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Biomedical subjects

M Cross

Publications and source records attributed to M Cross.

At least 19 recordsLinked to original sources
PubMed2001

Redox thermodynamics of mutant forms of the rubredoxin from Clostridiumpasteurianum: identification of a stable Fe(III)(S-Cys)3(OH) centre in the C6S mutant.

Redox thermodynamic data provide a detailed insight into control of the reduction potential E degrees' of the [Fe(S-Cys)4] site in rubredoxin. Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres. Those changes have been probed by resonance Raman spectroscopy. The decrease of approximately 200 mV in E degrees' observed for the [Fe(S-Cys)3(O-Ser)]-/2- couples in the surface ligand mutants C9S and C42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand olate. However, the pH dependence of the potentials below characteristic pKa(red) approximately equals 7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer Fe(II)-O(H)-Ser bond in the reduced form. The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pKa(red) > or = 9.3, but for pH pKa approximately 9: [Fe(III)(S-Cys)3(OH)]- + e- --> [Fe(II)(S-Cys)3(OH)]2-. pH [Fe(II)(S-Cys)3(OH2)]-.

Clostridium↗
PubMed2000

A chromatographic study of the reaction sequence and effect of ligand on the reaction of human hemoglobin with negatively charged isothiocyanates: characterization of an intermediate modified only on the amino termini of the alpha chains.

A HPLC investigation of the reaction of 4-isothiocyanatobenzoic acid and 4-isothiocyanatobenzenesulfonic acid with oxy- and deoxyhemoglobin was carried out. The initial reaction of aromatic isothiocyanato sulfonates and benzoates with either oxy- or deoxyhemoglobin is with the amino termini of the alpha chains followed by a much slower reaction with the amino termini of the beta chains. Both reactions are much faster with deoxyhemoglobin than with oxyhemoglobin. An intermediate reacted only at the termini of the alpha chains with 4-isothiocyanatobenzoic acid was isolated and purified and its functional properties determined. The intermediate showed a reduced oxygen affinity over a wide pH range and a reduced alkaline Bohr effect in the absence of chloride. The oxygen affinity of the intermediate showed a reduced but still significant response to chloride.

Benzenesulfonates↗
PubMed2000

Mutation of the surface valine residues 8 and 44 in the rubredoxin from Clostridium pasteurianum: solvent access versus structural changes as determinants of reversible potential.

The Pr(i) sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site. To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pr(i)), L (Bu(i)) and I (Bu(s)) at those positions. X-ray crystal structures of the Fe(III) forms of the V44A and V44I proteins are reported. Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the side-chains of ligands C9 and C42. Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH...Sgamma hydrogen bond at the active site and lead to a shift in potential of + 50 mV.

Base Sequence↗
PubMed2000

Tandemly repeated DNA is a target for the partial replacement of thymine by beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

In the DNA of African trypanosomes a small fraction of thymine is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J). The function of this large base is unknown. The presence of J in the silent variant surface glycoprotein gene expression sites and the lack of J in the transcribed expression site indicates that DNA modification might play a role in control of gene repression. However, the abundance of J in the long telomeric repeat tracts and in subtelomeric arrays of simple repeats suggests that J may also have specific functions in repetitive DNA. We have now analyzed chromosome-internal repetitive sequences in the genome of Trypanosoma brucei and found J in the minichromosomal 177-bp repeats, in the long arrays of 5S RNA gene repeats, and in the spliced-leader RNA gene repeats. No J was found in the rDNA locus or in dispersed repetitive transposon-like elements. Remarkably, the rDNA of T. brucei is not organized in long arrays of tandem repeats, as in many other eukaryotes. T. brucei contains only approximately 15-20 rDNA repeat units that are divided over six to seven chromosomes. Our results show that J is present in many tandemly repeated sequences, either at a telomere or chromosome internal. The presence of J might help to stabilize the long arrays of repeats in the genome.

Animals↗
PubMed2000

First, build the foundations: practical considerations in general practitioner-mental health service liaison.

The co-working relationship between general practitioners and local mental health services lies at the heart of the success of recent changes in mental health care. These changes have seen large-scale deinstitutionalisation, a shift to primary and community care, and a government policy of prevention and partnerships. This article outlines a liaison project developed jointly by the South-East New South Wales Division of General Practice and the Southern Area Mental Health Service to address these issues. We describe the development of a protocol of basic standards which were practical, pragmatic and possible and, when fully established, would be the foundation of effective, efficient and efficacious shared care in mental health.

Australia↗
PubMed2000

In the pipeline.

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Communication↗
PubMed1999

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans.

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.

Amino Acid Sequence↗
PubMed1999

Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei.

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10(-7) per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.

Animals↗