Search PubMed⌕ Search

Biomedical subjects

R C Miller

Publications and source records attributed to R C Miller.

At least 163 records · Page 9Linked to original sources
PubMed1987

Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains.

Glycosylated cellulases from Cellulomonas fimi were compared with their non-glycosylated counterparts synthesized in Escherichia coli from recombinant DNA. Glycosylation of the enzymes does not significantly affect their kinetic properties, or their stabilities towards heat and pH. However, the glycosylated enzymes are protected from attack by a C. fimi protease when bound to cellulose, while the non-glycosylated enzymes yield active, truncated products with greatly reduced affinity for cellulose.

Actinomycetales↗
PubMed1987

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster.

The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.

Animals↗
PubMed1987

Effect of endothelium and carbachol on alpha-adrenoceptor agonist stimulated uptake and efflux of 45Ca in rat isolated aorta.

Preincubation with carbachol (10 microM) did not affect basal 45Ca accumulation by rat isolated aortic segments complete with endothelium, although 45Ca accumulation was enhanced by removal of endothelium. This confirms the observation that in the presence of endothelium Ca2+ influx in rat aorta is antagonized, and indicates that the basal release of an endothelial derived factor might be sufficient to maximally antagonize basal Ca2+ influx, or alternatively that EDRF released as a result of muscarinic stimulation does not have identical effects to the factor released under basal conditions. Accumulation of 45Ca stimulated by B-HT 920 but not that stimulated by phenylephrine was antagonized in the presence of endothelium. Contractions elicited by B-HT 920 were abolished in the presence of endothelium while contractions evoked by phenylephrine were reduced by about 50%. Preincubation with 10 microM carbachol antagonized both phenylephrine (1 microM) stimulated 45Ca accumulation and contractile responses in the presence of endothelium to about the same extent. Therefore, it might be concluded that the inhibitory effect of EDRF in this tissue is due to an inhibition of stimulated Ca2+ influx. However, while addition of carbachol to tissues precontracted with phenylephrine elicited an immediate relaxation in the presence of endothelium, this relaxation could not be correlated with a reduction in tissue accumulation of 45Ca. Carbachol also antagonized the phenylephrine-induced reduction of tissue 45Ca content (i.e. efflux of Ca2+), in tissues preloaded with 45Ca. This implies that the initial endothelial-mediated relaxant effect of carbachol in precontracted tissues cannot be explained either by reduced influx or by an enhanced efflux of Ca2+ from the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1987

The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum.

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.

Brevibacterium↗
PubMed1987

A bifunctional exoglucanase-endoglucanase fusion protein.

A fusion was constructed between the cex gene of Cellulomonas fimi, which encodes an exoglucanase, and the cenA gene of the same organism, which encodes an endoglucanase. The cex-cenA fusion was expressed in Escherichia coli to give a fusion protein with both exoglucanase and endoglucanase activities. The fusion protein, unlike the cex and the cenA gene products from E. coli, did not bind to microcrystalline cellulose, presumably because it lacked an intact substrate-binding region. The fusion protein was exported to the periplasm in E. coli.

Cellulase↗
PubMed1987

Recovery of rat lens epithelial cells after total or partial X-irradiation.

Irradiation of whole lenses interferes with the normal mitogenic response of lens cells to stimulation by mechanical wounding. Radiation exposure causes a delay and partial suppression of cells entering the S phase of the cell cycle. When cells are irradiated 1 day before wounding, DNA synthesis begins 20 hr after wounding and peaks 10 hr later with 20% cell labelling. The return to a normal wound response is gradual when the time interval between irradiation and wounding is lengthened. By 28 days, essentially complete recovery of the wound response occurs. Examination of the beginning of mitosis shows a similar pattern of recovery. When half of the lens is irradiated 1 day before wounding, delay and suppression of the wound response in the irradiated half of the lens epithelium is observed. However, if 4 days elapse between irradiation and mechanical wounding, complete recovery of cells responding to the mitogenic stimulus occurs. Cells of the shielded half of the lens appear to compensate for the reduced number of irradiated cells entering S phase so that peak labelling of shielded cells is 70% compared with 50% for control lens cells. An evaluation of mitotic figures confirms faster recovery of the entire lens when only half the lens is irradiated. Complete recovery of the cellular response of partially irradiated lenses occurs in 4 days in contrast to almost 28 days for wholly irradiated lens.

Animals↗
PubMed1987

Regulation and initiation of cenB transcripts of Cellulomonas fimi.

