Biomedical subjects

R C Miller

Publications and source records attributed to R C Miller.

At least 73 records · Page 4Linked to original sources

PubMed1994

Ultrasonographic measurement of fetal nuchal skin to screen for chromosomal abnormalities.

OBJECTIVE: The purpose of this prospective investigation was to determine the utility of ultrasonographic measurement of the fetal nuchal skin in screening for chromosomal abnormalities. STUDY DESIGN: In 1510 patients undergoing genetic amniocentesis at 14 to 21 weeks' gestation, the fetal nuchal skin fold was measured. A measurement of > or = 6 mm was considered abnormal. RESULTS: In fetuses with normal karyotype the nuchal skin increased with advancing gestational age (r = 0.45, p < 0.001). Only 27 fetuses with normal karyotype (2.0%) had an abnormal nuchal skin thickness. Fifty-seven fetuses had abnormal karyotype. Eight of 14 fetuses with Down syndrome had abnormal ultrasonographic findings; four had increased nuchal skin as the only ultrasonographic abnormality. Increased nuchal skin alone, in the absence of other ultrasonographic dysmorphologic features, did not help to identify fetuses with other abnormal karyotypes. CONCLUSION: Nuchal skin thickness is a useful noninvasive measurement to screen for Down syndrome but does not help to identify fetuses with other chromosomal abnormalities.

Chromosome Aberrations↗

PubMed1994

Risk of eye splash in obstetric procedures.

A prospective cross-sectional study was designed to determine the rates of upper facial splash during obstetric deliveries. Macroscopic splashes on the attached eye shields of surgical masks, worn for obstetric deliveries were enumerated for a sample of parturients during a 3-month period. Masks were collected for 68 vaginal and 44 cesarean deliveries, 31% of all deliveries during the study period. For cesarean deliveries, 68% (30) of all masks worn by primary surgeons had at least one splash; 57% (17) of which were heavily contaminated (more than five splashes). For the first assistant, 68% (30) of all masks had at least one splash; 23% (7) of which were heavily contaminated. During vaginal deliveries 44% (30) of masks worn by the delivering physician had at least one splash; 20% (6) of which were heavily contaminated. The primary surgeon in a cesarean section was 1.6 times as likely to be splashed (P < 0.04) when compared with vaginal deliveries and 4.6 times as likely to be heavily contaminated (P < 0.003). Estimated blood loss and the rate of eye splash were linearly related. Method of delivery was not independently associated with eye splash. These findings suggest that the risk of contamination by splash during obstetric procedures is high, particularly for cesarean deliveries because these deliveries tend to have greater blood losses.

Adult↗

PubMed1994

Acute maternal and fetal cardiovascular effects of caffeine ingestion.

Little data exist regarding the acute maternal and fetal cardiovascular effects of caffeine ingestion. The purpose of this study is assess the cardiovascular effects of caffeine ingestion in pregnant subjects and their fetuses. We examined seven caffeine-naive gravidas longitudinally at 25.7 (SE +/- 0.7) and 36.1 (+/- 0.7) weeks' gestation, before and after ingestion of a caffeine citrate solution (100 mg/m-2 body surface area). Maternal pulse, blood pressure, fetal heart rate, and fetal heart rate accelerations were determined before and after caffeine ingestion. Uterine artery, fetal aorta, and umbilical artery flow velocity waveforms were examined at similar intervals. Significant before and after caffeine differences, regardless of gestational age, were noted for maternal pulse (85.1 vs 74.2 beats/min), diastolic blood pressure (65.9 vs 73.8 torr), mean arterial blood pressure (81.5 vs 87.5 torr), uterine artery systolic-to-diastolic (S/D) ratio (1.9 vs 2.4), fetal heart rate (144.6 vs 135.4 beats/min), and fetal heart rate accelerations (1.6 vs 4.2 per 30 minutes). Differences for fetal aortic and umbilical artery S/D ratios were not significant with respect to caffeine administration. Fetal aortic peak velocities increased with caffeine (85.5 vs 130.2 cm/sec) and the increase was augmented with advancing gestational age. We conclude that maternal ingestion of modest amounts of caffeine citrate in caffeine-naive subjects significantly affects both the fetal and maternal cardiovascular systems.

