Biomedical subjects

B A Young

Publications and source records attributed to B A Young.

At least 19 recordsLinked to original sources

PubMed2001

A coiled-coil from the RNA polymerase beta' subunit allosterically induces selective nontemplate strand binding by sigma(70).

For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the -10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the beta' subunit (aa 262--309) induces sigma(70) to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the beta' coiled-coil and region 2.2 of sigma(70) promotes an allosteric transition that allows sigma(70) to selectively recognize the nontemplate strand. As the beta' 262--309 peptide can function with the previously crystallized portion of sigma(70), nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

Allosteric Regulation↗

PubMed2001

Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits.

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.

Animals↗

PubMed2001

Health-related quality of life among dialysis patients in Seattle and Aichi.

We used the 36-item Short-Form Health Survey to compare health-related quality of life (HRQOL) between 104 dialysis patients in Seattle, WA, and 2,178 patients in Aichi, JAPAN: Compared with Aichi patients, Seattle patients had lower scores on three scales related to physical HRQOL: Physical Functioning (PF; P = 0.03), Role-Physical (RP; P = 0.004), and Vitality (VT; P < 0.001). However, scores related to mental HRQOL were higher for Seattle patients compared with those of Aichi patients, which included scores for Role-Emotional (RE; P = 0.005) and Mental Health (MH; P < 0.001). Scores for Bodily Pain, General Health Perception, and Social Functioning did not differ significantly between the two groups. These differences persisted even after potential confounding factors were controlled for. However, after taking into account national norm data for the United States and Japan, differences in PF and VT disappeared, whereas differences in RP, RE, and MH persisted. These results suggest that the higher scores for PF and VT in Aichi patients were partly explained by the higher physical HRQOL of the Japanese general population. Although these data may not be representative of the total dialysis populations in the United States and Japan, they suggest potential differences in HRQOL between patients in the two countries. Additional research is needed to confirm these results and understand the factors associated with these differences. The findings suggest the need for further attention to the physical limitations of US dialysis patients and the mental health of Japanese dialysis patients.

Adult↗

PubMed2001

Binding of the initiation factor sigma(70) to core RNA polymerase is a multistep process.

The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation.

Bacterial Proteins↗

PubMed2001

The cytoplasmic domain of the integrin alpha9 subunit requires the adaptor protein paxillin to inhibit cell spreading but promotes cell migration in a paxillin-independent manner.

The integrin alpha9 subunit forms a single heterodimer, alpha9beta1. The alpha9 subunit is most closely related to the alpha4 subunit, and like alpha4 integrins, alpha9beta1 plays an important role in leukocyte migration. The alpha4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the alpha9 cytoplasmic domain has similar effects, a series of chimeric and deleted alpha9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an alpha9beta1-specific ligand. Like alpha4, the alpha9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the alpha9 cytoplasmic domain and to a similar level as alpha4. In paxillin(-/-) cells, alpha9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the alpha9-paxillin interaction prevented alpha9 from inhibiting cell spreading but had no effect on alpha9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin alpha subunits enhance migration and inhibit cell spreading are distinct and that the alpha9 and alpha4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.

Amino Acid Motifs↗

PubMed2000

Exclusion limits on the WIMP-nucleon cross section from the cryogenic dark matter search.

The Cryogenic Dark Matter Search (CDMS) employs Ge and Si detectors to search for weakly interacting massive particles (WIMPs) via their elastic-scattering interactions with nuclei while discriminating against interactions of background particles. CDMS data, accounting for the neutron background, give limits on the spin-independent WIMP-nucleon elastic-scattering cross section that exclude unexplored parameter space above 10 GeV/c2 WIMP mass and, at >75% C.L., the entire 3sigma allowed region for the WIMP signal reported by the DAMA experiment.

Journal Article↗

PubMed2000

Functional subdivision of the venom gland musculature and the regulation of venom expulsion in rattlesnakes.

A combination of histology, whole muscle force physiology, glycogen depletion, and venom expulsion analyses using transonic probes to measure venom flow and fluid pressure transducers to measure venom pressure was performed on the m. compressor glandulae and m. pterygoideus glandulae. The m. pterygoideus glandulae has less than one-third the cross-sectional area of the m. compressor glandulae, and produces approximately one-fifth the total twitch and tetanic force; however, in situ surface stimulation of the muscle produces venom flow and pressure levels that are similar to those produced by the m. compressor glandulae. The similarity in venom output following stimulation reflects in part the functional role of the larger m. compressor glandulae in jaw adduction, but also the functional subdivisions within this muscle. The m. compressor glandulae is divided into a series of columnar fascicles that run from the surface of the muscle to the venom gland. The combined results of clearing and staining and glycogen depletion studies suggest that these fascicles may represent functional compartments. Identical stimulations applied to different regions of the m. compressor glandulae result in up to a six-fold difference in venom expulsion. This functional specialization may play a role in the regulation of venom flow during offensive and defensive strikes.