We characterized the in vivo transcription of the Cellulomonas fimi cenB gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4). By Northern blot (RNA blot) analysis, cenB mRNA was detected in C. fimi RNA preparations from glycerol-, glucose-, and carboxymethyl cellulose (CMC)-grown cells. The relative abundance of the specific mRNAs in these preparations appeared to depend on the carbon source provided, with the preparations from CMC-grown cells having the greatest amount of cenB transcripts, followed by glycerol- and glucose-grown cells. Therefore, the transcription of this gene could be regulated by the carbon source provided to C. fimi. High-resolution nuclease S1 protection studies were used to map cenB mRNA 5' termini with a unique 5'-labeled DNA probe and C. fimi RNA isolated in vivo. With this procedure, three 5' termini were found in abundance upstream of the translational initiation ATG codon in RNA preparations from C. fimi grown on CMC, while less-abundant 5' termini were found 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results are indicative of a tandem promoter arrangement, with the ATG-proximal promoter directing constitutive low-level cenB transcription and the more distal promoter directing higher levels of transcription under the inducing effects of the cellulosic substrate. The corresponding transcripts were not detected in S1 mapping experiments with RNA isolated in vivo from Escherichia coli clones harboring recombinant plasmids carrying C. fimi genomic inserts. Comparative analysis of the 5' -flanking DNA sequences of the cenB gene and the cenA and cex genes of C. fimi (N. M. Greenberg, R. A. J. Warren, D. G. Kilburn, and R. C. Miller, Jr., J. Bacteriol. 169:646-653, 1987) revealed a region of 50 bases in which these sequences displayed at least 64% homology.

Actinomycetales↗
PubMed1987

Regulation, initiation, and termination of the cenA and cex transcripts of Cellulomonas fimi.

We characterized the in vivo transcripts of two Cellulomonas fimi genes, the cenA gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4) and the cex gene, which encodes an extracellular exo-beta-1,4-glucanase (EC 3.2.1.91). By Northern blot analysis, cenA mRNA was detected in C. fimi RNA preparations from glycerol- and carboxymethyl cellulose-grown cells but not from glucose-grown cells. In contrast, cex mRNA was detected only in the preparations from carboxymethyl cellulose-grown cells. Therefore, the transcription of these genes is subject to regulation by the carbon source provided to C. fimi. By nuclease S1 protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, 5' termini were found 51 and 62 bases before the cenA translational initiation codon and 28 bases before the cex translational initiation codon. S1 mapping with unlabeled DNA probes and C. fimi RNA which had been isolated in vivo but which had been 5' labeled in vitro with guanylyltransferase and [alpha-32P]GTP confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. Comparative analysis of the DNA sequences immediately upstream of the initiation sites of the cenA and cex mRNAs revealed a 30-base-pair region where these two sequences display at least 66% homology. S1 mapping was also used to locate the 3' termini of the cenA and cex transcripts. Three 3' termini were found for cenA messages, whereas only one 3' terminus was identified for cex mRNA. The transcripts of both genes terminate in regions where their corresponding DNA sequences contain inverted repeats.

Bacteria, Anaerobic↗
PubMed1987

Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture.

Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and exposed to X irradiation to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5%, the average mean lethal dose Do was 97.6 cGy for HY cells and 96.7 and 94.3 cGy, respectively for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average Do of 85.0 cGy and PC cells a significantly (P = 0.05) lower average Do of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (P = 0.82) or sex (P = 0.10). These results suggest that malignant thyroid cells may be especially radiosensitive.

Adenoma↗
PubMed1986

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex.

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.

Cellobiose↗
PubMed1986

Alpha-adrenoceptor antagonistic and calcium antagonistic effects of nicergoline in the rat isolated aorta.

The activity of the alpha-adrenoceptor antagonist nicergoline, a molecule composed of two constituent parts, ergoline and bromonicotinic acid, was investigated in the rat isolated aorta. Nicergoline (10 nM-0.1 microM) displaced concentration-effect curves elicited by noradrenaline and phenylephrine to the right and inhibited maximal responses elicited by both alpha-adrenoceptor agonists without significantly affecting prostaglandin F2 alpha-induced contractions. Higher concentrations of nicergoline (1 microM-50 microM) displaced to the right the concentration-effect curves elicited by calcium in a depolarizing medium. This calcium antagonist activity was not shared by either of the constituent parts. Nicergoline 100 microM abolished the 45Ca influx induced into rat aorta by 100 mM K+-containing physiological solution. The selectivity of nicergoline for alpha 1-adrenoceptors seen in binding experiments also depends on the presence of the bromonicotinic moiety of the molecule. It is concluded that nicergoline, but not its substituent parts, displays both alpha 1-adrenoceptor and calcium antagonism. The latter property may account for some of the observed effects of this compound.

