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C J Howe

Publications and source records attributed to C J Howe.

At least 37 records · Page 2Linked to original sources
PubMed1998

Phylogenetic analysis of plastid origins based on secA sequences.

We have generated secA sequence data from a number of photosynthetic prokaryotes and carried out a phylogenetic analysis using secA sequences from prokaryotes, green plants, and red and brown algae. We have studied the substitution patterns that give rise to the apparent phylogenetic structure. We show that the high AT content of the plastid sequences significantly affects the amino-acid composition. We also show that most of the apparent evidence for an edge separating red and brown plastids from green plants within the phylogenetic tree is due to differences in nucleotide composition. The remaining apparent evidence is likely to be due, at least in part, to differences in the distribution of sites free to vary. We discuss the implications of this study for hypotheses of plastid origins.

Adenosine Triphosphatases↗
PubMed1998

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages.

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.

Markov Chains↗
PubMed1997

Use of filter paper for sample collection and transport in steroid pharmacology.

Field studies of androgen pharmacology are complicated by the necessity to collect, process, and store blood samples in a central facility. We have assessed the feasibility of using capillary blood spots collected by fingerprick and dried on filter paper for pharmacokinetics and pharmacodynamic measurements with nandrolone and testosterone RIAs modified for extracts from capillary blood spots. Assays on punched spots of 7.9-mm diameter (14.9 microL of dried blood) permitted accurate quantification of testosterone down to 0.4 nmol/L from a single spot and nandrolone down to 0.9 nmol/L from two spots. Stability of the steroids in dried blood spots to adverse environmental conditions, notably increased temperatures, was investigated both in the laboratory and in field studies of dried spots sent through the postal system. Storage or postal transport under moderate conditions appeared to have no deleterious effects on apparent androgen concentrations. However, under extreme conditions of storage at 50 degrees C for a week or more, or transport to a very hot tropical location, a rise in the final concentration of nandrolone, and, to a lesser extent, testosterone when corrected for tracer recovery, was noticed. These effects were largely due to apparent susceptibility of tritiated tracer, but not unlabeled androgen, to thermal degradation. In a pilot pharmacological study involving intramuscular injection of 100 mg of nandrolone decanoate in 1 mL of arachis oil, nandrolone concentrations in concurrently collected plasma as well as venous and capillary blood spots showed good agreement. Testosterone concentrations in contemporaneously collected plasma and venous blood spots also showed very good agreement. We propose that these methods may allow patients and experimental subjects to self-collect samples at remote or field locations for convenient mailing to a central laboratory for androgen assay. Applications of this methodology are now under way.

Blood Chemical Analysis↗
PubMed1996

Inhibition by penem of processing peptidases from cyanobacteria and chloroplast thylakoids.

Proteins targeted to the thylakoid lumen of plants and cyanobacteria and the periplasmic space of cyanobacteria are synthesised with N-terminal presequences which are removed following translocation across the membrane. These presequences are thought to direct translocation of the preprotein by a sec-type pathway. Detergent extracts of cyanobacterial and chloroplast membranes contain enzymes which are capable of processing precursors to the mature size. We show that the processing of a range of precursors by both cyanobacterial and chloroplast enzymes is inhibited by the penem SB216357. This is the first report of an inhibitor of these enzymes and indicates that they are type 1 signal peptidases.

Chloroplasts↗
PubMed1996

Some characteristics of cytochrome f in the cyanobacterium Phormidium laminosum: its sequence and charge properties in the reaction with plastocyanin.

