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Biomedical subjects

J L Smith

Publications and source records attributed to J L Smith.

At least 73 records · Page 4Linked to original sources
PubMed2000

Temperature-dependent function of the glutamine phosphoribosylpyrophosphate amidotransferase ammonia channel and coupling with glycinamide ribonucleotide synthetase in a hyperthermophile.

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90 degrees C, with half-lives of 65 min for GPAT and 60 h for GARS at 80 degrees C. GPAT activity is known to depend upon channeling of NH(3) from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH(3) for synthesis of PRA was found to increase from 34% at 37 degrees C to a maximum of 84% at 80 degrees C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90 degrees C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50 degrees C, and remained relatively constant up to 90 degrees C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.

Adenine Nucleotides↗
PubMed2000

ets-2 is a target for an akt (Protein kinase B)/jun N-terminal kinase signaling pathway in macrophages of motheaten-viable mutant mice.

The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase-stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase-Akt-JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival.

Animals↗
PubMed2000

Characteristics of veal calves upon arrival, at 28 and 84 days, and at end of the production cycle.

Holstein bull calves arriving at five special-fed veal farms (eight production groups) were scored for various physical condition traits and blood-sampled within 2 h after arrival and at 28 d, 84 d, and 1 wk prior to slaughter at 116 to 143 d. Of 1179 total calves in the production groups, 758 calves were scored and evaluated. Blood cell analyses (red and white blood cell counts, hemoglobin, and packed cell volume) were conducted at all four sampling times; total serum protein concentration was obtained at 0 and 28 d. The study was initiated in autumn and ended the following autumn. Mean initial and final body weights were 46.3+/-0.17 and 209.7+/-0.77 kg; mean mortality was 2.5%. Average daily gain of the eight groups ranged from 1.23 to 1.70 kg/d. Subjective scores of 5 = excellent to 1 = very poor condition were used to evaluate 16 different physical condition characteristics. With the exception of leg joint, hoof, and foot scores, most of the physical condition scores exhibited improvement during the first 28 d. Foot and leg impairments did not appear to hinder the ambulatory ability of the calves during the production period. Physical condition scores at d 0 and 28 were generally not related to numbers or types of medical treatments (enteric, respiratory, other, or total) or to average daily gain during the production period. Means for most erythrocytic and leukocytic traits upon arrival (d 0) were within normal ranges, although 27.4% of the calves were clinically or marginally anemic. Final mean hemoglobin and packed cell volume were 8.53 g/dl and 26.1%. Forty-three percent of the calves at d 0 were colostral deficient, assuming that total serum protein concentrations of <5.5 g/dl indicate colostral deficiency. No blood trait was consistently correlated with body weight gain when gain during the production period was divided into quartiles and the blood traits were averaged by gain quartile. Calves in the lowest serum total protein quartile (mean 4.58 g/dl) had more respiratory and total medical treatments than quartiles with higher total protein means. Dairy bull calves arriving at veal production units after transporting from the dairy farm to the auction market (or other collection facility) have several physical impairments. However, most of these physical impairments are improved early in the veal feeding period and are not generally related to subsequent growth rate or medical treatment.

Aging↗
PubMed2000

Spontaneous hepatic hemorrhage secondary to retained intraperitoneal gallstones.

We describe the first reported case of spontaneous hepatic hemorrhage as a result of retained gallstones in the region of Morison's pouch eroding into the liver parenchyma. The patient presented with acute right upper quadrant abdominal pain 7 months after laparoscopic cholecystectomy. She sustained an episode of limited spontaneous hepatic hemorrhage as noted by abdominal sonography, computerized tomography, and a 4-g/dL hemoglobin drop. Laparoscopic exploration revealed the source of hemorrhage to be an inflammatory source created by retained intraperitoneal gallstones.

Cholecystectomy↗
PubMed2000

Cholesterol, bile acid, and lipoprotein metabolism in two strains of hamster, one resistant, the other sensitive (LPN) to sucrose-induced cholelithiasis.

