Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Estimates of the Prevalence of Obesity in Canadian Children

Childhood obesity is becoming a topic of great concern due to the rising prevalence of this condition in North America. Studies conducted in the United States have indicated that the prevalence of obesity has increased dramatically over the past few decades. The purpose of this study was to estimate the prevalence of obesity in Canadian children between the ages of 5 and 12 years by examining data from two national and two regional surveys. The 85th percentiles of each of four anthropometric indices derived from large normative populations were used as diagnostic criteria for obesity. As expected, the resulting prevalences varied according to the criteria used. A significant increase in childhood obesity between the 1981 to 1988 national surveys was observed when the three indices which used skinfolds were applied. Weight-for-height percentiles did not indicate an increase in obesity in these samples. Regional samples showed a less than expected prevalence of obesity among the middle-class children and a higher than expected rate among the inner city boys. It can be concluded that there is a need for a defined criteria for identifying obesity in children in order to avoid confusion resulting from the wide variation in estimates of prevalence resulting from different standards and measurements. Using adiposity-based criteria for obesity it was clearly evident that the prevalence of obesity has increased in Canadian children.     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

Suppressor T cell growth and differentiation: production of suppressor T cell differentiation factor by the murine thymoma BW5147

Previous studies have identified a lymphokine, termed Ts differentiation factor (TsDF), in primary MLR supernatants that induces effector function of alloantigen-primed MLR-Ts. The present report describes constitutive production of TsDF by the murine thymoma BW5147, and its use to analyze alloantigen and TsDF requirements for MLR-Ts activation to TsF production. Serum-free supernatants of BW5147 restored the capacity of MLR-TsF production to alloantigen-primed MLR-Ts cultured with glutaraldehyde-fixed allogeneic stimulator cells, and were not themselves directly suppressive in the MLR assay. BW5147 supernatant induced MLR-TsF production from primed L3T4-Ly2+ MLR-Ts in the absence of concomitant proliferation, suggesting that the function of BW5147 supernatant, like that of MLR-derived TsDF, is a differentiative rather than a proliferative one, and is required for the synthesis or release of TsF. The differentiative activity of BW5147 supernatant was associated with a molecular species of approximately 14,500 m.w. by HPLC fractionation and was expressed independently of detectable IL 2, IL 3, IFN-gamma, and IL 1. The functional activity of BW5147 supernatant has therefore been provisionally designated BW5147-derived Ts differentiative factor, or BW-TsDF. By using BW-TsDF, it was demonstrated that MLR-Ts fail to respond to TsDF in the absence of, or preceding, reexposure to priming alloantigen. Instead, alloantigen binding by primed MLR-Ts appears to create a transient state of TsDF responsiveness. Primed MLR-Ts were fully sensitive to delayed addition of TsDF for approximately 12 hr after reexposure to alloantigen, but became TsDF-unresponsive within 24 to 36 hr. MLR-Ts cultured alone for 36 hr were fully responsive to the combined addition of TsDF and alloantigen. Thus, MLR-Ts activation to TsF release requires the sequential events of specific alloantigen binding, which induces a TsDF-responsive state, followed by interaction with TsDF. The transience of induced TsDF responsiveness suggests a precise mechanism for control of antigen-initiated Ts activation to effector function.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model

High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase-plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single-substrate, high-dilution-rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed-substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.