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Biomedical subjects

C M Chow

Publications and source records attributed to C M Chow.

At least 37 records · Page 2Linked to original sources
PubMed1994

Effects of REM sleep on the ventilatory response to airway occlusion in the dog.

We determined the effects of sleep state on the ventilatory response following transient airway occlusion and on the response to vagal blockade in the unanesthetized sleeping dog. Three tracheotomized dogs underwent repeated occlusions (159 trials) during rapid eye movement (REM) and nonrapid eye movement (NREM) sleep. In all sleep states we found significant but variable transient hyperventilation following release of occlusion. In NREM sleep, a significant central apnea [expiratory time (TE) prolonged 2-10 times control] followed the hyperpneic response, so long as the increase in tidal volume (VT) during the hyperpnea exceeded three times control VT, that is, a volume-dependent apneic threshold. In REM sleep with maintained levels of eye movement density, hyperventilation commonly followed release of obstruction but only very rarely did VT exceed the volume threshold, and central apnea was rare. Cervical vagal blockade was used to show that significant inhibitory pulmonary stretch receptor reflexes were present in both NREM and REM sleep, although the strength of the reflex was diminished in REM. We postulate that the phasic events of REM sleep inhibit the increase in VT in response to the chemical stimuli accumulated during airway occlusion and also interfere with the prolongation of TE in response to lung stretch and/or transient hypocapnia. The result is that central apnea occurs only very rarely in REM sleep.

Airway Obstruction↗
PubMed1994

The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source.

A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose. A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A. bisporus cDNA library. Two allelic cDNAs were isolated. They showed 98.8% identity of their nucleotide sequences. The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei. Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence. Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose. At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose. Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose. Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources. Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures. A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia.

Agaricus↗
PubMed1994

A volume-dependent apneic threshold during NREM sleep in the dog.

We determined the causes of central apnea that commonly follow the hyperpnea resulting from brief airway occlusion during non-rapid-eye-movement (NREM) sleep. Ventilation and end-tidal gases were measured before, during, and after 214 trials of 15-20 s of tracheal occlusion in three dogs during NREM sleep. Airway occlusion was accompanied by progressive increases in inspiratory effort and was followed by transient one- to four-breath hyperapneas, with subsequent central apnea [3-15 times eupneic control expiratory duration (TE)] in 62% of occlusion trials. Significant TE prolongation after hyperventilation did not occur until tidal volume (VT) was three times greater than control; i.e., there was a volume-dependent apneic threshold. Transient electroencephalogram arousal at the end of the occlusion often augmented VT, thereby contributing to the subsequent central apnea; however, arousal was not required for the apnea to occur. Significant transient hypocapnia (up to -12 Torr arterial PCO2) commonly occurred after release of airway occlusion but was not closely correlated with the length of central apnea. During vagal blockade, after release of airway occlusion, significant transient hyperventilation occurred but at VT < 40% greater than control, and TE prolongation was markedly reduced. In summary, after release of airway occlusion in NREM sleep, 1) VT greater than three times eupnea was necessary to cause central apnea, 2) transient arousal at the termination of airway occlusion caused longer apneas by augmenting VT, and 3) transient hypocapnia per se made a significant but minor contribution to the postocclusion central apnea.

Airway Obstruction↗
PubMed1993

Saccharomyces cerevisiae cytoplasmic tyrosyl-tRNA synthetase gene. Isolation by complementation of a mutant Escherichia coli suppressor tRNA defective in aminoacylation and sequence analysis.

Exploiting differences in tRNA recognition between prokaryotic and eukaryotic tyrosyl-tRNA synthetases (TyrRSs), we have isolated the gene for the cytoplasmic TyrRS of Saccharomyces cerevisiae by functional complementation in Escherichia coli of a mutant E. coli tRNA. The tRNA, derived from the E. coli initiator tRNA with changes to allow suppression of amber termination codons, is poorly aminoacylated in E. coli and hence, is only a weak amber suppressor. The same tRNA functions as a good suppressor in S. cerevisiae and is aminoacylated with tyrosine by yeast extracts. We expressed a yeast cDNA library in an E. coli strain carrying the mutant tRNA gene and several genes with amber mutations. cDNA clones were isolated which increased suppression and levels of aminoacylation of the mutant tRNA. Characterization of the gene identified a methionine-initiated open reading frame encoding a protein of 394 amino acids. Expression of this protein in E. coli demonstrated that tyrosine was incorporated during suppression and that yeast cytoplasmic TyrRS activity was produced. Yeast cytoplasmic TyrRS has sequences typical of class I aminoacyl-tRNA synthetases, but only weak overall sequence similarity to the corresponding eubacterial and mitochondrial TyrRSs. However, many of the residues known to line the tyrosyl-adenylate-binding pocket of the Bacillus stearothermophilus enzyme can be aligned in the yeast sequence. These include the aspartic acid and tyrosine residues thought to contact the tyrosine side chain to provide substrate specificity.