Animals↗

PubMed2000

Mycetoma.

Subcutaneous fungal infections are relatively uncommon in the lower extremity. Mycetoma begins as painless papules or nodules that increase in size and progresses to involve the connective tissue. Diagnosis is based on biopsy, with definitive identification of the organism needed for effective treatment. Treatment consists of antifungal medications and surgical debridement. This article provides an overview of this disorder and reports on a case of recurrent mycetoma in a 70-year-old woman.

Aged↗

PubMed1999

Comparative morphology of the cloacal scent gland in snakes (Serpentes: Reptilia).

The histology of the cloacal scent gland, or anal gland, was examined from a diverse group of 50 snakes. Extensive interspecific morphological variation was observed in the general structure of the gland and the glandular epithelium. Morphological variants were quantitatively scored from eight features: lobate nature of the gland; septa in the glandular epithelium; glandular epithelium having a rough or irregular outer surface; glandular epithelium having a rough or irregular inner surface; thickness of the glandular epithelium; PAS reactivity in the glandular epithelium; Periodic Acid-Schiff reactivity in the secretory product; and relative size of the cloacal scent gland. The distribution of the encoded morphological variation was compared to an established phylogeny for snakes and used as the basis for a cluster analysis. In both cases there was no apparent relationship between phylogeny and the morphological variation of the cloacal scent gland.

Animals↗

PubMed1999

Conserved extracellular cysteine residues in the inwardly rectifying potassium channel Kir2.3 are required for function but not expression in the membrane.

The mouse potassium channel Kir2.3 possesses conserved extracellular cysteine residues at positions 113 and 145. We have investigated the role of these cysteines in structure/function and membrane trafficking. Cysteine to serine mutations resulted in the absence of potassium currents in oocytes and co-expression of these mutants with wild-type channel showed a dominant negative inhibition of wild-type currents. FLAG-tagged channels expressed in oocytes were detected in the cell membrane by anti-FLAG antibody for wild-type and mutant channels. In vitro translation using the reticulocyte lysate system showed that mutation of these residues did not affect processing nor insertion into membranes. Cysteine residues at 113 and 145 are therefore required for function of the Kir2.3 channel but not for processing into the cell membrane; disulfide bonds between subunits are unlikely.

Animals↗

PubMed1999

The effects of oxidizing and cysteine-reactive reagents on the inward rectifier potassium channels Kir2.3 and Kir1.1.

The inwardly rectifying potassium channel Kir2.3 possesses extracellular cysteine residues at positions 113, 140, and 145, as well as at position 79 near the outer membrane boundary. In this study, we have investigated the roles of these extracellular cysteine residues in mediating inhibition of the Kir2.3 channel by the cysteine-reactive reagents para-chloromercuribenzenesulphonate (PCMBS) and thimerosal, and the oxidizing agent hydrogen peroxide (H2O2). We have also compared the effects of these reagents with those on Kir1.1 channels (which do not possess cysteine residues equivalent to 140 and 79 in Kir2.3 channels). Mutant channels were made in which cysteine residues were mutated to serine by site-directed mutagenesis. Wild-type or mutant cRNA was injected into Xenopus oocytes and voltage-clamp recordings made 1-2 days later. Wild-type Kir2.3 currents were significantly inhibited by PCMBS, thimerosal and H2O2. Currents for mutants Kir2.3 C79S and C140S were also inhibited by PCMBS, thimerosal and H2O2. These mutations affected the time course of inhibition by all three reagents. For PCMBS, a slow component of inhibition was absent for the C79S mutation, and a fast component was absent for C140S. For the double mutation C79S/C140S, PCMBS no longer had any effect. For thimerosal, there was a slower time course for C140S, a faster time course for C79S, and a delayed onset for C79S/C140S. For H2O2, the main effect was a delayed onset for the double mutant. The reducing agent dithiothreitol (DTT) reversed the inhibition by both PCMBS and thimerosal of wild-type and mutant currents, but not the inhibition due to H2O2. Finally, wild-type Kir1.1 currents were not significantly inhibited by the applications of either PCMBS or thimerosal, while H2O2 produced small inhibition. The results taken together indicate that inhibition by the cysteine-reactive reagent PCMBS is mediated through cysteine residues 79 and 140 in Kir2.3 channels, with C79 mediating a slow component of inhibition and C140 a faster component, and that both residues are extracellularly exposed. The data indicate that these two cysteine residues are also main sites for inhibition by thimerosal and H2O2 but, unlike for PCMBS, an additional non-extracellular inhibitory site(s) must also be involved.

