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Biomedical subjects

C J Howe

Publications and source records attributed to C J Howe.

At least 19 recordsLinked to original sources
PubMed2006

Influence of demographic factors and sport type on growth hormone-responsive markers in elite athletes.

CONTEXT: GH-responsive markers of the IGF system and of collagen turnover hold promise as the basis of a GH doping test. OBJECTIVE: The purpose of this study was to determine the influence of age, gender, body mass index (BMI), ethnicity, and sporting type on GH-responsive serum markers in a large cohort of elite athletes from different ethnic backgrounds. DESIGN: The study was designed as a cross-sectional study. PARTICIPANTS: A total of 1103 elite athletes (699 males, 404 females), aged 22.2 +/- 5.2 yr, from 12 countries and 10 major sporting categories participated in this study. MAIN OUTCOME MEASURES: Serum IGF-I, IGF binding protein-3 (IGFBP-3), acid labile subunit (ALS), and collagen markers [N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), N-terminal propeptide of type III procollagen (PIIINP)] were measured. RESULTS: There was a significant negative correlation (r = -0.14 to -0.58, P < 0.0005) between age and each of the GH-responsive markers. Serum IGF-I, IGFBP-3, and ALS were all lower (P < 0.05), whereas the collagen markers PINP, ICTP, and PIIINP were higher (P < 0.05) in men than in women. Multiple regression analysis indicated that age, gender, BMI, and ethnicity accounted for 23-54% of total between-subject variability of the markers. Age and gender cumulatively accounted for 91% of the attributable variation of IGF-I and more than 80% for PINP, ICTP, and PIIINP. Gender exerted the greatest effect on ALS (48%), and BMI accounted for less than 12% attributable variation for all markers. The influence of ethnicity was greatest for IGFBP-3 and ALS; however, for the other markers, it accounted for less than 6% attributable variation. Analysis of 995 athletes indicated that sporting type contributed 5-19% of attributable variation. CONCLUSIONS: Age and gender were major determinants of variability of GH-responsive markers except for IGFBP-3 and ALS. Ethnicity is unlikely to confound the validity of a GH doping test based on IGF-I and these collagen markers.

Adolescent↗
PubMed2006

Distinctive organization of genes for light-harvesting proteins in the cryptophyte alga Rhodomonas.

Cryptophyte algae contain two kinds of light-harvesting protein, phycobiliproteins and chlorophyll a,c-binding proteins. The beta subunit of the phycobiliprotein phycoerythrin (PE) is encoded in the chloroplast. Genes for the other PE polypeptides are located in the nucleus but little is known of their organization. We cloned and sequenced six cpeA genes encoding the phycoerythrin alpha subunit from a genomic library of the cryptophyte Rhodomonas CS24. Derived peptide sequences of the cpeA genes show that alpha subunits occur in at least two forms, a longer alpha1 form and a shorter alpha2 form. Remarkably, all six cpeA genes occur in divergent pairs encoding one alpha1 and one alpha2 subunit. Four cac genes encoding chlorophyll a,c-binding proteins were cloned and sequenced and also found to occur in divergent pairs comprising one cac1 and one cac2 gene. Inspection of the predicted targeting sequences of the alpha1 and alpha2 phycoerythrin polypeptides shows that only the alpha1 polypeptides have a thylakoid lumen targeting sequence, corresponding to the TAT pathway. Given the previously reported lack of a lumen-targeting sequence on the beta subunit, we propose a novel import mechanism in which the entire alpha1alpha2 betabeta phycoerythrin complex is assembled in the stroma and transported into the thylakoid under the direction of the single targeting sequence on the alpha1 protein. The FAP motif implicated in plastid targeting in diatoms appears to be conserved in this cryptophyte.

Algal Proteins↗
PubMed2005

Terminal oxidases of cyanobacteria.

The respiratory chain of cyanobacteria appears to be branched rather than linear; furthermore, respiratory and photosynthetic electron-transfer chains co-exist in the thylakoid membrane and even share components. This review will focus on the three types of terminal respiratory oxidases identified so far on a genetic level in cyanobacteria: aa3-type cytochrome c oxidase, cytochrome bd-quinol oxidase and the alternative respiratory terminal oxidase. We summarize here their genetic, biochemical and biophysical characterization to date and discuss their interactions with electron donors as well as their physiological roles.

Bacterial Proteins↗
PubMed2005

Erythropoietin administration does not influence the GH--IGF axis or makers of bone turnover in recreational athletes.

