducens internuclear projection is predominantly, if not exclusively, contralateral, whereas the ATD projection is exclusively ipsilateral, as demonstrated by anterograde autoradiographic and biocytin labeling. Both populations of synaptic endings contain spheroidal synaptic vesicles and establish synaptic contacts with modest postsynaptic densifications. In addition, ATD synaptic endings frequently are associated with subjunctional dense bodies and subsurface cisternae. The two populations of excitatory inputs differ, however, in their soma-dendritic distribution. The majority of abducens internuclear synaptic endings contact distal dendrites, whereas the majority of ATD synaptic endings contact proximal dendrites or somata. Abducens internuclear synaptic endings furthermore have a higher density of mitochondria than ATD synaptic endings. The more proximal location of ATD synaptic endings is consistent with the faster rise time and earlier reversal to polarizing currents of ATD excitatory postsynaptic potentials in comparison to those evoked by the abducens internuclear pathway as determined electrophysiologically. Given the differences in the physiologic signals conveyed by the abducens internuclear (eye velocity and eye position) and ATD (head velocity) pathways, the findings in this study suggest that the soma-dendritic stratification of the two inputs to medial rectus motoneurons may provide a means for the separate control of visuomotor and vestibular functions, respectively.

Abducens Nerve↗
PubMed1999

Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.

Aequorin↗
PubMed1999

Gene structure, expression and chromosomal localization of murine theta class glutathione transferase mGSTT1-1.

We have isolated and characterized a cDNA and partial gene encoding a murine subfamily 1 Theta class glutathione transferase (GST). The cDNA derived from mouse GSTT1 has an open reading frame of 720 bp encoding a peptide of 240 amino acids with a calculated molecular mass of 27356 Da. The encoded protein shares only 51% deduced amino acid sequence identity with mouse GSTT2, but greater than 80% deduced amino acid sequence identity with rat GSTT1 and human GSTT1. Mouse GSTT1-1 was expressed in Escherichia coli as an N-terminal 6x histidine-tagged protein and purified using immobilized-metal affinity chromatography on nickel-agarose. The yield of the purified recombinant protein from E. coli cultures was approx. 14 mg/l. Recombinant mouse GSTT1-1 was catalytically active towards 1, 2-epoxy-3-(p-nitrophenoxy)propane, 4-nitrobenzyl chloride and dichloromethane. Low activity towards 1-menaphthyl sulphate and 1-chloro-2,4-dinitrobenzene was detected, whereas mouse GSTT1-1 was inactive towards ethacrynic acid. Recombinant mouse GSTT1-1 exhibited glutathione peroxidase activity towards cumene hydroperoxide and t-butyl hydroperoxide, but was inactive towards a range of secondary lipid-peroxidation products, such as the trans-alk-2-enals and trans,trans-alka-2,4-dienals. Mouse GSTT1 mRNA is most abundant in mouse liver and kidney, with some expression in intestinal mucosa. Mouse GSTT1 mRNA is induced in liver by phenobarbital, but not by butylated hydroxyanisole, beta-napthoflavone or isosafrole. The structure of mouse GSTT1 is conserved with that of the subfamily 2 Theta class GST genes mouse GSTT2 and rat GSTT2, comprising five exons interrupted by four introns. The mouse GSTT1 gene was found, by in situ hybridization, to be clustered with mouse GSTT2 on chromosome 10 at bands B5-C1. This region is syntenic with the location of the human Theta class GSTs clustered on chromosome 22q11.2. Similarity searches of a mouse-expressed sequence tag database suggest that there may be two additional members of the Theta class that share 70% and 88% protein sequence identity with mouse GSTT1, but less than 55% sequence identity with mouse GSTT2.

Amino Acid Sequence↗
PubMed1999

Shaping the modern pharmaceutical development facility.

The modern pharmaceutical development facility is vastly different from previous designs. In this review, the authors describe the trends and types of facility currently used in the development of pharmaceutical products, and discuss some of the trends that will shape those facilities of the future.

Journal Article↗
PubMed1999

The effect of botulinum toxin A on gastrocnemius length: magnitude and duration of response.

Thirty-nine ambulant children (22 with hemiplegia, 17 with diplegia) with spastic cerebral palsy receiving isolated gastrocnemius muscle injection with botulinum toxin A were studied prospectively. The children had a mean age of 6 years (range 3 to 13 years). Measurement of gastrocnemius muscle length was used to estimate the dynamic component of each child's spasticity and to quantify the response. There was a strong correlation between the dynamic component of spasticity before injection and the corresponding magnitude of the response after injection. Children undergoing repeated injections showed similar correlations. A strong correlation was found between the duration of response and the dynamic component. Children with hemiplegia showed twice the duration for a given dynamic component compared with those with diplegia when injected with the same total dose per unit body weight. Long-term lengthening did not occur for the cohort, although some patients showed a response at a 12-month follow-up. By delaying shortening, the injections may have a role in delaying the need for surgery. Injections were well tolerated with few side effects.