4-Chloromercuribenzenesulfonate↗

PubMed1999

Protein variation in the venom spat by the red spitting cobra, Naja pallida (Reptilia: Serpentes).

The venom spat by red spitting cobras (Naja pallida) was analyzed to document variations in protein composition occurring over short temporal periods (less than 5 min). These cobras exhibited distinct control of venom flow with spits averaging 1.7% of the volume of the venom gland, thus enabling the cobras to rapidly expel over 40 consecutive spits. Variations in the low and high molecular weight proteins were observed when comparing the 1st, 20th and 40th spits produced by the same specimens. The first few spits were characterized by a distinctive 9 kDa protein which was never observed beyond the 7th spit, was present in milked venom and was present when the spitting behavior was preceded by a 5 min period of induced defensive behaviors.

Animals↗

PubMed1999

Thrombospondin-1 induces tyrosine phosphorylation of adherens junction proteins and regulates an endothelial paracellular pathway.

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC-EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations >/=1 microg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 microg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, gamma-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.

Animals↗

PubMed1999

Extent and economic effect of heat loads on dairy cattle production in Australia.

OBJECTIVE: To investigate the extent of heat load problems, caused by the combination of excessive temperature and humidity, in Holstein-Friesian cows in Australia. Also, to outline how milk production losses and consequent costs from this can be estimated and minimised. PROCEDURES: Long-term meteorological data for Australia were analysed to determine the distribution of hot conditions over space and time. Fifteen dairy production regions were identified for higher-resolution data analysis. Both the raw meteorological data and their integration into a temperature-humidity thermal index were compiled onto a computer program. This mapping software displays the distribution of climatic patterns, both Australia-wide and within the selected dairying regions. Graphical displays of the variation in historical records for 200 locations in the 15 dairying regions are also available. As a separate study, production data from research stations, on-farm trials and milk factory records were statistically analysed and correlated with the climatic indices, to estimate production losses due to hot conditions. RESULTS: Both milk yields and milk constituents declined with increases in the temperature-humidity index. The onset and rate of this decline are dependent on a number of factors, including location, level of production, adaptation, and management regime. These results have been integrated into a farm-level economic analysis for managers of dairy properties. CONCLUSION: By considering the historical patterns of hot conditions over time and space, along with expected production losses, managers of dairy farms can now conduct an economic evaluation of investment strategies to alleviate heat loads. These strategies include the provision of sprinklers, shade structures, or combinations of these.

Animals↗

PubMed1999

Effect of recombinant human growth hormone and insulin-like growth factor-1 administration on IGF-1 and IGF-binding protein-3 levels in brain injury.

STUDY OBJECTIVE: To assess the effect of recombinant human growth hormone (rhGH) on the insulin-like growth factor-1 (IGF-1) plasma concentration versus time profile during continuous infusion of recombinant human (rh)IGF-1 to patients with traumatic brain injury (TBI). SETTING: University of Kentucky Chandler Medical Center. PATIENTS: Twenty-three patients with TBI (aged 18-59 yrs) with Glascow Coma Scale scores of 4-10. INTERVENTION: Patients were randomized to receive rhIGF-1 0.01 mg/kg/hour and daily subcutaneous doses of rhGH 0.05 mg/kg/day or saline for 14 days. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of IGF-1 and IGF-binding protein (BP)-3 were quantified by radioimmunoassay. Patients receiving rhIGF-1/rhGH reached a peak IGF-1 concentration (1199.3+/-84.0 microg/L) at 72 hours and maintained it throughout the study. Levels of IGF-1 in the control group did not change significantly above baseline throughout the study. Concentrations of IGFBP-3 were significantly higher after 48 hours in the treated group (5.1+/-0.4 mg/L) than in controls (2.9+/-0.5 mg/L) and continued until the end of the study (p<0.05). CONCLUSION: Infusion of rhIGF-1 in conjunction with rhGH effectively achieved and maintained supraphysiologic IGF-1 plasma concentrations throughout the dosing period in patients with TBI. It appears that rhGH alters the IGF-1 plasma concentration versus time profile during continuous administration. Although speculative, changes in protein binding of IGF-1 are the most likely mechanism.