Animals↗
PubMed1986

Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi.

Cellulomonas fimi produces an endoglucanase and an exoglucanase which bind strongly to cellulose. Each enzyme contains three distinct regions: a short sequence of about 20 amino acids containing only proline and threonine (the Pro-Thr box); an irregular region, rich in hydroxyamino acids, of low charge density, and which is predicted to have little secondary structure; and an ordered region of higher charge density which contains a potential active site, and which is predicted to have secondary structure. The Pro-Thr box is conserved almost perfectly in the two enzymes. The irregular regions are 50% conserved, and the conserved sequences include four Asn-Xaa-Ser/Thr sites. The ordered regions appear not to be conserved, but the potential active sites both have the sequence Glu-Xaa7-Asn-Xaa6-Thr; they occur at widely separated sites in the two regions. The order of the regions is reversed in the two enzymes: irregular-Pro-Thr box-ordered in the endoglucanase; ordered-Pro-Thr box-irregular in the exoglucanase. The genes for the two enzymes appear to have arisen by shuffling of two conserved sequences and either one or two other sequences.

Actinomycetales↗
PubMed1986

Effect of endothelium on basal and alpha-adrenoceptor stimulated calcium fluxes in rat aorta.

The rate of unstimulated influx of Ca2+ into rat aorta smooth muscle, measured as uptake of 45Ca, was inhibited in the presence of endothelium as compared to influx in the absence of endothelium. Efflux of 45Ca from unstimulated prelabelled tissues was also reduced in the presence of endothelium. In normal physiological solution the rate of influx and efflux of Ca2+ stimulated by B-HT 920 (1 and 10 microM), but not that stimulated by phenylephrine (30 nM and 1 microM), was also reduced in the presence of endothelium. In the presence of the calcium entry blocker flunarizine (3 microM), phenylephrine (1 microM) stimulated efflux of Ca2+ was inhibited by the presence of endothelium. A correlation between inhibition of Ca2+ influx and modulation of alpha-adrenoceptor agonist-induced contractions by endothelium could not be demonstrated, and methylene blue, an antagonist of endothelium mediated inhibition of B-HT 920 contractions, did not affect Ca2+ influx stimulated by the agonist. The effects of endothelium on Ca2+ influx and efflux are unlikely to be due to alterations by endothelium of diffusion of 45Ca or the agonists in the vessel. The results demonstrate that an endothelial derived factor or factors can reduce calcium influx into smooth muscle cells and also modulate the release of calcium from cells, perhaps by affecting intracellular calcium pumping mechanisms. A reduction of calcium influx cannot be the sole explanation for the modulatory effect of endothelium on alpha-adrenoceptor agonist-induced contractions but an effect on intracellular calcium metabolism may be important.

Animals↗
PubMed1986

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi.

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.

Amino Acid Sequence↗
PubMed1986

Structure of the gene encoding the exoglucanase of Cellulomonas fimi.

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.

Amino Acid Sequence↗
PubMed1986

Secretion of Cellulomonas fimi exoglucanase by Escherichia coli.

A leader sequence of 41 amino acids (aa) has been proposed as the signal sequence for the exoglucanase (Exg) from Cellulomonas fimi. The ability of this 41-aa peptide to function as a leader sequence has been shown here by gene fusion experiments in Escherichia coli. A hybrid leader sequence containing C-terminal 37 aa of the leader peptide and N-terminal 6 aa of beta-galactosidase (beta Gal) directed export of the Exg into the periplasm of E. coli. In contrast, hybrid beta Gal-Exg proteins in which the leader sequence is not present are retained in the cytoplasm.

Amino Acid Sequence↗
PubMed1986

Epidermoid cyst of the spleen manifested as an abdominal abscess.

We have reported a case of epidermoid cyst of the spleen, an uncommon congenital anomaly that usually occurs as an asymptomatic abdominal mass. The diagnosis of splenic cyst may be established by ultrasonography. Uncomplicated cysts should be resected, while infected cysts should be drained and resected after resolution of the infection. Splenic salvage should be achieved when possible.

Abdomen↗