A comprehensive study of cholesterol, bile acid, and lipoprotein metabolism was undertaken in two strains of hamster that differed markedly in their response to a sucrose-rich/low fat diet. Under basal conditions, hamsters from the LPN strain differed from Janvier hamsters by a lower cholesterolemia, a higher postprandial insulinemia, a more active cholesterogenesis in both liver [3- to 4-fold higher 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) activity and mRNA] and small intestine, and a lower hepatic acyl-coenzyme A:cholesterol acyltransferase activity. Cholesterol saturation indices in the gallbladder bile were similar for both strains, but the lipid concentration was 2-fold higher in LPN than in Janvier hamsters. LPN hamsters had a lower capacity to transform cholesterol into bile acids, shown by the smaller fraction of endogenous cholesterol converted into bile acids prior to fecal excretion (0.34 vs. 0.77). In LPN hamsters, the activities of cholesterol 7alpha-hydroxylase (C7OHase) and sterol 27-hydroxylase (S27OHase), the two rate-limiting enzymes of bile acid synthesis, were disproportionably lower (by 2-fold) to that of HMG-CoAR. When fed a sucrose-rich diet, plasma lipids increased, dietary cholesterol absorption improved, hepatic activities of HMG-CoA reductase, C7Ohase, and S27OHase were reduced, and intestinal S27OHase was inhibited in both strains. Despite a similar increase in the biliary hydrophobicity index due to the bile acid enrichment in chenodeoxycholic acid and derivatives, only LPN hamsters had an increased lithogenic index and developed cholesterol gallstones (75% incidence), whereas Janvier hamsters formed pigment gallstones (79% incidence). These studies indicate that LPN hamsters have a genetic predisposition to sucrose-induced cholesterol gallstone formation related to differences in cholesterol and bile acid metabolism.

Animals↗
PubMed1999

Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase.

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Trp(482) in the C-terminal helix (residues 471-492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile(335) and Tyr(74) mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased the K(d) for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.

Amidophosphoribosyltransferase↗
PubMed1999

Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family.

The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-A crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins.

Amino Acid Sequence↗
PubMed1999

Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis.

Single tryptophan residues were incorporated into each of three peptide segments that play key roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substrate binding were used to calculate binding constants and to detect the structural rearrangements that coordinate reactions at active sites for glutamine hydrolysis and PRTase catalysis. Pre-steady state kinetics of enzyme.PRPP and enzyme.PRPP.glutamine complex formation were determined from stopped-flow fluorescence measurements. The kinetics of the formation of the enzyme.PRPP complex were consistent with a model with two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzyme isomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely the rate-limiting step in the reaction of PRPP with NH(3). The pre-steady state kinetics for binding glutamine to the binary enzyme. PRPP complex could also be fit to a model involving rapid equilibrium binding of glutamine followed by an enzyme isomerization step. The changes monitored by fluorescence account for the interconversions between "end state" structures determined previously by X-ray crystallography and define an intermediate enzyme.PRPP conformer.

Amidophosphoribosyltransferase↗
PubMed1999

Structure of the soluble domain of cytochrome f from the cyanobacterium Phormidium laminosum.

Cytochrome f from the photosynthetic cytochrome b(6)f complex is unique among c-type cytochromes in its fold and heme ligation. The 1. 9-A crystal structure of the functional, extrinsic portion of cytochrome f from the thermophilic cyanobacterium Phormidium laminosum demonstrates that an unusual buried chain of five water molecules is remarkably conserved throughout the biological range of cytochrome f from cyanobacteria to plants [Martinez et al. (1994) Structure 2, 95-105]. Structure and sequence conservation of the cytochrome f extrinsic portion is concentrated at the heme, in the buried water chain, and in the vicinity of the transmembrane helix anchor. The electrostatic surface potential is variable, so that the surface of P. laminosum cytochrome f is much more acidic than that from turnip. Cytochrome f is unrelated to cytochrome c(1), its functional analogue in the mitochondrial respiratory cytochrome bc(1) complex, although other components of the b(6)f and bc(1) complexes are homologous. Identical function of the two complexes is inferred for events taking place at sites of strong sequence conservation. Conserved sites throughout the entire cytochrome b(6)f/bc(1) family include the cluster-binding domain of the Rieske protein and the heme b and quinone-binding sites on the electrochemically positive side of the membrane within the b cytochrome, but not the putative quinone-binding site on the electrochemically negative side.

Bacterial Proteins↗
PubMed1999

Distinct dendritic cell subsets differentially regulate the class of immune response in vivo.