Adolescent↗

PubMed1998

The counteradhesive protein SPARC regulates an endothelial paracellular pathway through protein tyrosine phosphorylation.

SPARC (Secreted Protein Acidic and Rich in Cysteine) regulates the transendothelial flux of macromolecules through a paracellular pathway. We now have demonstrated that SPARC-induced increments in albumin flux across postconfluent endothelial cell (EC) monolayers are mediated, in part, through protein tyrosine phosphorylation. SPARC increased tyrosine phosphorylation of EC proteins up to 12-fold within 1 h. The phosphotyrosine-containing proteins were immunolocalized to the intercellular boundaries. Two substrates for SPARC-induced tyrosine phosphorylation were identified as beta-catenin and paxillin. Inhibition of tyrosine kinases with herbimycin A or genistein reversed the barrier dysfunction induced by SPARC by 71% and 49%, respectively. Herbimycin A also protected against SPARC-induced intercellular gap formation. In contrast, inhibition of tyrosine phosphatases with sodium orthovanadate or phenylarsine oxide enhanced the loss of barrier function associated with SPARC treatment by 120% and 88%, respectively. These data indicate that SPARC influences EC-EC interactions through a tyrosine phosphorylation-dependent signaling pathway.

Albumins↗

PubMed1998

Effects of solar radiation, dietary energy, and time of feeding on thermoregulatory responses and energy balance in cattle in a hot environment.

Ten growing heifers were either exposed to or protected from solar radiation, offered a diet of either high (H) or low (L) ME, and fed either in the morning or afternoon during a hot summer. Heifers that consumed the H diet had a greater water intake, DMI, metabolizable energy intake, energy expenditure, and retained energy than heifers that consumed the L diet. Solar radiation did not have an effect on any of these variables. Furthermore, dietary energy and time of measurement had an effect on rectal temperature (Tr), respiration rate (RR), heart rate (HR), and rate of oxygen uptake (VO2); solar radiation had an effect on Tr and RR but not on HR and VO2; and time of feeding had an effect only on VO2. Heifers coped with greater heat loads by increasing RR and the difference in Tr between morning and afternoon. It seems that a lowered body temperature in the morning is a physiological mechanism used by animals to prepare for the heat load that develops during the day. Heat production (HP) and HR throughout the day were affected mainly by the time of feeding and not by the environmental heat load. Feeding in the afternoon increased HP in the cooler hours of the day when heat losses from the animal through conduction and radiation were more efficient. With a pending high heat load situation, reducing feed quality and(or) changing the time of feeding to the late afternoon could be beneficial to the animals in reducing their heat loads.

Animals↗

PubMed1997

Cytoplasmic domain of E-selectin contains a non-tyrosine endocytosis signal.

E-selectin is an activation-dependent, endothelial cell-restricted adhesion molecule that is internalized and degraded rapidly once expressed on the cell surface. Tyrosine-containing structural motifs play an important role in the internalization of a number of integral proteins, and the membrane-proximal E-selectin cytoplasmic tyrosine residue (Tyr582) conforms to the endocytosis motif proposed previously. To determine the endocytosis motif in E-selectin, we selectively introduced truncation, substitution, and deletion mutations to the cytoplasmic tail of E-selectin. We analyzed the internalization kinetics of surface-expressed wild-type and mutant E-selectin constructs in transiently transfected Chinese hamster ovary cells using 125I-labeled E-selectin monoclonal antibody (125I-P6E2) in an acid elution assay. Interestingly, truncation immediately membrane proximal to Tyr582 (DeltaDGS construct) did not alter internalization kinetics significantly (DeltaDGS versus wild-type, mean surface half-life = 42 versus 45 min, respectively). Thus, it appears that the tyrosine residues are not required for internalization of E-selectin. Additional analyses indicated that Ser581 was necessary but alone was insufficient for surface E-selectin endocytosis. Thus, we conclude that there exists a novel non-tyrosine-containing endocytosis signal in the cytoplasmic tail which involves Ser581 and residues membrane-proximal to it.

Amino Acid Sequence↗