Adult↗

PubMed1994

An internal cellulose-binding domain mediates adsorption of an engineered bifunctional xylanase/cellulase.

A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA. A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene. The gene fusion was overexpressed in Escherichia coli and the fusion polypeptide purified from the cell extracts using the polyhistidine tail. The hybrid protein behaved like the parental endoglucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrates or xylans. The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates. However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin. Like the parental endoglucanase, bound XynCenA could not be eluted from these polysaccharides with either low or high salt buffer or distilled water. More stringent conditions, such as 1% SDS or 8 M guanidinium hydrochloride, fully desorbed the protein. The fusion protein did not adsorb significantly to insoluble xylan.

Adsorption↗

PubMed1994

The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding.

Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA). Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA. The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.

Amino Acid Sequence↗

PubMed1994

Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a beta-1,4-exocellobiohydrolase analogous to Trichoderma reesei CBH II.

The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85,349 Da. It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA catalytic domain are closely related to those of CBH II, an exocellobiohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesei. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.

Amino Acid Sequence↗

PubMed1994

Streptomyces lividans glycosylates the linker region of a beta-1,4-glycanase from Cellulomonas fimi.

The beta-1,4-glycanase Cex of the gram-positive bacterium Cellulomonas fimi is a glycoprotein comprising a C-terminal cellulose-binding domain connected to an N-terminal catalytic domain by a linker containing only prolyl and threonyl (PT) residues. Cex is also glycosylated by Streptomyces lividans. The glycosylation of Cex produced in both C. fimi and S. lividans protects the enzyme from proteolysis. When the gene fragments encoding the cellulose-binding domain of Cex (CBDCex), the PT linker plus CBDCex (PT-CBDCex), and the catalytic domain plus CBDCex of Cex were expressed in S. lividans, only PT-CBDCex was glycosylated. Therefore, all the glycans must be O linked because only the PT linker was glycosylated. A glycosylated form and a nonglycosylated form of PT-CBDCex were produced by S. lividans. The glycosylated form of PT-CBDCex was heterogeneous; its average carbohydrate content was approximately 10 mol of D-mannose equivalents per mol of protein, but the glycans contained from 4 to 12 alpha-D-mannosyl and alpha-D-galactosyl residues. Glycosylated Cex from S. lividans was also heterogeneous. The presence of glycans on PT-CBDCex increased its affinity for bacterial microcrystalline cellulose. The location of glycosylation only on the linker region of Cex correlates with the properties conferred on the enzyme by the glycans.

Adsorption↗

PubMed1994

Oncogenic transformation through the cell cycle and the LET dependent inverse dose rate effect.

Synchronised populations of mouse C3H/10T-1/2 cells were obtained by a stringent mitotic dislodgment procedure. Mitotic cells rapidly attach and progress sequentially through the cell cycle. Irradiation (3 Gy of X rays) was carried out at intervals from 0 to 18 h after initiating cell cycle progression of the mitotic cells. Oncogenic transformation was enhanced 10-fold over cells irradiated soon after replating (G1 and S phases) for cells in a near 2 h period corresponding to cells in G2 phase but not in mitosis. The cell surviving fraction had a 2-1/2-fold variation with resistant peaks corresponding to the late G1 and late S phases. These findings provide experimental support for the hypothesis initiated by Rossi and Kellerer and developed by Brenner and Hall to explain the LET dependent inverse dose rate effect for oncogenic transformation.

Animals↗

PubMed1994

Fetal oculocerebrorenal syndrome of Lowe associated with elevated maternal serum and amniotic fluid alpha-fetoprotein levels.

