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Biomedical subjects

M J Berry

Publications and source records attributed to M J Berry.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of critical amino acids for 3,5,3'-triiodothyronine deiodination by human type 1 deiodinase based on comparative functional-structural analyses of the human, dog, and rat enzymes.

The selenoenzyme, type 1 iodothyronine deiodinase (type 1 DI), catalyzes the activation of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) but 3,3',5'-triiodothyronine (rT3) is the preferred substrate for the human enzyme. Since the dog type 1 DI has a significantly lower affinity for rT3, we cloned the dog type 1 DI to identify amino acids critical for rT3 binding. The Km of the transiently expressed dog enzyme for rT3 5'-deiodination is 25-fold higher than that of the human enzyme. However, the Ki of T4 for rT3 deiodination by dog type 1 DI is only 3-fold higher than that for the human, suggesting that the differences between the two proteins affect binding of rT3 more than that of T4. Comparative competition studies in which rT3 or T4 is used to block covalent bromoacetyl T3 binding to the two proteins support this. Mutational studies showed that the critical differences between the dog (D) and human (H) enzymes are Asn (D) 45 Gly (H), Gly (D) 46 Glu (H), and Leu (D) 60:Phe (H) 65. Substitution of the human residues for those of the dog at these positions causes the predicted changes in the Km (rT3) and vice versa. A Phe65 to Leu mutation alone in the human enzyme increases the Km (rT3) 10-fold. We speculate that Phe65 is especially important for rT3 binding due to an interaction between the tyrosyl ring of rT3 and the aromatic ring of Phe65.

Amino Acid Sequence↗
PubMed1994

Activation and inactivation of thyroid hormone by type I iodothyronine deiodinase.

The prohormone thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3',5-triiodothyronine (T3) and both hormones are degraded by inner ring deiodination (IRD) to 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine, respectively. Indirect evidence suggests that the type I iodothyronine deiodinase (ID-I) in liver has both ORD and IRD activities, with preference for rT3 and sulfated iodothyronines as substrates. To establish this, we have compared the ORD of rT3 and IRD of T3 and T3 sulfate by homogenates of cells transfected with rat ID-I cDNA and by rat liver microsomes. In both preparations rT3 is the preferred substrate, while deiodination of T3 is markedly accelerated by its sulfation. Kinetic analysis provided similar Km and Vmax values in cell homogenates and liver microsomes. These data demonstrate unequivocally that ID-I is capable of both activating and inactivating thyroid hormone by ORD and IRD, respectively.

Animals↗
PubMed1994

Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.

Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.

Amino Acid Sequence↗
PubMed1994

A comparison between aero and standard racing handlebars during prolonged exercise.

The use of aero style handlebars has gained popularity in triathlons and individual time trial events recently. The effect of using these handlebars has only been examined during short term exercise. It was the aim of this investigation to compare time to exhaustion and selected measures of ventilation, gas exchange and pulmonary function when using aero style handlebars versus standard racing style handlebars during long term exercise. Eleven well trained cyclists with a VO2max of 61.7 +/- 2.9 ml.kg-1.min-1 completed two continuous rides at 80 to 95 percent of their VO2max. Subjects rode at 80% of their VO2max for one hour and the work rate was increased by 5% of their VO2max every 15 min thereafter. The rides were completed with the subject riding his/her personal bicycle on a Velodyne trainer and differed in that during one ride the subjects rode with aero style handlebars and during the other they rode with standard racing style handlebars. During the ride with standard racing style handlebars, subjects rode with their hands on top of the bars or on the hoods of the brake levers. The subjects rode with the standard racing handlebars for 69.3 +/- 5.3 minutes as compared to 59.8 +/- 7.0 minutes with the aero style handlebars. This difference was not statistically significant. The average work rates when the cyclists terminated the exercise bouts with the aero and standard racing handlebars were 268.1 +/- 38.4 and 276.4 +/- 39.3 watts and were not significant from one another. Gas exchange and ventilatory parameters were measured every 15 min during the exercise bouts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Stability of immunoadsorbents comprising antibody fragments. Comparison of Fv fragments and single-chain Fv fragments.