Dendritic cells (DCs) are unique in their ability to stimulate T cells and initiate adaptive immunity. Injection of mice with the cytokine Flt3-ligand (FL) dramatically expands mature lymphoid and myeloid-related DC subsets. In contrast, injection of a polyethylene glycol-modified form of granulocyte/macrophage colony-stimulating factor (GM-CSF) into mice only expands the myeloid-related DC subset. These DC subsets differ in the cytokine profiles they induce in T cells in vivo. The lymphoid-related subset induces high levels of the Th1 cytokines interferon gamma and interleukin (IL)-2 but little or no Th2 cytokines. In contrast, the myeloid-related subset induces large amounts of the Th2 cytokines IL-4 and IL-10, in addition to interferon gamma and IL-2. FL- or GM-CSF-treated mice injected with soluble ovalbumin display dramatic increases in antigen-specific antibody titers, but the isotype profiles seem critically dependent on the cytokine used. Although FL treatment induces up to a 10, 000-fold increase in ovalbumin-specific IgG2a and a more modest increase in IgG1 titers, GM-CSF treatment favors a predominantly IgG1 response with little increase in IgG2a levels. These data suggest that distinct DC subsets have strikingly different influences on the type of immune response generated in vivo and may thus be targets for pharmacological intervention.

Adoptive Transfer↗
PubMed1999

Interdependence of serum concentrations of vitamin K1, vitamin E, lipids, apolipoprotein A1, and apolipoprotein B: importance in assessing vitamin status.

Vitamin E (alpha-tocopherol) and vitamin K1 (phylloquinone) are fat-soluble vitamins and are important nutrients in health and disease. In this study serum concentrations of vitamin E and vitamin K1, lipids and apolipoproteins A1 and B were measured in neonates, normal and hyperlipidaemic individuals in an attempt to establish their interrelationships. A high degree of correlation was observed between the concentrations of the vitamins and those of lipids and apolipoproteins (r ranged from 0.42 to 0.92; p<0.001). Stepwise linear regression methods determined that serum concentrations of both vitamin E and vitamin K1 could best be predicted by using equations excluding lipids but containing only apolipoprotein A1 and B concentrations. Correlation coefficients between predicted and measured values were 0.89 for serum vitamin E, and 0.83 for serum vitamin K1 concentrations. To test the validity of the derived formulae, measured and estimated vitamin K1 and vitamin E concentrations in serum were determined in another group of neonates, normal adults and hypercholesterolemic adults and the comparisons were shown to be very good. These results indicate that the serum levels of both vitamins depend critically on the concentration of the lipoprotein carriers, apolipoproteins A1 and B. Hence, in order to identify variations in serum vitamin K1 and vitamin E concentrations, which are independent of variations in carrier concentration, it will be necessary to express these serum vitamins as ratios of vitamins to apolipoprotein A1 and B carriers.

Adult↗
PubMed1999

Increased carriage of resistant non-pneumococcal alpha-hemolytic streptococci after antibiotic therapy.

OBJECTIVE: We compared colonization and resistance rates of non-pneumococcal alpha-hemolytic streptococci (AHS) and Streptococcus pneumoniae in children receiving antibiotic therapy for acute otitis media. STUDY DESIGN: Between December 1997 and September 1998, children 6 months to 6 years of age, diagnosed with acute otitis media were randomly assigned to receive amoxicillin/clavulanate (Augmentin) 45 mg/kg/d in 2 divided doses for 10 days or azithromycin (Zithromax), 10 mg/kg, once on the first day, followed by 5 mg/kg daily for 4 days. Nasopharyngeal swabs for culture were obtained before and at 2 weeks and 2 months after the start of therapy. Streptococci were identified by species, and antibiotic susceptibility was determined by the epsilometric test. RESULTS: One hundred six children completed the 2-week follow-up and 2-month follow-up, respectively. The nasopharyngeal carriage rate of non-pneumococcal AHS increased from 14% before treatment to 32% at the 2-week follow-up (P =.02) and was similar in both treatment groups. In contrast, the carriage of S pneumoniae decreased from 51% before therapy to 27% at the 2-week follow-up (P =.002). The carriage of penicillin-resistant AHS strains (minimum inhibitory concentration > 1 microg/mL) increased from 9% before treatment to 26% at 2 weeks and 36% at 2 months. CONCLUSIONS: Amoxicillin/clavulanate and azithromycin therapy resulted in increased isolation of nasopharyngeal non-pneumococcal AHS, many of which were multidrug-resistant, in contrast to a decrease in pneumococcal carriage. This suggests that the competitive balance between these 2 groups of organisms was disturbed as a result of differential antibiotic susceptibility. The importance of drug-resistant AHS as a reservoir for resistance genes for S pneumoniae warrants further investigation.

