Biomedical subjects

C Colson

Publications and source records attributed to C Colson.

At least 19 recordsLinked to original sources

PubMed2001

Subcortical neglect as a consequence of a remote parieto-temporal dysfunction. A quantitative EEG study.

In patients with a right-sided deep-seated lesion, a causal relationship between a cortical dysfunction in the right temporo-parietal region and the occurrence of neglect has been suggested. In the present study we tried to correlate clinical and quantitative EEG data from a sample of 33 right stroke patients divided into two subgroups according to the presence or absence of neglect. A 20-channel EEG cartography system was used for EEG mapping. Delta and theta activities were calculated in sixteen regions of interest. The analysis of raw values stressed the importance of the right parieto-temporal cortex to discriminate between the two subgroups of patients. These results suggest that in patients with right subcortical damage, a remote cortical parieto-temporal dysfunction within an intra-hemispheric network is necessary to provoke neglect.

Adult↗

PubMed1997

Pathogenesis of subcortical visuo-spatial neglect. A HMPAO SPECT study.

The pathophysiology of neuropsychological disorders due to right deep-seated hemispheric lesions remains a debated point. We undertook this study to check the hypothesis according to which remote cortical dysfunction could be responsible for the occurrence of neglect. Twenty-eight patients presenting with a right-sided subcortical stroke were studied. A neuropsychological battery of tests suitable for assessment of possible visuo-spatial neglect was performed as well as HMPAO SPECT. Neglect was observed in 15 cases out of 28. The lesion's site (at CT and/or MRI) did not allow discrimination between patients without neglect and patients with neglect. The latter however could be distinguished from the former by the presence of a remote decrease in cortical blood flow in the right temporo-parietal region. By suggesting that cortical involvement is necessary for the occurrence of neglect, the results were interpreted according to a network approach in which subcortical neglect is attributed to a cortical deprivation from afferent input in the posterior part of the brain.

Aged↗

PubMed1995

Remote cortical dysfunction as a possible cause of subcortical neglect? A regional cerebral blood flow study.

Cortical remote effects of right deep-seated lesions were studied with two cerebral blood flow measurement methods (two-dimensional xenon-133 inhalation and 99mTc HMPAO SPECT) in a population of 13 right-handed stroke patients. A neuropsychological battery of tests suitable for assessment of possible visual neglect was performed. Neglect was present in 7 cases. A regional cortical hypoperfusion was observed in all patients. However, in neglect patients it was more extended and involved the right inferior parietal region suggesting a causal relationship between cortical dysfunction and neuropsychological deficit. This finding supports the model attributing neglect to a unilateral attention-arousal defect in a cortico-limbic-reticular loop.

Aged↗

PubMed1994

Crystallization and preliminary X-ray analysis of a lipase from Bacillus subtilis.

Single crystals of the lipase from Bacillus subtilis have been obtained using a mixture of polyethylene glycol 4000 and sodium sulphate solution as the precipitant. The crystals grow at room temperature in two to three weeks in the presence of n-octyl-beta-D-glucoside. They belong to the monoclinic space group C2 with a = 121.20 A, b = 93.19 A, c = 80.96 A, and beta = 110.67 degrees, with four protein molecules per asymmetric unit. The crystals diffract to at least 2.5 A resolution and are suitable for an X-ray structure analysis.

Bacillus subtilis↗

PubMed1994

The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis.

Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of approximately 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure.

Amino Acid Sequence↗

PubMed1994

Bacterial lipases.

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis.

The previously cloned Bacillus subtilis lipase gene (lip) was mapped on the chromosome and used in the construction of a B. subtilis derivative totally devoid of any lip sequence. Homologous overexpression was performed in this strain by subcloning the lip open reading frame on a multicopy plasmid under the control of a strong gram-positive promoter. A 100-fold overproducing strain was obtained, which should facilitate purification of the secreted protein. Furthermore, the delta lip strain BCL1050 constitutes an ideal host for the cloning of heterologous lipase genes.

Alleles↗

PubMed1994

The H-NS protein is involved in the biogenesis of flagella in Escherichia coli.

The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway.

Bacterial Outer Membrane Proteins↗

PubMed1993

Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme.

The extracellular lipase of Bacillus subtilis 168 was purified from the growth medium of an overproducing strain by ammonium sulfate precipitation followed by phenyl-Sepharose and hydroxyapatite column chromatography. The purified lipase had a strong tendency to aggregate. It exhibited a molecular mass of 19,000 Da by SDS-PAGE and a pI of 9.9 by chromatofocusing. The enzyme showed maximum stability at pH 12 and maximum activity at pH 10. The lipase was active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups. Using trioleyl glycerol as substrate, the enzyme preferentially cleaved the 1(3)-position ester bond. No interfacial activation effect was observed with triacetyl glycerol as substrate.

Ammonium Sulfate↗

PubMed1993

Sequential elution of denatured proteins, hydrolyzed RNA, and plasmid DNA of bacterial lysates adsorbed onto stacked DEAE-cellulose membranes.

Modified membrane chromatography is emerging as a widely applicable technique for the separation of macromolecules. The use of DEAE-cellulose membranes (MemSep) for the purification of bacterial plasmid DNA has been assessed. Cleared bacterial lysates were injected directly onto the membranes without any prior sample cleanup. A single phenol extraction of adsorbed DNA was carried out and about 60 times less phenol was required to achieve the same extent of deproteination for DNA adsorbed onto the membranes as compared to DNA in solution. After chloroform and ethanol wash, (oligo)ribonucleotides resulting from RNase treatment were desorbed with 0.3 M LiCl, 5 mM LiOH. Finally, DNA was eluted with 0.5 M NaCl, 5 mM NaOH. The complete elution of DNA required NaOH in addition to NaCl and the latter salt was a better eluent than LiCl. Without RNase treatment, plasmid DNA-RNA complex required 2 M NaCl, 5 mM NaOH to be completely desorbed. The whole procedure took less than 40 min. The DEAE-cellulose membranes can withstand more than 100 cycles of regenerations and uses without any noticeable decrease of their binding capacity. No cross-contamination of successive DNA preparations was observed. Plasmid DNA was a good substrate for DNA endonucleases. Restriction fragments repurified by this procedure were amenable to ligation. Transformation of Escherichia coli and Saccharomyces cerevisiae with plasmid DNA was observed.