Part of the petCA operon was cloned and the sequence of the cytochrome f gene from the moderately thermophilic cyanobacterium Phormidium laminosum determined. A partial sequence of the petC gene encoding the Rieske iron-sulphur protein was also obtained. The cytochrome f gene encodes a mature protein of 385 residues and a leader sequence of 45 residues. The mature protein contains several acidic or neutral residues corresponding to basic residues in the turnip protein. Some of the latter are thought to be important for the interaction with plastocyanin via its "eastern' face. Many of the corresponding residues on the eastern face of P. laminosum plastocyanin are either basic or neutral instead of acidic. These comparisons suggested that the local charges on P. laminosum cytochrome f that are important for its interaction with the homologous plastocyanin may be negative rather than positive. The importance of acidic groups was confirmed by measuring the rates of reduction of horse heart cytochrome c and P. laminosum and spinach plastocyanins by the cytochrome bf complex isolated from P. laminosum. P. laminosum plastocyanin gave the highest rates, which decreased at high ionic strength, confirming the importance of positive local charges on this protein. When extrapolated to infinite ionic strength the rates observed with the two kinds of plastocyanin were similar, but cytochrome c became unreactive. An optimum was observed in the ionic strength response with P. laminosum plastocyanin.

Amino Acid Sequence↗
PubMed1996

Establishing the minimum effective dose and additive effects of depot progestin in suppression of human spermatogenesis by a testosterone depot.

Hormonally induced azoospermia induced by weekly im injections of testosterone enanthate provides effective and reversible male contraception, but more practical regimens are needed. Given our previous findings that six 200-mg pellets implanted subdermally produced more stable, physiological T levels and reduced the delivered T dose by more than 50% while maintaining equally effective suppression of sperm output with fewer metabolic side-effects than weekly 200-mg testosterone enanthate injections, we sought in this study to determine 1) whether further dose-sparing could be achieved by lower testosterone doses while maintaining efficacy and 2) the efficacy of adding a depot progestin to a suboptimally suppressive depot testosterone dose as a model depot progestin/androgen combination male contraceptive. Healthy volunteers were randomized into groups (n = 10) who received either of two lower T doses (two or four 200-mg T pellets) or four 200-mg T pellets plus a single im injection of 300 mg depot medroxyprogesterone acetate (DMPA). Two T pellets (400 mg, 3 mg/day) had a negligible effect on sperm output. Four T pellets (800 mg, 6 mg/day) suppressed sperm output between the second to fourth postimplant months; output returned to normal by the seventh postimplant month, although only 4 of 10 men became azoospermic or severely oligozoospermic (< 3 mol/L/mL). The addition of a depot progestin markedly increased the extent, but not the rate, of sperm output suppression, with 9 of 10 becoming azoospermic and 10 of 10 becoming severely oligozoospermic. There were no serious adverse effects during the study. Plasma total and free testosterone levels remained within the eugonadal range at all times with each treatment. Plasma epitestosterone was suppressed by all 3 regimens, consistent with a dose-dependent inhibition of endogenous Leydig cell steroidogenesis. Plasma LH and FSH measured by a two-site immunoassay were suppressed in a dose-dependent fashion by T and further suppressed by the addition of DMPA. Sex hormone-binding globulin levels were decreased by DMPA, but not by either T dose. Prostate-specific antigen and lipids (total, low or high density lipoprotein cholesterol, and triglycerides) were not significantly changed in any group. Thus, a depot testosterone preparation with zero order release must be delivered at between 6-9 mg/day to provide optimal (but not uniform) efficacy at inducing azoospermia. The addition of a single depot dose of a progestin to a suboptimal testosterone dose (6 mg/day) markedly enhances the extent, but not the rate, of spermatogenic suppression, with negligible biochemical androgenic side-effects. These findings provide a basis for the use of a progestin/androgen combination depot for hormonal male contraception.

Adult↗
PubMed1996

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle.

Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane. To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particle-like chaperone Ffh. We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species. We have cloned and sequences ffh from Synechococcus sp. PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts.

Bacterial Proteins↗
PubMed1996

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle.

Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane. To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particlelike chaperone Ffh. We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species. We have cloned and sequenced ffh from Synechococcus sp. PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts.

Amino Acid Sequence↗
PubMed1995

Location and sequence of rearranged immunoglobulin genes in human thymus.

