Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.     

An investigation of seated postural stability

Results of tests undertaken to identify which measures of seated posture control are most effective in two areas, distinguishing differences in the x and y direction control strategies for a given task and distinguishing differences in overall control strategies for pairs of different tasks, are presented. The test platform, calibration tests, test protocol, and data analysis method are described. The results of statistical analyses performed on the data are summarized     

Changes in mitochondrial calcium concentration during the cardiac contraction cycle

OBJECTIVE: The aim was to examine whether mitochondrial Ca2+ fluxes are high enough to change mitochondrial and cytosolic calcium concentration during the contraction cycle. METHODS: Isolated guinea pig ventricular myocytes were stimulated with paired voltage clamp pulses until contractions were maximal (2 mM [Ca2+]o, 36 degrees C). At defined times of diastole or systole, the cells were shock frozen. Electron-probe microanalysis measured the concentration of total calcium in mitochondria (sigma Ca(mito)) and surrounding cytosol (sigma Cac). Other experiments were performed to evaluate DNP sensitive mitochondrial Ca2+ uptake from depolarisation induced [Ca2+]c transients (K5indo-1 fluorescence). RESULTS: At end of diastole, sigma Ca(mito) was 446 mumol.litre-1. During systole, sigma Ca(mito) increased with a 20 ms delay. A peak sigma Ca(mito) of 1050 mumol.litre-1 was measured 40 ms after start of systole, while 95 ms after start of systole sigma Ca(mito) had fallen to 530 mumol.litre-1. From the changes in sigma Ca(mito) the rates of net mitochondrial Ca2+ flux were estimated at 100 nmol.s-1 x mg-1 protein for Ca2+ influx and 36 nmol.s-1 x mg-1 protein for Ca2+ egress. Decay of sigma Ca(mito) was coupled to a rise in sigma Na(mito). sigma Cl(mito) and sigma K(mito) rose and fell in parallel with sigma Ca(mito), suggesting Ca2+ activation of mitochondrial anion and cation channels. Activation of the non-specific permeability can be excluded. Block of mitochondrial Ca2+ uptake with DNP (100 microM) or FCCP (10 microM) increased the amplitude of the [Ca2+]c transients for 1-3 min by about 50%; evaluation of mitochondrial Ca2+ uptake from DNP sensitive difference signals, however, was hampered by sequestration of mitochondrial Ca2+ into the sarcoplasmic reticulum. CONCLUSIONS: Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.     

Assay conditions for the mitochondrial NADH:coenzyme Q oxidoreductase

The assay of Complex I activity requires the use of artificial acceptors, such as short-chain coenzyme Q homologs and analogs, because the physiological quinones, such as CoQ10, are too insoluble in water to be added as substrates to the assay media. The medical interest raised in the last years on the pathological changes of Complex I activity has focussed on the requirement of easy reliable assays for its analysis. We have undertaken a systematic examination of the assay conditions of Complex I in mitochondrial membranes, using a series of quinones as electron acceptors, particularly the coenzyme Q homologs CoQ0, CoQ1 and CoQ2, and the analogs duroquinone and decylubiquinone. Our findings have pointed out that the most suitable electron acceptor for the NADH:CoQ reductase assay is the homolog CoQ1. The analog DB, commercially available, although yielding a high activity, nevertheless causes some problems for the standardization of the assay conditions.     

Localization of interferon-gamma and Ia-antigen in T cell line-mediated experimental autoimmune encephalomyelitis

This study reports the cellular localization of interferon-gamma (IFN-gamma) and MHC class II antigen (Ia) in the spinal cord of rats with experimental autoimmune encephalomyelitis induced by adoptive transfer of myelin basic protein-specific T cells. Numerous IFN-gamma-positive cells, stained with two different monoclonal antibodies against IFN-gamma, were present from days 3 to 7 after cell transfer. Their number was greatly reduced on day 10. A subpopulation of T cells was IFN-gamma positive. Moreover, a large number of ED1-positive macrophages contained IFN-gamma immunoreactivity. The transient presence of immune cells containing IFN-gamma immunoreactivity in experimental autoimmune encephalomyelitis suggests a pathogenic role of this cytokine in immune-mediated demyelination of the central nervous system.     

Local synthesis of immunoglobulins in neuropathies

It is essential to know how the immune system acts in different neurological diseases, some of them non very well known or of unknown etiology at all. It was applied Reiber and Felgenhauer's formula in 56 patients with different diseases. IgA, IgM, IgG and albumin were quantified in sera and cerebrospinal fluid by simple immunodiffusion. It was observed more frequent IgG local synthesis and IgA in this sample.     

Fractionation of wheat proteins by counter-current distribution using aqueous two-phase systems containing propionic acid

Wheat proteins, soluble in diluted acid (glutenins), have been fractionated by counter-current distribution (CCD) using an aqueous two-phase system. The phase system is based on poly(ethylene glycol) and dextran but contains also 1% propionic acid and 6 mM magnesium sulfate. Approximately half of the bulk proteins partitioned to the upper phase while starch and other particles were recovered only into the lower phase. Whole wheat flour could be applied as sample for the CCD and 57 transfers were carried out. Starch and insoluble proteins remained stationary, while proteins followed the mobile phase to various degrees giving rise to a distribution pattern. The CCD pattern of the proteins showed distinct differences when various kinds of wheat flour were analysed. The patterns indicate that at least six subpopulations of proteins can be obtained by using two-phase extraction.     

Designing for parallel fuzzy computing

As the number of fuzzy logic applications increases, demand for faster architectures will grow. Our design for a VLSI fuzzy processor uses fuzzy inference techniques that optimize processing time. Preprocessing that reduces the number of rules to be processed, parallel computation of active rule degrees of activation, and scalability are major features of this architecture. The journal issue contains a concise summary of this article. The complete article is linked to Micro's home page on the World Wide Web (http://www.computer.org/pubs/micro/micro.htm)