Immunoadsorbents comprising Fv fragments specific for hen egg lysozyme were used to recover the enzyme from a 20-fold excess of bovine albumin. We designed automatic equipment to run this model purification system for 100 cycles non-stop and monitored the deterioration of the immunoadsorbents during the cycling procedure. Only minor losses (approximately 25%) in the immunoadsorbents' capacity were detected; this correlated well with ligand loss (measured by enzyme-linked immunosorbent assay) which was approximately 0.2% per cycle. A surprising finding was that the use of "single-chain" Fv fragments conferred only a minor advantage with respect to stability of the immunoadsorbents.

Animals↗
PubMed1993

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.

Animals↗
PubMed1993

Molecular cloning of the selenocysteine-containing enzyme type I iodothyronine deiodinase.

It has proved impossible to purify the three types of iodothyronine deiodinase to homogeneity by use of standard physiochemical techniques. However, using expression cloning with Xenopus oocytes, we were successful in isolating a cDNA coding for the rat type I iodothyronine deiodinase. An extremely puzzling aspect of this cDNA was an in-frame thymine-guanine-adenine (TGA) codon at position 126. By mutagenesis studies we showed that this coded for selenocysteine, consistent with the previously described requirement for selenium for hepatic and renal thyronine deiodination. The cloned enzyme can be expressed transiently in JEG, a human cell line, or COS, a monkey cell line, by gene-transfer techniques, and the expressed protein has the appropriate biochemical characteristics of type I iodothyronine deiodinase. These include a newly identified extreme sensitivity to competitive inhibition of reverse-3,5,3'-triiodothyronine (rT3) deiodination by gold. The identification of this elusive protein should prove helpful in understanding the process of thyroid hormone activation.

Animals↗
PubMed1993

Cardiovascular responses in black and white males during exercise.

Previous investigations have shown blacks to have a significantly lower resting heart rate (HR) compared with whites. Our purpose was to determine if this difference exists during submaximal exercise and to compare other cardiovascular responses during submaximal exercise in black and white males. Sixteen black and 16 white males matched on age, body surface area, and maximal O2 consumption exercised at 0, 50, and 100 W on a cycle ergometer. HR, O2 consumption, and cardiac output via CO2 rebreathing were measured at rest and at each work rate. Stroke volume was then calculated. O2 consumption was not significantly different between blacks and whites at rest or at work rates of 0, 50, or 100 W. Cardiac output increased from rest with 0, 50, and 100 W work for both blacks and whites (6.1 to 13.0, 14.4, and 16.9 l/min and 5.7 to 12.2, 14.3, and 16.3 l/min, respectively). The differences in cardiac output between blacks and whites at rest and all work rates were not statistically significant. At rest and work rates of 0, 50, and 100 W, HR was significantly lower in blacks compared with whites (71, 99, 108, and 119 beats/min vs. 80, 107, 114, and 127 beats/min, respectively). The lower HR in blacks compared with whites was accompanied by a trend toward a higher stroke volume at rest and work rates of 0, 50, and 100 W (85.2, 130.3, 134.7, and 142.9 ml vs. 72.5, 114.9, 126.4, and 127.4 ml, respectively). No differences in resting blood pressures were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Physiological and genetic analyses of inbred mouse strains with a type I iodothyronine 5' deiodinase deficiency.

Inbred mouse strains differ in their capacity to deiodinate iododioxin and iodothyronines, with strains segregating into high or low activity groups. Metabolism of iododioxin occurs via the type I iodothyronine 5'deiodinase (5'DI), one of two enzymes that metabolize thyroxine (T4) to 3,5,3'-triiodothyronine (T3). Recombinant inbred strains derived from crosses between high and low activity strains exhibit segregation characteristic of a single allele difference. Hepatic and renal 5'DI mRNA in a high (C57BL/6J) and low (C3H/HeJ) strain paralleled enzyme activity and concentration, in agreement with a recent report. 5'DI-deficient mice had twofold higher serum free T4 but normal free T3 and thyrotropin. Brown adipose tissue 5'DII was invariant between the two strains. Southern analyses using a 5'DI probe identified a restriction fragment length variant that segregated with 5'DI activity in 33 of 35 recombinant inbred strains derived from four different pairs of high and low activity parental strains. Recombination frequencies using previously mapped loci allowed assignment of the 5'DI gene to mouse chromosome 4 and identified its approximate chromosomal position. We propose the symbol Dio1 to denote the mouse 5'DI gene. Conserved linkage between this segment of mouse chromosome 4 and human HSA1p predicts this location for human Dio1.