Adolescent↗
PubMed1999

Identification and characterization of two missense mutations causing factor XIIIA deficiency.

In this study, two amino acid substitutions, Arg260His and Val414Phe, have been identified in the factor XIIIA subunits of factor XIII deficient patients of Syrian and Indian descent, respectively. To confirm the deleterious effects of these substitutions, both variant sequences have been engineered into cDNA clones and the mutant enzymes expressed in yeast. Determination of the transglutaminase activity and immuno detection of the mutant enzymes together with mRNA hybridization revealed that the mutations dramatically reduce both the catalytic activity and the level of enzyme expressed in yeast. The mutations Arg260His and Val414Phe occur within the 'core' domain of the enzyme. Computer modelling of the mutant enzymes reveals that the substitution of the Arg260 by His results in the loss of a conserved electrostatic interaction whereas the effect of the Val414Phe substitution is a consequence of the large increase in side-chain volume. Although both mutations do not effect the active site directly, they are predicted to reduce the stability of the enzyme. The effects of these two amino acid substitutions on enzyme expression and three-dimensional structure strongly confirm that residues which are located outside of the active site can have a significant effect on protein stability and function.

Adult↗
PubMed1999

Cortisol metabolism, cortisol sensitivity and the pathogenesis of leprosy reactions.

The concentration of cortisol in a tissue is regulated by a reversible enzyme 'shuttle' that can deactivate cortisol by converting it to cortisone, or activate cortisone by converting it to cortisol. The activity of this shuttle, and the direction in which it operates, is regulated by numerous factors including cytokines. This results in large swings in the effective cortisol concentration in sites of inflammation at different phases of an inflammatory response. Thus changes in local cortisol concentration can be largely independent of circulating cortisol levels. The relevant shuttle enzymes are present in skin, blood vessels and nervous tissue, and inhibition of the enzymes in skin enhances the local anti-inflammatory effect of cortisol. We therefore suggest that changes in the activity or direction of action of the shuttle in leprosy lesions may predispose to reactions, requiring exogenous steroid supplements to regain control of the inflammation.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Control of vancomycin-resistant enterococci at a community hospital: efficacy of patient and staff cohorting.

OBJECTIVE: To evaluate the efficacy of patient and staff cohorting to control vancomycin-resistant enterococci (VRE) at an Indianapolis community hospital. DESIGN: To interrupt transmission of VRE, a VRE point-prevalence survey of hospital inpatients was conducted, and VRE-infected or -colonized patients were cohorted on a single ward with dedicated nursing staff and patient-care equipment. To assess the impact of the intervention, staff compliance with contact isolation procedures was observed, and the VRE point-prevalence survey was repeated 2 months after the cohort ward was established. RESULTS: Following the establishment of the cohort ward, VRE prevalence among all hospitalized inpatients decreased from 8.1% to 4.7% (25 positive cultures among 310 patients compared to 13 positive cultures among 276 patients, P=.14); VRE prevalence among patients whose VRE status was unknown before cultures were obtained decreased from 5.9% to 0.8% (18 positive cultures among 303 patients compared to 2 positive cultures among 262 patients, P=.002); and observed staff-patient interactions compliant with published isolation recommendations increased (5 [22%] of 23 interactions compared to 36 [88%] of 41 interactions, P<.0001). CONCLUSIONS: Our data suggest that, in hospitals with endemic VRE or continued VRE transmission despite implementation of contact isolation measures, establishing a VRE cohort ward may be a practical and effective method to improve compliance with infection control measures and thereby to control epidemic or endemic VRE transmission.

Adult↗
PubMed1999

Altered body shape in HIV disease: a side effect of therapy?

An interactive teleconference on HIV-associated lipodystrophies and altered body habitus was held on January 20, 1999, part of a series known as HIV Treatment LIVE!. It was moderated by Ronald Baker, Ph.D., and sponsored by the AIDS Wasting Foundation (AWF). AWF, located at 15 East 26th Street, New York, NY 10010, was founded in 1997 as an education and advocacy organization, under the direction of Carola Burroughs, Executive Director. This tele-conference brought together three AIDS experts to address important issues concerning the etiology, diagnosis, and treatment of this novel and enigmatic syndrome. The resulting report is an edited, selected transcript of that discussion.

Anti-HIV Agents↗