Thymic B cells are a proliferating B cell population concentrated in normal thymic medulla. They are large cells, some with dendritic morphology, and are not associated with any organized follicular structure. Previous work in this laboratory has shown that most of these B cells are surrounded by tightly adherent thymocytes. The literature on human thymic B cells contains many inconsistencies. There is no consensus on whether they express CD5. Even the existence of thymic B cells has been questioned. In this study we have undertaken the first analysis of rearranged immunoglobulin (Ig) genes, looking in particular for evidence of affinity maturation. The Ig VH genes of human thymic B cells in this study are those of the fetal repertoire, though the resemblence to fetal Ig genes is limited in other respects. They are mostly unmutated, but the presence of mutated sequences suggests that this is not a uniform population, as has been previously indicated by phenotypic studies.

Antigens, CD↗
PubMed1995

Characterization of plastocyanin from the cyanobacterium Phormidium laminosum: copper-inducible expression and SecA-dependent targeting in Escherichia coli.

Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene cloned and sequenced. The derived amino acid sequence indicates that the plastocyanin protein is initially synthesized with an N-terminal leader sequence of 34 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a hydrophobic region and a cleavage site, which are characteristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides. The petE gene and flanking regions have been cloned in Escherichia coli, and the plastocyanin protein is expressed and directed to the periplasmic space, with concomitant processing to the mature form. Targeting to the periplasm and processing of the plastocyanin protein in E. coli appears to be dependent on components of the Sec apparatus, since the unprocessed precursor accumulates in the cytoplasm of a secA mutant. Expression of plastocyanin in E. coli is copper-inducible and apparently controlled at the level of transcription, leading to the conclusion that copper-regulated promoters exist in the regions flanking the gene and are recognized in a heterologous system. Possible implications for gene expression and protein targeting in the cyanobacterium are discussed.

Adenosine Triphosphatases↗
PubMed1995

Cloning and sequence analysis of a signal peptidase I from the thermophilic cyanobacterium Phormidium laminosum.

Type I signal peptidases are a widespread family of enzymes which remove the presequences from proteins translocated across cell membranes, including thylakoid and cytoplasmic membranes of cyanobacteria and thylakoid membranes of chloroplasts. We have cloned and sequenced a signal peptidase gene from the thermophilic cyanobacterium Phormidium laminosum which is believed to encode an enzyme common to both membrane systems. The deduced amino acid sequence is 203 residues long and although the overall similarity among signal peptidases is rather low there are a number of identifiable conserved regions present. The P. laminosum enzyme is predicted to have a single transmembrane domain, in contrast to other Gram-negative bacterial sequences, but similar to other type I signal peptidases.

Amino Acid Sequence↗
PubMed1994

The extrinsic proteins of photosystem II in photosynthetic organisms: distribution, properties and evolutionary implications.

Photosystem II from higher plant chloroplasts contains extrinsic proteins of molecular weights 33, 23 and 16 kDa. Cyanobacteria, which are thought to be closely related to the evolutionary progenitor of chloroplasts, have a protein homologous to the largest of these, but lack homologues of the smaller ones. Here we report immunological evidence for the occurrence of the 33 kDa extrinsic protein in red and chromophyte algae and its absence from anoxygenic photosynthetic bacteria. The 23 kDa protein was absent from all three groups. The red algal protein was found to be hydrophilic and rather more loosely associated with the thylakoid membrane than its higher plant equivalent. These findings support an origin for all plastids among the oxygenic prokaryotes, in contrast to the phylogeny inferred from ribulose bisphosphate carboxylase-oxygenase sequence data.

Biological Evolution↗
PubMed1994

Characterization of the stability of human and murine thymic B cell/thymocyte rosettes and investigation of unusual immunophenotypic findings.

In this study we have used immunohistochemistry to study the controversial expression of CD2 and cytokeratin by thymic B cells and also the expression of the adhesion molecules CD40 and CD80. The B cells were clearly CD2 and cytokeratin negative, however the majority expressed CD40 and some coexpression of CD80 was observed. We have previously observed that human thymic B cells are surrounded by rosetting thymocytes; a relationship which is maintained when cells are cultured. Here we show that the rosettes are remarkably stable in both man and mouse. They are dependent on intact surface protein but not on divalent cations, active metabolism, or components of the cytoskeleton. The rosettes could not be dissociated with antibodies to CD40 or CD80. Failure to consider such a stable rosette structure would result in sampling biases when purifying thymic B cell populations and could also contribute to some of the more unusual phenotypic findings such as the expression of CD2.