OBJECTIVE: To report an association between fetal oculocerebrorenal syndrome of Lowe and elevations in maternal serum alpha-fetoprotein (MSAFP) and amniotic fluid alpha-fetoprotein (AFAFP). METHODS: Case 1 was identified during routine MSAFP screening. Cases 2-5 were identified through review of a data base of individuals with oculocerebrorenal syndrome enrolled at the National Institutes of Health. To estimate the frequency of this association, only those whose mothers would have been in the early second trimester from February 1987 to August 1993 were enumerated. The MSAFP was assumed to be normal unless explicitly reported or unless information outside the data base confirmed that MSAFP was not determined. RESULTS: An elevated MSAFP (2.5 multiples of the median [MoM] or greater) was detected in five of 20 pregnancies with a fetus affected by oculocerebrorenal syndrome. Maternal serum alpha-fetoprotein was greater than 5.0 MoM in three pregnancies undergoing amniocentesis, and all had an elevated AFAFP without significant acetylcholinesterase activity. No abnormalities were found by ultrasound, and there was no other cause of elevated AFP identified postnatally. Family history was positive in three of the five cases. The mothers were carriers in four of the five cases, whereas the fifth case appeared to be a spontaneous mutation. CONCLUSIONS: Elevated MSAFP and AFAFP appear to occur at a higher than expected frequency in pregnancies carrying an oculocerebrorenal syndrome fetus. The mechanism of elevation of AFP may be related to fetal renal tubular dysfunction. A directed interview, focusing on a maternal family history of male relatives with unexplained mental retardation, early institutionalization, or congenital rubella, is appropriate with unexplained MSAFP elevations and, particularly, with unexplained AFAFP elevations without acetylcholinesterase activity.

Acetylcholinesterase↗

PubMed1994

Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation.

Misoprostol, a PGE1 analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostol, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clonogenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events.

Animals↗

PubMed1994

Role of acidic amino acids in the allosteric modulation by gallamine of antagonist binding at the m2 muscarinic acetylcholine receptor.

The purpose of this study was to explore the role of acidic amino acids in the allosteric behavior of gallamine at the m2 receptor. This was achieved by first mutating the acidic residues to neutral residues by site-directed mutagenesis. Both the parent and mutated receptors were expressed in mouse fibroblast A9L cells and characterized pharmacologically. The two main methods used were (i) Schild analysis of equilibrium binding data and (ii) study of the effect of gallamine on the dissociation kinetics of N-methylscopolamine. The Schild analysis gave an estimate of the affinity of gallamine for the allosteric site (KdA) and also a measure of the level of cooperativity (alpha) between the allosteric and primary binding sites. For the receptors studied, a good agreement was found between the alpha KdA values calculated from the Schild analysis and the IC50 values for the effect of gallamine on the N-methylscopolamine off-rate. One mutated receptor, in which the acidic EDGE (Glu-Asp-Gly-Glu) sequence of the putative third outer domain was changed to the neutral LAGQ (Leu-Ala-Gly-Gin) sequence, displayed an 8-fold reduction in affinity for gallamine at the allosteric site, in comparison with the parent receptor. The level of cooperatively between the allosteric and primary binding sites in this mutant was 46% of that of the parent receptor. A second mutated receptor, in which Asp-97 (near the top of putative transmembrane domain 3) was changed to asparagine, was found to have a level of cooperativity between sites 58% of that of the parent but was found not to be affected with respect to the affinity of gallamine for the allosteric site. When all of the acidic groups on the outer side were changed to neutral residues, there was still only an 8.6-fold reduction in gallamine affinity for the allosteric site, but the level of cooperativity was reduced to 19% of that found in the parent receptor. The results suggest that the allosteric site for gallamine binding in the m2 receptor residues at or near the putative third outer domain and that both the EDGE motif and Asp-97 play an essential role in the interaction between the two sites. However, none of the acidic amino acids mutated were found to be critical for binding at the allosteric site.

Allosteric Regulation↗

PubMed1993

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.

Base Sequence↗

PubMed1993

Endoglucanase CasA from alkalophilic Streptomyces strain KSM-9 is a typical member of family B of beta-1,4-glucanases.

CasA is an endo-beta-1,4-glucanase from Streptomyces KSM-9 belonging to family B of beta-1,4-glucanases. A previous analysis of a portion of the corresponding gene (casA) revealed sequencing errors in a region encoding part of the catalytic site. Additional errors in the original sequence were suspected, based on sequence comparison of the C terminus of CasA with other members of its family. Re-sequencing of the remainder of the casA coding region showed that CasA is a typical member of family B.

Amino Acid Sequence↗

PubMed1993

A non-flight muscle isoform of Drosophila tropomyosin rescues an indirect flight muscle tropomyosin mutant.