Animals↗
PubMed1993

The type I iodothyronine 5'-deiodinase messenger ribonucleic acid is localized to the S3 segment of the rat kidney proximal tubule.

A complementary DNA clone encoding the type I iodothyronine 5'-deiodinase (5'DI), which converts T4 to T3, has been isolated recently. The 5'DI messenger RNA (mRNA) is most abundant in kidney and liver. To gain insight into the function of 5'DI in the kidney, Northern blot analysis was used to localize the expression of this mRNA. Our results show that 5'DI mRNA was expressed in the cortex and outer medulla, but not inner medulla and papilla, indicating that there are regional differences in its expression. Using in situ hybridization, we demonstrate that the 5'DI hybridization signal is localized predominantly over tubules in the outer stripe of the outer medulla and in medullary rays, suggesting localization to proximal tubules. To identify which tubular cells express 5'DI mRNA, we compared the profile of 5'DI message from in situ hybridization with the immunostaining of adjacent sections with proximal tubular S1, S2, and S3 segment-specific antibodies. Most of the 5'DI antisense complementary RNA hybridized to the same proximal tubular cells with which the S3-specific antibody, anti-ecto-ATPase, reacted. Cells staining with an S1 or S2 segment antibody showed little, if any, 5'DI mRNA. We conclude that the expression of 5'DI mRNA is restricted to the tubular cells of the proximal S3 segment. The S3 segment is also known to express high levels of proteins required for glutathione synthesis consistent with the requirement for a reduced thiol cofactor for iodothyronine deiodination by the 5'DI pathway.

Animals↗
PubMed1993

Effects of body mass on exercise efficiency and VO2 during steady-state cycling.

Oxygen uptake (VO2) and exercise efficiency during cycle ergometer exercise are considered to be independent of body mass. To determine the validity of this assumption, 50 females ranging in body mass from 41.5-98.9 kg exercised on a cycle ergometer with no load at 60 rpm and at 25, 50, 75, and 100 W at 60 and 90 rpm. Gross VO2 and efficiency, net VO2 and efficiency, work VO2 and efficiency, and delta efficiency were computed. Gross and net VO2 were significantly and positively correlated with body mass at all work rates and pedal frequencies. Gross efficiency was significantly and negatively correlated with body mass at all work rates and pedal frequencies. Work VO2 and body mass were not significantly correlated. The correlations between work and delta efficiency and body mass were not significant. Since body mass was found to be significantly correlated with gross VO2, the following equation was developed using stepwise multiple regression to predict gross VO2: VO2 (ml.min-1) = 10.9 (work rate, W) + 8.2 (pedal rate, rpm) + 8.3 (body mass, kg) - 559.6. These data suggest that body mass should be considered when estimating the oxygen uptake during cycle ergometer exercise.

Adult↗
PubMed1993

Relationship of lactate and ventilatory thresholds in cardiac transplant patients.

The ventilatory threshold (VT) has been suggested as a method for determining exercise training intensity in cardiac transplant patients (CTPs). Since the VT has not been validated against the more accepted marker of the anaerobic threshold, the lactate threshold (LT), in CTPs, the purpose of this investigation was to compare the VT to the LT within, as well as between, CTPs and normal subjects (NLs). Ten male orthotopic CTPs and 10 age, size, and gender matched NLs were exercised to symptom-limited maximal levels following incremental treadmill protocols. The VT was determined using the V-slope method, and the LT was identified using the log-log transformation method. The NLs and significantly higher absolute levels of VO2 at both the VT (1298.6 +/- 78.5 vs 919.0 +/- 57.2 ml.min-1) and LT (1561.1 +/- 144.2 vs 921.6 +/- 47.6 ml.min-1) compared with the CTPs. However, there was no significant difference in the relative VO2 (% peak) between CTPs and NLs at the VT (57.2 +/- 3.0 vs 49.0 +/- 3.5%) or LT (58.2 +/- 3.3 vs 58.5 +/- 4.9%), respectively. Within groups there was no significant difference between the VT and LT for either CTPs (919.0 +/- 57.2 vs 921.6 +/- 47.6 ml.min-1 or NLs (1298.6 +/- 78.5 vs 1561.1 +/- 144.2 ml.min-1). From the results of this investigation it appears that the VT may be used as an indicator of LT in CTPs and is within a range acceptable for clinical application.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Identification of essential histidine residues in rat type I iodothyronine deiodinase.

