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Biomedical subjects

M J Berry

Publications and source records attributed to M J Berry.

At least 91 records · Page 5Linked to original sources
PubMed1992

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Affinity Labels↗
PubMed1992

A comparison between two forms of aerobic dance and treadmill running.

Aerobic dance has been reported to result in a disproportionately higher heart rate than running at a similar percent of VO2max. It has been suggested that the extensive use of the arms overhead during aerobic dance results in an increase in sympathetic outflow thereby disproportionately increasing the heart rate. To compare the hemodynamic and sympathetic nervous system activity responses during aerobic dance and treadmill running, nine healthy females exercised at approximately 50% of their VO2max during each of the following three exercise trials: aerobic dance where the arms were used extensively overhead (ABOVE), aerobic dance where the arms were kept below the shoulders (BELOW), and treadmill running (TR). Mean heart rate values during the ABOVE, BELOW, and TR trials were 136 beats.min-1 for all three trials. Mean VO2 values during the ABOVE, BELOW, and TR trials were 1.48, 1.51, and 1.47 l.min-1, respectively, and were not significantly different. Mean cardiac output for the ABOVE, BELOW, and TR trials were 13.5, 14.0, and 13.0 1. min-1, respectively, and were not significantly different. Postexercise blood lactate and norepinephrine values were not significantly different among the three trials. These results suggest a similar relationship between heart rate and VO2 during low intensity aerobic dance and running and do not support the contention that the use of the arms overhead during aerobic dance exercise elicits a disproportionately greater increase in heart rate as compared with running. Additionally these results demonstrate similar cardiovascular and sympathetic nervous system responses between aerobic dance exercise and running.

Adult↗
PubMed1991

Immobilization of Fv antibody fragments on porous silica and their utility in affinity chromatography.

Recent advances in molecular biology have allowed antibody binding domains to be cloned and expressed in Escherichia coli. The use of Fv antibody fragments as ligands in immunoaffinity chromatography is reported. Fv fragments specific for hen-egg lysozyme were immobilized on porous silica and used to recover antigen from spiked serum in a single step. Comparison with a conventional immunoadsorbent (whole antibodies immobilized on silica) showed the Fv-silica to have a fivefold superior capacity. Analysis of sectioned Fv-silica particles by immunoelectron microscopy indicated that captured antigen was evenly distributed throughout the internal porous structure of the particle.

Animals↗
PubMed1991

Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region.

Selenocysteine is incorporated cotranslationally at UGA codons, normally read as stop codons, in several bacterial proteins and in the mammalian proteins glutathione peroxidase (GPX), selenoprotein P and Type I iodothyronine 5' deiodinase (5'DI). Previous analyses in bacteria have suggested that a stem-loop structure involving the UGA codon and adjacent sequences is necessary and sufficient for selenocysteine incorporation into formate dehydrogenase and glycine reductase. We used the recently cloned 5'DI to investigate selenoprotein synthesis in eukaryotes. We show that successful incorporation of selenocysteine into this enzyme requires a specific 3' untranslated (3'ut) segment of about 200 nucleotides, which is found in both rat and human 5'DI messenger RNAs. These sequences are not required for expression of a cysteine-mutant deiodinase. Although there is little primary sequence similarity between the 3'ut regions of these mRNAs and those encoding GPX, the 3'ut sequences of rat GPX can substitute for the 5'DI sequences in directing selenocysteine insertion. Computer analyses predict similar stem-loop structures in the 3'ut regions of the 5'DI and GPX mRNAs. Limited mutations in these structures reduce or eliminate their capacity to permit 5'DI translation. These results identify a 'selenocysteine-insertion sequence' motif in the 3'ut region of these mRNAs that is essential for successful translation of 5'DI, presumably GPX, and possibly other eukaryotic selenocysteine-containing proteins.

Animals↗
PubMed1991

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.

Animals↗
PubMed1991

Type I iodothyronine deiodinase is a selenocysteine-containing enzyme.

Although thyroxine (3,5,3',5'-tetraiodothyronine, T4) is the principal secretory product of the vertebrate thyroid, its essential metabolic and developmental effects are all mediated by 3,5,3'-triiodothyronine (T3), which is produced from the prohormone by 5'-deiodination. The type-I iodothyronine deiodinase, a thiol-requiring propylthiouracil-sensitive oxidoreductase, is found mainly in liver and kidney and provides most of the circulating T3(1) but so far this enzyme has not been purified. Using expression cloning in the Xenopus oocyte, we have isolated a 2.1-kilobase complementary DNA for this deiodinase from a rat liver cDNA library. The kinetic properties of the protein expressed in transient assay systems, the tissue distribution of the messenger RNA, and its changes with thyroid status, all confirm its identity. We find that the mRNA for this enzyme contains a UGA codon for selenocysteine which is necessary for maximal enzyme activity. This explains why conversion of T4 to T3 is impaired in experimental selenium deficiency and identifies an essential role for this trace element in thyroid hormone action.