OBJECTIVE: Measurement of biochemical markers of the IGF-system and of collagen turnover is a potential approach to detect GH abuse in sport. These markers are increased in patients on dialysis treated with recombinant human erythropoietin (r-HuEPO), mimicking the effects of GH. The aim was to determine whether r-HuEPO induces similar effects on the IGF-system and collagen turnover in healthy athletes. SUBJECTS AND MEASUREMENTS: Young male Caucasian recreational athletes were administered 50 U/kg r-HuEPO (n=14) or placebo (n=16) three times a week for 25 days, followed by a 4-week wash-out period. IGF-I, IGFBP-3, the acid labile subunit (ALS), N-terminal propeptide of type I collagen (PINP), C-terminal telopeptide of type I collagen (ICTP) and N-terminal propeptide of type III collagen (PIIINP) were measured in samples collected at baseline (two samples), after 10, 22 and 24 days of r-HuEPO treatment and at the end of the 4-week wash-out period. RESULTS: Treatment with r-HuEPO resulted in approximately threefold elevation of serum EPO and marked elevation of markers of erythropoiesis. There was no significant treatment effect of r-HuEPO compared to baseline on IGF-I, IGFBP-3, ALS, PINP, ICTP or PIIINP. CONCLUSIONS: r-HuEPO administration did not change markers of the IGF-system and of collagen turnover in young healthy male athletes. Therefore, use of r-HuEPO in athletes should not affect the validity of a GH doping test using these GH-responsive markers.

Adult↗
PubMed2002

Novel cyanobacterial biosensor for detection of herbicides.

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.

Biosensing Techniques↗
PubMed2001

Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum.

We show using PCR that psbC, atpA and petB genes are present in the plastid DNA minicircles from the dinoflagellate Amphidinium operculatum, extending the set of plastid genes identified from this organism. Unusually, the petBand atpA genes are located on the same minicircle. PCR using primers based on the "core" region found on all coding minicircles revealed the existence of a number of DNA minicircles with no apparent coding function. Northern analysis of total RNA from A. operculatum showed that the petB and atpA genes are represented on separate transcripts, despite being encoded in close proximity on the same minicircle. The possibility of transcript editing was investigated by RT-PCR, but psaA, psbA, psbB and atpB transcripts showed no evidence of editing, indicating that GUA can be used as an initiation codon in A. operculatum.

Amino Acid Sequence↗
PubMed2001

Manuscript evolution.

Frequently, letters, words and sentences are used in undergraduate textbooks and the popular press as an analogy for the coding, transfer and corruption of information in DNA. We discuss here how the converse can be exploited, by using programs designed for biological analysis of sequence evolution to uncover the relationships between different manuscript versions of a text. We point out similarities between the evolution of DNA and the evolution of texts.

Evolution, Molecular↗
PubMed2001

Manuscript evolution.

Frequently, letters, words and sentences are used in undergraduate textbooks and the popular press as an analogy for the coding, transfer and corruption of information in DNA. We discuss here how the converse can be exploited, by using programs designed for biological analysis of sequence evolution to uncover the relationships between different manuscript versions of a text. We point out similarities between the evolution of DNA and the evolution of texts.

DNA↗
PubMed2000

Tryptophan-heme pi-electrostatic interactions in cytochrome f of oxygenic photosynthesis.

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.

Amino Acid Sequence↗
PubMed2000

Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum.

Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3-2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a "core" region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles.

Amino Acid Sequence↗
PubMed2000

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.

Aspartic Acid↗
PubMed1999

Structure of the soluble domain of cytochrome f from the cyanobacterium Phormidium laminosum.

Cytochrome f from the photosynthetic cytochrome b(6)f complex is unique among c-type cytochromes in its fold and heme ligation. The 1. 9-A crystal structure of the functional, extrinsic portion of cytochrome f from the thermophilic cyanobacterium Phormidium laminosum demonstrates that an unusual buried chain of five water molecules is remarkably conserved throughout the biological range of cytochrome f from cyanobacteria to plants [Martinez et al. (1994) Structure 2, 95-105]. Structure and sequence conservation of the cytochrome f extrinsic portion is concentrated at the heme, in the buried water chain, and in the vicinity of the transmembrane helix anchor. The electrostatic surface potential is variable, so that the surface of P. laminosum cytochrome f is much more acidic than that from turnip. Cytochrome f is unrelated to cytochrome c(1), its functional analogue in the mitochondrial respiratory cytochrome bc(1) complex, although other components of the b(6)f and bc(1) complexes are homologous. Identical function of the two complexes is inferred for events taking place at sites of strong sequence conservation. Conserved sites throughout the entire cytochrome b(6)f/bc(1) family include the cluster-binding domain of the Rieske protein and the heme b and quinone-binding sites on the electrochemically positive side of the membrane within the b cytochrome, but not the putative quinone-binding site on the electrochemically negative side.