Amoxicillin-Potassium Clavulanate Combination↗
PubMed1999

Comparison of the cytochrome bc1 complex with the anticipated structure of the cytochrome b6f complex: Le plus ça change le plus c'est la même chose.

Structural alignment of the integral cytochrome b6-SU IV subunits with the solved structure of the mitochondrial bc1 complex shows a pronounced asymmetry. There is a much higher homology on the p-side of the membrane, suggesting a similarity in the mechanisms of intramembrane and interfacial electron and proton transfer on the p-side, but not necessarily on the n-side. Structural differences between the bc1 and b6f complexes appear to be larger the farther the domain or subunit is removed from the membrane core, with extreme differences between cytochromes c1 and f. A special role for the dimer may involve electron sharing between the two hemes b(p), which is indicated as a probable event by calculations of relative rate constants for intramonomer heme b(p) --> heme b(n), or intermonomer heme b(p) --> heme b(p) electron transfer. The long-standing observation of flash-induced oxidation of only approximately 0.5 of the chemical content of cyt f may be partly a consequence of the statistical population of ISP bound to cytfon the dimer. It is proposed that the p-side domain of cyt f is positioned with its long axis parallel to the membrane surface in order to: (i) allow its large and small domains to carry out the functions of cyt c1 and suVIII, respectively, of the bc1 complex, and (ii) provide maximum dielectric continuity with the membrane. (iii) This position would also allow the internal water chain ("proton wire") of cyt f to serve as the p-side exit port for an intramembrane H+ transfer chain that would deprotonate the semiquinol located in the myxothiazol/MOA-stilbene pocket near heme b(p). A hypothesis is presented for the identity of the amino acid residues in this chain.

Animals↗
PubMed1999

The clinical utility of the Revised European-American Lymphoma (R.E.A.L.) Classification: preliminary results of a prospective study in patients with non-Hodgkin's lymphoma from a single centre.

BACKGROUND: The clinical applicability of the Revised European-American Lymphoma (R.E.A.L.) Classification has been demonstrated in several retrospective studies. The present, ongoing study was initiated to evaluate the clinical and pathological utility of the R.E.A.L. Classification compared with the Working Formulation (WF) in a prospective fashion, in an unselected patient population treated at a single institution. PATIENTS AND METHODS: Prospective data were collected on 596 biopsies from 557 patients referred with an initial diagnosis of lymphoma. After initial histologic review, 465 biopsies from 441 patients were confirmed as non-Hodgkin's lyphoma (NHL), 412 of which could be classified in R.E.A.L. and WF. RESULTS: According to WF criteria, 25% were low grade, 58% intermediate grade and 2% high grade, 14% could not be allocated to a WF subtype. According to R.E.A.L., 46% were diffuse large B cell, 19% follicle centre lymphoma, 6% marginal zone, 6% small lymphocytic, 4% mantle cell, and 3% T-cell anaplastic large cell. For those with B-cell NHL, 7% were unclassifiable in WF compared with 1% in R.E.A.L. Corresponding figures for T-cell NHL were 68% and 3%, respectively. CONCLUSIONS: Preliminary results confirm the clinical utility of the R.E.A.L. Classification in a single institution setting, demonstrating that cases were more readily sub-typed in R.E.A.L. compared with WF. Frequencies are comparable with I.L.S.G. data. Further follow up with large patient numbers is on-going to analyse survival data with reference to clinical prognostic factors.

Adolescent↗
PubMed1999

The impact of dependent-care responsibility and gender on work attitudes.

On the basis of a survey of 18,120 federal employees in dual-income households, six 5-stage hierarchical multiple regression analyses, controlling for 10 demographic variables, assessed the impact of child care, elder care, and gender on work-family balance and various facets of job satisfaction. Elder-care responsibility was associated with lower levels of satisfaction with perceived organizational support, pay, leave benefits, and work-family balance, whereas the negative main effects of child care were limited to leave benefits and work-family balance. However, child-care responsibility also interacted with gender: Its negative influence was greater on women's work-family balance and leave satisfaction. Decrements in satisfaction associated with dependent care on the "sandwich generation" were additive, not interactive.

Adult↗