Amino Acid Sequence↗
PubMed1991

Evidence that cysteine, not selenocysteine, is in the catalytic site of type II iodothyronine deiodinase.

Recent cloning of the cDNA for Type I iodothyronine deiodinase revealed that the mRNA contains a UGA codon encoding the amino acid selenocysteine. Mutagenesis of the selenocysteine codon to a cysteine codon produced a protein with lower deiodinase activity. The presence or absence of selenocysteine in Type II deiodinase, which differs from the Type I enzyme in a number of parameters, has not been determined. Gold inhibits the activity of both the Type I deiodinase and the only other known eukaryotic selenocysteine-enzyme, glutathione peroxidase. Substitution of cysteine for selenocysteine in Type I deiodinase reduced its sensitivity to inhibition by gold 500-fold. We found that gold thioglucose was a competitive inhibitor with respect to the iodothyronine substrate of both deiodinases. However, the Type II enzyme from brown fat and pituitary was 100 to 1000-fold less sensitive to gold than was Type I activity in liver and pituitary, similar to the results with the cysteine-substituted Type I enzyme. This suggests that Type II deiodinase contains cysteine instead of selenocysteine in the active site.

Adipose Tissue, Brown↗
PubMed1991

Dissociation of the ventilatory and lactate thresholds following caffeine ingestion.

Caffeine ingestion prior to the start of exercise has been shown to have an effect on ventilatory parameters and substrate utilization. Changes in either substrate utilization or ventilatory parameters may influence the determination of the lactate threshold (LT) and/or the ventilatory threshold (VT). Therefore, it was the purpose of this investigation to determine whether the VT and LT occur at similar metabolic rates and what effect caffeine ingestion will have on these two measures. Ten male subjects completed two maximal exercise bouts on the treadmill using a single blind procedure. One trial was performed 45 min after the ingestion of caffeine citrate (CC) in an amount equal to 7.0 mg of anhydrous caffeine.kg-1 body weight. The second trial was performed 45 min after the ingestion of a gelatin powdered placebo (P). Ventilatory parameters were monitored on a breath-by-breath basis, and blood for lactate determination was obtained from an antecubital vein every minute. Maximal oxygen consumption did not differ significantly between the CC (60.3 +/- 5.2 ml.kg-1.min-1) and P (59.7 +/- 5.6 ml.kg-1.min-1) trials. Oxygen consumption (VO2) values during the P trial at the VT (40.2 +/- 6.1 ml.kg-1.min-1) and the LT (38.6 +/- 3.3 ml.kg-1.min-1) were not significantly different (P less than 0.05). During the CC trial, VO2 values at the VT (44.4 +/- 6.6 ml.kg-1.min-1) and the LT (39.7 +/- 5.8 ml.kg-1.min-1) were significantly different. When comparing the VO2 at the LTs between the CC and P trials, there was no significant difference. There was, however, a significant difference in VO2 at the VTs when comparing the two trials. These data demonstrate a dissociation between the VT and LT following caffeine ingestion and suggest that the use of the VT as an indicator of the LT may be inappropriate following ingestion of moderate dosages of caffeine.

Adult↗
PubMed1990

Cytochrome c'' isolated from Methylophilus methylotrophus. An example of bis-histidine-co-ordinated Fe3+ haem, with near-perpendicular orientation of the ligands.

Cytochrome c'' (Methylophilus methylotrophus) is a soluble protein, Mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. Low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 M-1.cm-1 at 5 T, 4.2 K) charge-transfer band at 1560 nm, establishing that the oxidized haem is co-ordinated by two His residues in a near-perpendicular orientation. This conformation is well established for transmembrane b cytochromes, but this appears to be the first example in a water-soluble cytochrome. The low-temperature m.c.d. spectra of the reduced form of the protein confirms that the haem contains a high-spin Fe2+ ligated by one His residue. The redox-linked spin-state change releases a His group. Since this residue is likely to bind a proton at pH values less than 6.5, this cytochrome may provide a useful model of a molecular mechanism of a redox-linked proton uptake and release process.

Bacteria↗
PubMed1990

The thermoregulation of pregnant women during aerobic exercise in the water: a longitudinal approach.

Twelve women early in their pregnancies were recruited to examine thermoregulation during immersion and exercise in the water (30 degrees C). Their responses were compared at 15, 25 and 35 weeks of pregnancy as well as 10-12 weeks post pregnancy to determine whether the responses differ between the gravid and non-gravid woman or were modified during pregnancy. Rectal temperature, mean skin temperature, heat storage, and evaporation were similar during immersion or exercise during the 15th, 25th and 35th weeks of pregnancy. Compared to 10 weeks post partum, pregnancy reduced heat storage, lowered skin temperature and increased evaporative heat loss during immersion and exercise (P less than 0.05). The results suggest that pregnancy causes subtle changes in the mechanism of thermoregulation which tend to increase heat production and improve heat conservation.