Amino Acid Sequence↗
PubMed1993

Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.

We have isolated and characterized a gene, fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the leu-5 gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ: fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while leu-5 mRNA appears during germination and mycelial growth. The structure of the fdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.

Amino Acid Sequence↗
PubMed1989

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.

Amino Acid Sequence↗
PubMed1989

Regulation of the nuclear genes encoding the cytoplasmic and mitochondrial leucyl-tRNA synthetases of Neurospora crassa.

We show that the nuclear genes for the cytoplasmic and mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa are distinct in their encoded proteins, codon usage, mRNA levels, and regulation. The 4.2-kilobase-pair region representing the structural gene for cytoplasmic LeuRS and flanking regions has been sequenced. The positions of the 5' and 3' ends of mRNA and of a single 62-base-pair intron have been mapped. The methionine-initiated open reading frame encoded a protein of 1,123 amino acids and displayed a strong codon bias. Although cytoplasmic LeuRS shares with mitochondrial LeuRS some general features common to most aminoacyl-tRNA synthetases, there is little amino acid sequence similarity between them, mRNA levels for cytoplasmic LeuRS were much higher than those for mitochondrial LeuRS. This observation and the strong codon bias in the cytoplasmic LeuRS gene may contribute to a greater abundance of cytoplasmic LeuRS than mitochondrial LeuRS. The genes for cytoplasmic and mitochondrial LeuRS are regulated independently. The cytoplasmic LeuRS gene is regulated by the cross-pathway control system in N. crassa, which is analogous to general amino acid control in Saccharomyces cerevisiae. The cytoplasmic LeuRS mRNA levels are induced by amino acid starvation resulting from the addition of aminotriazole. Part of this increase is due to utilization of new transcription start sites. In contrast, the mitochondrial LeuRS gene is not induced by amino acid limitation. However, the mitochondrial LeuRS mRNA levels did increase dramatically upon inhibition of mitochondrial protein synthesis by chloramphenicol or ethidium bromide or in the temperature-sensitive strain leu-5 carrying a mutation in the mitochondrial LeuRS structural gene.

Amino Acid Sequence↗
PubMed1988

Cytoplasmic leucyl-tRNA synthetase of Neurospora crassa is not specified by the leu-5 locus.

We generated a lambda gt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, lambda NCLRSC1 and lambda NCLRSC2, were obtained which have inserts of approximately 2 kbp and approximately 1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that lambda NCLRSC1 and lambda NCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by lambda NCLRSC1 or lambda NCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an approximately 115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is approximately 3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located.

Amino Acid Sequence↗
PubMed1986

Influences of endogenous dopamine on carotid body discharge and ventilation.

Ventilatory and carotid body responses to hypoxia have been related to the endogenous release of dopamine by use of the antagonist drug haloperidol. The published studies have produced conflicting data for ventilation. However, antagonist drugs can act at multiple anatomical sites, on multiple pharmacological receptors, often at different dosages, and have nonspecific actions at high dosage. For these reasons, we have undertaken a systematic study of haloperidol dose-response curves with particular emphasis on the lowest possible concentrations of drug. In five cats anesthetized with pentobarbital sodium (30-35 mg/kg), single- or few-fiber afferent recordings of the carotid body showed that haloperidol increased the discharge during both basal and asphyxic conditions, the increments being proportional to haloperidol dosage (0.1-1,000 micrograms/kg). Increments of ventilation were also produced, these increments increasing only over the lower range of dosage; at the highest haloperidol dosage, the dose response showed a tendency to plateau or inflect downward, suggesting the appearance of an opposing inhibitory mechanism.

Animals↗
PubMed1984

Effects of naloxone on the hering-breuer apnea in sleeping kittens.

