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PubMed1995

The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP-dependent transport systems and a novel polA locus associated with genomic variability.

In the framework of the mycobacterial genome sequencing project, a continuous 37,049 bp sequence from the Mycobacterium leprae chromosome has been determined. Computer analysis revealed 10 complete open reading frames, and nine of their products show similarity to known proteins. Seven of these were identified as the enzyme isocitrate lyase, two P-type ATPase cation transporters, two AMP-binding proteins, the ribosomal protein S1, and DNA polymerase I. Interestingly, the polA gene, encoding DNA polymerase, is flanked by two inverted copies of a new class of the M. leprae specific repetitive sequence, RLEP, and this structure resembles a transposable element. A second copy of this element was found at another locus in the genome, but the two copies were not present in equal amounts and could not be found in all isolates of M. leprae. This is the first evidence for genomic variability in the leprosy bacillus and might ultimately be useful for developing a molecular test capable of distinguishing between strains of M. leprae.

Adenosine Triphosphatases↗
PubMed1988

Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators.

The 5' regulatory region (-345 to +1) of the rat insulin I gene (Ins-I) was examined for binding to cellular factors with short oligodeoxynucleotide probes. Over 40 binding species were detected. The binding profiles were specific for each cell type studied. We characterized the factors binding two elements crucial for enhancer activity (the Nir and Far boxes) which bear sequence similarity to the microE1, microE2, and microE3 elements of the immunoglobulin heavy-chain enhancer. The Nir box binds three cellular factors that display preferential affinities for microE1, microE2, or microE3, and the Far box binds two factors related to microE2 or microE3. The insulin gene enhancer was mutated at the Nir box element to reflect the sequences of microE1, microE2, or microE3. Ins-microE2 was fully active, Ins-microE3 was partially active, and Ins-microE1 was inactive. Thus, factors similar or identical to nuclear factor NF-microE1, NF-microE2, or NF-microE3 may play a role in the activity of the insulin gene enhancer.

Animals↗
PubMed2000

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5.

Structural and biochemical data indicate the importance of the phosphate-binding loop residues Gly(12) and Gly(13) of Ras both in the GTP hydrolysis reaction and in biological activity, but these two residues are not conserved in other Ras-related GTPases. To gain a better understanding of this region in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPase as a model for comparison, and substituted the Ala(30) residue (the equivalent of Gly(13) of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp contrast with the observation that a proline substitution at the neighbouring position (Gly(12) of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution surprisingly stimulated the GTPase activity by 5-fold. Molecular modelling suggests that this built-in arginine mimics the catalytic arginine residues found in trimeric GTPases and GTPase-activating proteins in providing the positive charge to facilitate the GTP hydrolysis reaction. We investigated further the biological activity of the Rab5 mutants in relation to stimulating endocytosis. When expressed in cultured baby hamster kidney cells, both arginine and proline mutants, like wild-type Rab5, stimulated endocytosis. However, the arginine mutant was a more potent stimulator than the proline mutant (3-fold stimulation as against 1.7-fold). The tryptophan mutant, on the other hand, was completely deficient in activity in terms of the stimulation of endocytosis, demonstrating the importance of the phosphate-binding loop in Rab GTPase function.

Alanine↗
PubMed1992

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

Barbiturates↗
PubMed1992

Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit.

A library of mutants of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase was screened in vitro for mutants defective in the recognition of the regulatory subunit. The mutations identified were mapped onto the three-dimensional structure of the mouse catalytic subunit with a peptide inhibitor. Mutations defective in the recognition of both the regulatory subunit and the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) mapped to the peptide-binding site shared by all substrates and inhibitors of the catalytic subunit and functionally define the binding site for the autoinhibitor sequence in the hinge region of the regulatory subunit. Mutants defective only in the recognition of the regulatory subunit identified residues that comprise additional binding sites for the regulatory subunit. The majority of these residues are clustered on the surface of the catalytic subunit in a region flanking the distal portion of the autoinhibitor/peptide-binding site. The simultaneous substitution of Lys233, Asp237, Lys257, and Lys261 in this region caused a 260-fold decrease in affinity for the regulatory subunit, whereas the catalytic efficiency toward Kemptide decreased by only 1.8-fold. The substitution of autophosphorylated Thr241, also in this region, and the 3 residues interacting with the phosphate also caused an unregulated phenotype.

Amino Acid Sequence↗
PubMed1992

A single-column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine.

A single-column solid-phase extraction procedure was developed for the screening of acidic, neutral, and basic drugs from plasma. The recoveries of all 25 tested drugs exceeded 82%. After the plasma had been diluted with phosphate buffer (pH 6.0), the drugs were extracted using a single Bond Elut Certify column. The acidic and most of the neutral drugs were eluted by acetone/chloroform (1:1) and the basic drugs were eluted by 2% ammoniated ethyl acetate. Some neutral drugs appeared in both fractions. The two fractions were collected separately and evaporated until approximately 100 microL of solvent remained in the tube. Both fractions were analyzed separately on a gas chromatograph equipped with a wide-bore capillary column and a flame ionization detector. The procedure could also be used for urine samples.

Animals↗
PubMed1991

A systematic molecular analysis of the T cell-stimulating antigens from Mycobacterium leprae with T cell clones of leprosy patients. Identification of a novel M. leprae HSP 70 fragment by M. leprae-specific T cells.

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1989

Histochemical study, morphometric analysis and systematic distribution of fiber types in the rectus femoris muscle of the albino rats.

The histochemical and morphometric characteristics as well as the systematic distribution of fiber types in the muscle rectus femoris (RF) of adult male rats were studied from ATPase (pre-incubated at pH 9'4, 4'6 and 4'2) and NADH stained sections. The muscle fiber types were classified as I type, IIA type and IIB-type. In 20 RF muscles 90-95% of total fibers were sampled and the mean diameter, length and irregularity-degree analysed. The data were grouped in four quadrants and the percent of fiber types was determined. The type IIB fibers predominate in all quadrants. The largest fibers were the IIB type and the smallest ones the type I, for all the quadrants. Our findings support a functional compartmentalization as proposed by many authors.

Animals↗