Adult↗
PubMed1990

Renal responses to immersion and exercise in pregnancy.

Twelve healthy pregnant women were studied at 15, 25, and 35 weeks' gestation and at 8 to 12 weeks postpartum. Women were immersed for 20 minutes at 30 degrees C. They then exercised at 60% maximum oxygen capacity on a modified ergometer. Substantial diuresis and natriuresis occurred without changes in osmolarity or serum sodium. The diuresis was significantly greater during pregnancy than postpartum. The natriuresis was similar. Diuresis and natriuresis were greater than would be expected from investigations in nonpregnant subjects. This study suggests that immersion may be a beneficial therapy for edema without decreasing plasma volume.

Diuresis↗
PubMed1990

Parental influenza virion nucleocapsids are efficiently transported into the nuclei of murine cells expressing the nuclear interferon-induced Mx protein.

The interferon-induced murine Mx1 protein, which is localized in the nucleus, most likely specifically blocks influenza virus replication by inhibiting nuclear viral mRNA synthesis, including the mRNA synthesis catalyzed by inoculum (parental) virion nucleocapsids (R. M. Krug, M. Shaw, B. Broni, G. Shapiro, and O. Haller, J. Virol. 56:201-206, 1985). We tested two possible mechanisms for this inhibition. First, we determined whether the transport of parental nucleocapsids into the nucleus was inhibited in murine cells expressing the nuclear Mx1 protein. To detect the Mx1 protein, we prepared rabbit antibodies against the Mx1 protein with a CheY-Mx fusion protein expressed in bacteria. The fate of parental nucleocapsids was monitored by immunofluorescence with an appropriate dilution of monoclonal antibody to the nucleocapsid protein. The protein synthesis inhibitor anisomycin was added to the cells 30 min prior to infection, so that the only nucleocapsids protein molecules in the cells were those associated with nucleocapsids of the parental virus. These nucleocapsids were efficiently transported into the nuclei of murine cells expressing the Mx1 protein, indicating that this protein most likely acts after the parental nucleocapsids enter the nucleus. The second possibility was that the murine Mx1 protein might act in the nucleus to inhibit viral mRNA synthesis indirectly via new cap-binding activities that sequestered cellular capped RNAs away from the viral RNA transcriptase. We show that the same array of nuclear cap-binding proteins was present in Mx-positive and Mx-negative cells treated with interferon. Interestingly, a large amount of a 43-kDa cap-binding activity appeared after interferon treatment of both Mx-positive and Mx-negative cells. Hence, the appearance of new cap-binding activities was unlikely to account for the Mx-specific inhibition of viral mRNA synthesis. These results are most consistent with the possibility that the Mx1 protein acts directly to inhibit the viral transcriptase in the nucleus.

Animals↗
PubMed1990

Thyroid hormone regulates type I deiodinase messenger RNA in rat liver.

Conversion of the prohormone T4 to the active hormone T3 is catalyzed by 5'-deiodinases, enzymes that have not been purified. Previous studies have shown that modulating thyroid status results in changes in type I deiodinase activity in the rat liver. We have quantitated type I deiodinase mRNA in liver by an expression assay using Xenopus laevis oocytes. We report here that changes in enzyme activity correlate closely with changes in levels of the mRNA for this enzyme, indicating that thyroid hormone regulates type I deiodinase at a pretranslational step. Using the oocyte system to express size-fractionated mRNA, we have also determined that the mRNA coding for this protein is between 1.9-2.4 kilobases in length. It has been proposed that protein disulfide isomerase (PDI) is closely related to the rat type I 5'-deiodinase. Our results indicate that this is not the case, since injection of in vitro transcribed PDI mRNA into oocytes did not result in expression of deiodinase activity, and the deiodinase mRNA could be physically separated from the 2.8-kilobase mRNA species hybridizing to rat PDI cRNA by size fractionation.

Animals↗
PubMed1990

The beta-endorphin responses of pregnant women during aerobic exercise in the water.