Bacterial Proteins↗
PubMed1999

Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides.

The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.

Algal Proteins↗
PubMed1999

Adrenal and gonadal contributions to urinary excretion and plasma concentration of epitestosterone in men--effect of adrenal stimulation and implications for detection of testosterone abuse.

OBJECTIVE: The ratio of urinary testosterone (T) to epitestosterone (EpiT) is used to detect T abuse in sport. Also, plasma or urinary concentrations of EpiT have been measured to assess testicular steroidogenesis during hormonal male contraception. Further investigations are required to evaluate the relative contributions of the testis and adrenal to EpiT production. To this purpose, we have compared basal urinary EpiT glucuronide and plasma EpiT and the response to synthetic adrenocorticotrophic hormone (ACTH) stimulation between eugonadal and hypogonadal men. DESIGN AND SUBJECTS: The basal urinary excretion rate of EpiT glucuronide was determined in 34 eugonadal men. Six men, clinically diagnosed as hypogonadal, and 6 out of the 34 eugonadal men previously described, received an intramuscular injection of synthetic ACTH depot (1 mg) at 0800 h on two consecutive days. Blood samples were collected prior to and then at 1.5, 8, 24, 25.5, 32 and 48 h with respect to the first administration (0 h). 24-h urine specimens were collected from 0800 h on days 1 and 2 (baseline) and 3 and 4 (stimulation). MEASUREMENTS: Plasma EpiT, T and cortisol were measured by RIA and urinary EpiT and T, following glucuronide hydrolysis, by gas chromatography-mass spectrometry (extract combines aglycones with a minor amount of urinary free steroids). RESULTS: Basal excretion rates of EpiT glucuronide in eugonadal men (range: 62-751 nmol/24 h) were considerably greater than in hypogonadal men (range: 3-34 nmol/24 h). Mean basal plasma EpiT in eugonadal men (1.32 +/- 0.08 nmol/l) were greater than in hypogonadal men (0.68 +/- 0.04 nmol/l). In each group, synthetic ACTH stimulation increased plasma cortisol 4-fold. In eugonadal men, plasma and urinary EpiT were unchanged whereas plasma and urinary T glucuronide decreased in response to ACTH. In hypogonadal patients, ACTH increased plasma and urinary EpiT while plasma T remained unchanged. CONCLUSION: The testes are the major source of epitestosterone, the adrenal contribution being relatively modest. Following adrenal stimulation, urinary epitestosterone glucuronide increases considerably in hypogonadal men but this increase is masked in eugonadal men because testicular production is probably suppressed by the ACTH-induced rise in cortisol. Activation of the adrenal cortex results in no change or only a small decrease in the urinary T/EpiT ratio in eugonadal men.

Adrenal Glands↗
PubMed1999

The structure of plastocyanin from the cyanobacterium Phormidium laminosum.

The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P. laminosum plastocyanin crystallizes in space group P43212 with unit-cell dimensions a = 86.57, c = 91.47 A and with three protein molecules per asymmetric unit. The final residual R is 19.9%. The structure was solved using molecular replacement with a search model based on the crystal structure of a close homologue, Anabaena variabilis plastocyanin (66% sequence identity). The molecule of P. laminosum plastocyanin has 105 amino-acid residues. The single Cu atom is coordinated by the same residues - two histidines, a cysteine and a methionine - as in other plastocyanins. In the crystal structure, the three molecules of the asymmetric unit are related by a non-crystallographic threefold axis. A Zn atom lies between each pair of neighbouring molecules in this ensemble, being coordinated by a surface histidine residue of one molecule and by two aspartates of the other.

Amino Acid Sequence↗
PubMed1998

Characterization of a cDNA encoding the thylakoidal processing peptidase from Arabidopsis thaliana. Implications for the origin and catalytic mechanism of the enzyme.

We have identified and sequenced a cDNA containing a complete open reading frame for a putative 340-amino acid precursor of the thylakoidal processing peptidase from Arabidopsis thaliana. The predicted amino acid sequence of the protein includes regions highly conserved among Type I leader peptidases and indicates that the enzyme uses a serine-lysine catalytic dyad mechanism. Phylogenetic analysis indicated a common ancestry of the enzyme with those from oxygenic photosynthetic prokaryotes, suggesting that the cDNA encoded the chloroplast enzyme. The catalytic domain was overexpressed in Escherichia coli, generating a product capable of cleaving the thylakoid-transfer domain from a chloroplast protein. Antibodies to the overexpressed polypeptide cross-reacted with a 30-kDa thylakoid membrane protein.

Amino Acid Sequence↗