Animals↗
PubMed1994

Adverse events associated with childhood vaccines other than pertussis and rubella. Summary of a report from the Institute of Medicine.

In September 1993, the Institute of Medicine released a report entitled Adverse Events Associated With Childhood Vaccines: Evidence Bearing on Causality. The report examined putative serious adverse consequences associated with administration of diphtheria and tetanus toxoids; measles, mumps, and measles-mumps-rubella vaccines; oral polio vaccine and inactivated polio vaccine; hepatitis B vaccines; and Haemophilus influenzae type b (Hib) vaccines. The committee spent 18 months reviewing all available scientific and medical data, from individual case reports (published and unpublished) to controlled clinical trials. The committee found that the evidence favored the rejection of a causal relation between diphtheria and tetanus toxoids and encephalopathy, infantile spasms, and sudden infant death syndrome, and between conjugate Hib vaccines and susceptibility to Hib disease. The committee found that the evidence favored acceptance of a causal relation between diphtheria and tetanus toxoids and Guillain-Barré syndrome and brachial neuritis, between measles vaccine and anaphylaxis, between oral polio vaccine and Guillain-Barré syndrome, and between unconjugated Hib vaccine and susceptibility to Hib disease. The committee found that the evidence established causality between diphtheria and tetanus toxoids and anaphylaxis, between measles vaccine and death from measles vaccine-strain viral infection, between measles-mumps-rubella vaccine and thrombocytopenia and anaphylaxis, between oral polio vaccine and poliomyelitis and death from polio vaccine-strain viral infection, and between hepatitis B vaccine and anaphylaxis. For five vaccine-related adverse events, there was no evidence identified. For the remaining 33 vaccine-related adverse events, the evidence was inadequate to accept or reject a causal relation.

Child↗
PubMed1994

Cloning and characterisation of genes for tetrapyrrole biosynthesis from the cyanobacterium Anacystis nidulans R2.

The genes for 5-aminolevulinic acid dehydratase (ALAD) and uroporphyrinogen III synthase (UROS), two enzymes in the biosynthetic pathway for tetrapyrroles, were independently isolated from a plasmid-based genomic library of Anacystis nidulans R2 (also called Synechococcus sp. PCC7942), by their ability to complement Escherichia coli strains carrying mutations in the equivalent genes (hemB and hemD respectively). The identity of the genes was confirmed by comparing the appropriate enzyme activities in complemented and mutant strains. Subclones of the original plasmids that were also capable of complementing the mutants were sequenced. The inferred amino acid sequence of the cyanobacterial HemB protein indicates a significant difference in the metal cofactor requirement from the higher-plant enzymes, which was confirmed by overexpression and biochemical analysis. The organisation of the cyanobacterial hemD locus differs markedly from other prokaryotes. Two open reading frames were found immediately upstream of hemD. The product of one shows considerable similarity to published sequences from other organisms for uroporphyrinogen III methylase (UROM), an enzyme involved in the production of sirohaem and cobalamins (including vitamin B-12). The product of the other shows motifs which are similar to those found in proteins responsible for metabolic regulation in yeast and indicates that this family of transcription control proteins, which has previously been reported only from eukaryotes, is also represented in prokaryotes.

Amino Acid Sequence↗
PubMed1993

Components of the protein translocation machinery in the thermophilic cyanobacterium Phormidium laminosum.

Cyanobacteria possess cytoplasmic membranes, which are common to all eubacteria, and an additional internal network of thylakoid membranes. We have investigated the distribution of the protein translocation machinery between the two membrane systems. We have demonstrated that leader peptidase activity exists in both membrane systems, suggesting that newly synthesized protein precursors are targeted directly rather than sorted after translocation. Southern blot analysis suggests that the leader peptidase is likely to be a single enzyme common to both membrane systems. We have also shown that cyanobacteria possess a single SecA homologue. These results indicate that the protein translocation machinery in the two membrane systems is likely to be the same and leaves open the question of what mechanism enables the cell to sort proteins between them.

Adenosine Triphosphatases↗