The tropomyosin I(TmI) gene of Drosophila melanogaster encodes two isoforms of tropomyosin. The Ifm-TmI isoform is expressed only in indirect flight and jump muscles; the Scm-TmI isoform is found in other muscles of the larva and adult. The level of Ifm-TmI is severely reduced in the flightless mutant Ifm(3)3, which also is unable to jump. To explore the functional significance of tropomyosin isoform diversity in Drosophila, we have used P element-mediated transformation to express Scm-TmI in the indirect flight and jump muscles of Ifm(3)3 flies. Transformants gained the ability to jump and fly. The mechanical properties of isolated indirect flight muscle myofibres, and the ultrastructure of indirect flight and jump muscles from the transformants were comparable to wildtype. Thus, the Scm-TmI isoform can successfully substitute for Ifm-TmI in the indirect flight and jump muscles of the Ifm(3)3 strain.

Adenosine Triphosphatases↗

PubMed1993

Visualization of the adsorption of a bacterial endo-beta-1,4-glucanase and its isolated cellulose-binding domain to crystalline cellulose.

Endo-beta-1,4-glucanase A (CenA), a cellulase from the bacterium Cellulomonas fimi, is composed of two domains: a catalytic domain and a cellulose-binding domain. Adsorption of CenA and its isolated cellulose-binding domain (CBD.PTCenA) to Valonia cellulose microcrystals was examined by transmission electron microscopy using an antibody sandwich technique (CenA/CBD.PTCenA-alpha CenA IgG-protein A-gold conjugate). Adsorption of both CenA and CBD.PTCenA occurred along the lengths of the microcrystals, with an apparent preference for certain crystal faces or edges. CenA or CBD.PTCenA, but not the isolated catalytic domain, were shown to prevent the flocculation of microcrystalline bacterial cellulose. The cellulose-binding domain may assist crystalline cellulose hydrolysis in vitro by promoting substrate dispersion.

Actinomycetales↗

PubMed1993

The structure and specificity of endothelin receptors: their importance in physiology and medicine.

In addition to involvement in vascular endothelium-smooth muscle communication, the secretion of and receptors for, endothelins are widely distributed. Two cloned receptor subtypes are G-protein-coupled to several intracellular messengers, predominantly inositol phosphates. From a knowledge of structure-activity relationships and peptide conformations, details of receptor architecture and selective agents, including nonpeptides and antagonists, have been discovered. From the nature of the actions of endothelins, receptor distributions (including CNS) and plasma levels, it is concluded that they are paracrine factors normally involved in long-term cellular regulation, but which may be important in several pathologies, many of which are stress-related.

Amino Acid Sequence↗

PubMed1993

Endothelins--from receptors to medicine.

Since the discovery of endothelins, peptides with exceptional vasoconstrictor potency that were originally suggested to act by causing the opening of Ca2+ channels, it has emerged that these agents are important in intercellular communication in many tissues. They exert their effects through G protein-coupled receptors, of which two classes have been cloned. Robert Miller, John Pelton and John Huggins review the progress made towards a molecular understanding of ligand recognition by endothelin receptors. Receptor-selective agonists and antagonists have emerged from attempts to understand the three-dimensional structure of the endothelin pharmacophore, from structure-activity studies and from rapid-screening programmes. From the nature of the secretion and action of endothelins, it would seem that these peptides are involved in long-term changes rather than in acute responses to stimuli, and that they are likely to be important in a number of pathological states. Evidence suggests that receptor antagonists with appropriate affinity and selectivity may be useful in the treatment of conditions as diverse as hypertension, ulcerogenesis and ciclosporin toxicity.

Amino Acid Sequence↗

PubMed1993

A bifunctional affinity linker to couple antibodies to cellulose.

We have constructed a fusion protein between the staphylococcal A protein and the cellulose binding domain of an exoglucanase (Cex) from Cellulomonas fimi that can be directly immobilized on cellulose while retaining its capacity to bind immunoglobulin G molecules. The cellulose domain provides binding that does not interfere with the biological activity of the fusion partner, does not involve hazardous chemicals and the matrix does not need to be chemically activated which reduces its cost. We have tested some of the possible applications of the fusion protein and show that it can be used in immunoassays, affinity chromatography and immunoprecipitations.

Antibodies↗