The modulatory role of endogenous opiates on the Hering -Breuer inflation reflex was examined in 11 newborn kittens, aged 10-31 days, during active and quite sleep. The Hering -Breuer apnea duration was significantly shortened by naloxone (1 mg/kg, intraperitoneally; P less than 0.05). This effect was abolished by 100% O2 breathing. The duration of apnea, and its shortening by naloxone, did not differ significantly in the two sleep states. Hering -Breuer apnea is a result of the inspiratory inhibition and expiratory excitation of medullary neurons in response to lung stretch; the apnea is terminated by the opposing influences of chemoreceptors, which respond to hypercapnia and hypoxia. The results suggest that opioid influences on the Hering -Breuer reflex are due to an opioid modulation of the carotid body discharge in hypoxia, or of its central integration, and that sleep state is not implicated in such modulation in kittens.

Animals↗
PubMed1982

Proteolytic modifications of the carboxyl-terminal region of H-2Kk.

Conditions were established for the generation of limited proteolysis products from purified H-2Kk in high yield (greater than 70%). Chymotrypsin, trypsin, or papain treatment in buffer containing Nonidet P-40 resulted in removal of discrete segments from the H-2 heavy chain without detectable alteration of the beta 2-microglobulin. The Mr = 47,400 heavy chain was converted to products with Mr = 44,200, 42,800, or 40,600 by treatment with chymotrypsin, trypsin, or papain, respectively. Papain digestion removed both the hydrophilic carboxyl terminus and the hydrophobic regions. The size, detergent binding properties, and products resulting from subsequent papain treatment demonstrated that chymotrypsin or trypsin removed segments of the hydrophilic carboxyl-terminal region of the heavy chain while leaving the hydrophobic (membrane-spanning) and glycosylated NH2-terminal regions intact. Chymotrypsin and trypsin caused rapid and extensive degradation of the H-2Kk heavy chain when treatment was done in buffer containing deoxycholate, suggesting that the protein undergoes partial, but readily reversible, denaturation in this detergent. This may account for the elution of H-2K and D antigens from monoclonal antibody affinity columns by deoxycholate-containing buffers.

Animals↗
PubMed1982

Ipratropium bromide and fenoterol by aerosolized solution.

Ipratropium bromide (0.5 mg) and fenoterol (2 mg) produced equivalent peak bronchodilatation between 1 and 2 h after administration to eight patients with chronic partially reversible airways obstruction. The duration of action compared with saline was 6 h for ipratropium and 4 h for fenoterol. Both drugs in combination produced greater bronchodilatation than either drug alone. The increase in FVC was disproportionately greater than FEV1 with both drugs and saline, suggesting relief of obstruction of small airways.

Aerosols↗
PubMed1981

Comparison of ipratropium bromide and salbutamol by aerosolized solution.

Ipratropium bromide (0.125 mg, 0.25 mg and 0.5 mg) and salbutamol (5 mg) by aerosolized solution produced equivalent peak bronchodilatation between one and two hours after administration to ten patients with chronic partially reversible airways obstruction. The duration of action of the two higher doses of ipratropium bromide (0.25 mg--6 hours; 0.5 mg--5 hours) was significantly greater than salbutamol (4 hours). FVC increases with both drugs and saline were greater than FEV1 increases which may indicate dilatation of small peripheral airways or removal of bronchial mucus from these sites after coughing. A dose of 0.25 mg ipratropium bromide as an aerosolised solution is recommended for clinical use.

Aerosols↗
PubMed1980

Serum total IgE in normal subjects and the influence of a family history of allergy.

The purpose of this study was to establish the range of serum total immunoglobulin E (IgE) in a healthy population free of personal and family history of allergy, and to determine whether a family history of allergy influenced serum IgE levels. Using commercially available Phadebas reagents, the mean serum IgE in eighty-four adults aged 17-30 years with no personal or family history of allergy was found to be 38.8 iu/ml. Seventy-five percent of subjects had a serum IgE below 50 iu/ml and in only one subject was serum IgE in excess of 150 iu/ml, which we suggest be taken as the upper limit of normal. When healthy subjects with no personal history but with a family history of allergy were included with this normal population, the mean serum IgE in 207 subjects was 95.4 iu/ml and the upper limit of the distribution curve over 200 iu/ml, suggesting an influence of genetic factors on IgE production.

Adolescent↗