Adsorption↗

PubMed1993

Sequence of the Salmonella typhimurium StyLT1 restriction-modification genes: homologies with EcoP1 and EcoP15 type-III R-M systems and presence of helicase domains.

The StyLT1 restriction-modification (R-M) system of Salmonella typhimurium has recently been suggested to belong to the type-III R-M systems [De Backer and Colson, Gene 97 (1991) 103-107]. The nucleotide sequences of StyLT1 mod and res have been determined. Two closely adjacent open reading frames were found 12 bp apart with coding capacities of 651 (Mod) and 982 (Res) amino acids (aa), respectively. The genes, lying in the same direction of transcription in the mod-res order, are transcribed as distinct units. The deduced aa sequences reveal homologies with known type-III enzymes from the Escherichia coli P1 prophage, E. coli P15 plasmid and Bacillus cereus chromosome. In addition, the StyLT1 restriction endonuclease (ENase), like other type-I and type-III ENases, contains sequence motifs characteristic of superfamily-II helicases, which may be involved in DNA unwinding at the cleavage site.

Amino Acid Sequence↗

PubMed1992

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.

Amino Acid Sequence↗

PubMed1992

Mutations in bglY, the structural gene for the DNA-binding protein H1 of Escherichia coli, increase the expression of the kanamycin resistance gene carried by plasmid pGR71.

bglY mutants of Escherichia coli K12 which show higher levels of kanamycin resistance (Kmr) in the presence of plasmid pGR71 have been previously described. In this work, we show that this increased resistance to an aminoglycoside antibiotic is not due either to low drug uptake or to alteration of its target, the ribosome. The copy number of plasmid pGR71 is not modified. The fact that increased antibiotic resistance is observed with only some of the Kmr determinants used in this study suggests a specific role for the bglY gene product. Moreover, for one such determinant, a higher level of resistance was observed when it was inserted in the chromosome but not when harbored by a plasmid. This discrepancy can be explained by the twin transcriptional-loop model, which proposes that transcription can lead to local variation in topology. A kan-lacZ fusion was constructed from the Kmr gene of plasmid pGR71 and inserted into a low copy number vector. Assay of beta-galactosidase in wild-type and mutant strains showed that expression of the antibiotic resistance gene was directly affected by H1 protein, the bglY gene product.

Aminoglycosides↗

PubMed1991

Identification of the recognition sequence for the M.StyLTI methyltransferase of Salmonella typhimurium LT7: an asymmetric site typical of type-III enzymes.

The StyLTI restriction-modification (R-M) system is encoded by chromosomal genes of Salmonella typhimurium LT7. We report here the identification of the nucleotide (nt) sequence methylated by the StyLTI modification methyltransferase (M.StyLTI). This enzyme was partially purified from an Escherichia coli strain expressing the cloned M.StyLTI-encoding gene, but lacking StyLTI restriction activity, and used to methylate DNAs of known sequence, using S-adenosyl-[methyl-3H]-methionine as the methyl donor. The [3H]methylated DNA was then digested with various endonucleases. Examination of labelled and unlabelled restriction fragments allowed us to map the M.StyLTI sites in perfectly defined regions of the DNA. Comparison of the nt sequences of DNA segments with or without M.StyLTI sites permitted us to identify the asymmetric and nondegenerate pentanucleotide, 5'-CAGAG-3', 3'-GTCTC-5' as the StyLTI sequence. M.StyLTI was found to methylate only the 3' A (see asterisk) in the upper strand of this sequence. Thus, M.StyLTI recognises and methylates the DNA in a manner very similar to that of the three known type-III MTases, M.EcoPI, M.EcoP15, and M.HinfIII. This strongly suggests that StyLTI constitutes a fourth type-III R-M system.

Adenine↗

PubMed1991

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.

Cloning, Molecular↗

PubMed1991

Transfer of the genes for the StyLTI restriction-modification system of Salmonella typhimurium to strains lacking modification ability results in death of the recipient cells and degradation of their DNA.

The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.

Conjugation, Genetic↗

PubMed1989

Partial purification and characterization of the specific protein-lysine N-methyltransferase of YL32, a yeast ribosomal protein.

YL23 and YL32 are two of the three most heavily methylated ribosomal proteins of Saccharomyces cerevisiae. Using an in vitro assay, it was determined that they are methylated by two distinct enzymes. The protein-lysine N-methyltransferase that methylates YL32 was partially purified by affinity and ion-exchange chromatography. Its molecular mass was estimated to be 82 kDa, and its isoelectric point to be 4.45. Optimum activity was expressed at pH 7.5, and the enzyme was irreversibly inactivated at pH lower than 5.0. The Km of the enzyme for AdoMet is 1.7 +/- 0.4 microM, and the Ki toward AdoHcy was 0.71 microM. Formation of epsilon-N-dimethyllysine was observed to occur in two steps via epsilon-N-monomethyllysine. Like other protein-lysine N-methyltransferases, the methylase of YL32 exhibits a high substrate specificity.

Chromatography, Affinity↗

PubMed1989

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells.

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.

Bacillus subtilis↗