To determine the effect of pregnancy on the plasma beta-endorphin response to exercise in the water, 12 women were tested during the 15th, 25th, and 35th wk of pregnancy and 10 wk post partum. Each trial consisted of 20 min of lateral supine rest on land, 20 min of immersion to the level of the xiphoid in 30 degrees C water, 20 min of exercise at 60% predicted maximal capacity, and 20 min of lateral supine recovery. Resting beta-endorphin concentrations were elevated during the 15th wk (137.4 +/- 77.4 pg.ml-1) compared to post partum levels (19.8 +/- 17.6). However, neither was different from the 25th (65.6 +/- 68.4) or 35th (48.0 +/- 54.4) wk. Immersion increased beta-endorphin during the post partum trials but resulted in no consistent change during pregnancy. Conversely, exercise had no effect on beta-endorphin post partum but significantly elevated it during pregnancy, the greatest increase occurring during the 15th wk compared to the 25th or 35th wk. Twenty minutes after exercise, beta-endorphin returned to resting levels during all trials. It was concluded that the effect of pregnancy on plasma beta-endorphin is greater than the effect of exercise. Furthermore, pregnancy seems to accentuate the exercise-induced increase in beta-endorphin.

Adult↗
PubMed1990

The effects of elastic tights on the post-exercise response.

It has previously been demonstrated that graduated compression stockings will affect the post-exercise venous lactate profile. To determine the effects of elastic tights on venous lactate levels and the post-exercise response, eight males completed three exercise bouts on a motor driven treadmill. Each subject ran on the treadmill for up to three minutes at 110% of his VO2max. The conditions for the three exercise bouts were: elastic tights worn during exercise and recovery, elastic tights worn only during exercise, and no elastic tights worn during exercise or recovery. Oxygen consumption, heart rates and venous blood samples, for lactate and hematocrit determination, were obtained at rest and at 5, 15 and 30 min post-exercise. Analysis revealed no significant differences (p greater than 0.05) in any of the above variables between the three trials at any of the measurement times. These results indicate that the use of elastic tights will not significantly affect the post-exercise response or circulating lactate levels.

Adult↗
PubMed1989

Ratings of perceived exertion in individuals with varying fitness levels during walking and running.

It was the purpose of this investigation to: 1) compare the ratings of perceived exertion (RPEs) in high and low fit individuals when walking and running at comparable exercise intensities and 2) to determine if ventilation (VE) provides a central signal for RPEs. Nine high fit and nine low fit male subjects completed two exercise bouts on a treadmill, one uphill walking and the other level running. Workloads for each bout were set at 90% of each subject's ventilatory threshold (VT) as determined from a graded exercise test. Oxygen consumption (Vo2), heart rate (HR), and VE were all similar between the walk and run trials for the low fit subjects (P greater than 0.05). HR were found to be significantly greater during the walk trial vs. the run trial (P less than 0.05) for the high fit subjects, whereas, VE was significantly greater during the run trial. Oxygen consumption was similar for the high fit subjects during both trials (P greater than 0.05). During the walk and run trials, central (12.1 +/- 1.6 vs. 11.4 +/- 1.5), local (14.0 +/- 1.3 vs. 13.9 +/- 1.1) and overall (12.8 +/- 1.2 vs. 12.4 +/- 1.4) RPEs were not found to be significantly different for the low fit group (P greater than 0.05). In contrast, during the walk vs. the run trial there was a significant increase in central (10.7 +/- 2.0 vs. 9.2 +/- 1.9), local (11.5 +/- 2.0 vs. 9.8 +/- 1.8) and overall (11.2 +/- 2.4 vs. 9.6 +/- 2.3) RPEs for the high fit group (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Ventilatory responses during varied stride and pedal frequencies.

The effects of limb movement frequency during walking and running and cycling at 60 and 90 rpm on the ventilatory responses were studied in 19 male subjects. Ten of the subjects were trained runners whereas nine of the subjects were trained cyclists. The runners completed walk and run exercise trials at equal levels of CO2 excretion (VCO2) and low (approximately 60) rpm and high (approximately 90) rpm cycling trials at equal VCO2 levels. The cyclists completed low and high rpm cycling trials at equal VCO2 levels. The cyclists were not tested on the walk/run trials as they had been tested previously and had been shown to have similar ventilatory responses when walking and running at equal VCO2 levels. Minute ventilation (VE), tidal volume (VT), breathing frequency (f), end-tidal CO2 (PETCO2) and O2 (PETO2) tensions, and inspiratory (TI) and expiratory (TE) times were not found to differ significantly between the low and high pedal frequency trials for either the cyclists or the runners. No significant differences were found in estimated arterial CO2 tensions (PaCO2) or estimated alveolar ventilation (VA) between the cycling trials for either the runners or the cyclists. When comparing running to walking, the runners were found to have a greater VE and estimated VA. This was mediated by an increase in f as VT was found to decrease. The increased f was associated with a shortened TI as TE was not significantly different between the walk and run trials. End-tidal CO2 tension and estimated PaCO2 was significantly lower during the run trial as compared to the walk trial. These results suggest some form of neurogenic stimuli influencing ventilation in the runners while running. This same neurogenic influence is not present when cyclists run and when either cyclists or runners exercise on the bicycle. A possible source for the neurogenic stimuli is discussed.

Adult↗