Deiodination is required for conversion of thyroxine, the inactive prohormone secreted by the thyroid gland, to 3,5,3'-triiodothyronine, the biologically active thyroid hormone. The principal enzyme catalyzing this reaction, Type I iodothyronine 5' deiodinase, was shown recently to contain the amino acid, selenocysteine, and site-directed mutagenesis showed that this amino acid confers the biochemical properties characteristic of this enzyme. Previous studies suggest that a histidine residue may also be critical for activity. To further our understanding of the biochemical mechanism of this reaction, we have used in vitro mutagenesis to examine the contribution of each of the 4 histidines in this enzyme to the deiodination process. Two of the histidines (185 and 253) are not involved in deiodination, as their removal had no effect on activity. Mutagenesis of histidine 158 resulted in complete loss of activity, suggesting a role in either protein conformation or catalysis. The most informative results were obtained from the studies of histidine 174. Mutagenesis of this histidine to asparagine or glutamine altered reactivity with substrate and reduced inhibition by diethylpyrocarbonate and rose bengal. These results demonstrate that histidine 174 is critical to function and appears to be involved in binding of hormone.

Amino Acid Sequence↗
PubMed1992

Use of antibody fragments in immunoaffinity chromatography. Comparison of FV fragments, VH fragments and paralog peptides.

Some new antibody fragments have recently been described: FV fragments (Mr 25,000), VH fragments or "dAbs" (12,500) and paralog peptides (1000-2000). FV fragments, VH fragments and a paralog peptide that had been derived from a parent antibody with a specificity for hen lysozyme were produced. All three reagents were immobilized on Sepharose and evaluated for their ability to recover hen lysozyme from "spiked" serum and to separate hen lysozyme from turkey lysozyme. The FV column had excellent specificity for hen lysozyme, the VH column had significantly reduced specificity and the paralog peptide column did not bind lysozyme at all.

Animals↗
PubMed1992

Substitution of cysteine for selenocysteine in type I iodothyronine deiodinase reduces the catalytic efficiency of the protein but enhances its translation.

Type I iodothyronine 5' deiodinase (5'DI) contains selenocysteine, encoded by a UGA codon, and this amino acid is essential for maximum catalytic efficiency in this enzyme. We recently showed that translation of UGA as selenocysteine in this protein requires a specific sequence of about 250 nucleotides in the 3' untranslated region of the messenger RNA. Translation of a 5'DI cysteine mutant does not require the 3' untranslated region. To examine both the efficiency of UGA codon recognition and the relative catalytic efficiency of selenocysteine vs. cysteine in 5'DI, we used bromoacetyl 125I-T3 labeling to quantitate transiently expressed selenocysteine (wild type) and cysteine containing type I iodothyronine deiodinases in transfected COS-7 and JEG-3 cell lines. Kinetic analyses of the same cell sonicates were performed to determine the apparent maximum velocity and Michaelis-Menten constant values for reverse T3 5' deiodination. COS-7 cells express the cysteine mutant protein at about 20-fold and JEG-3 cells about 400-fold higher levels than the selenoenzyme. However, in both cell types, the apparent catalytic constant values were at least 100-fold higher for the wild-type enzyme, compared with the cysteine mutant. These results indicate that cell lines differ markedly in their capacity to translate UGA-containing messenger RNAs. The much higher catalytic constant values for the selenium-containing enzyme illustrate the biochemical advantage of this element as compared with sulfur in the catalysis of iodothyronine deiodination.

Catalysis↗