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Biomedical subjects

M Smith

Publications and source records attributed to M Smith.

At least 613 records · Page 34Linked to original sources
PubMed1994

Identification of marker chromosomes in thirteen patients using FISH probing.

Fourteen marker chromosomes were studied by FISH (fluorescence in-situ hybridization) in cytogenetic preparations from 13 patients. The derived markers were identified as one isodicentric bisatellited mar(22), one fragment sized r(X), one fragment sized r(Y), one i(18p), small autosomal ring markers in three different patients derived from chromosomes 2, 8, and 8, a marker comprised of 9p and part of 9qh, and 3 bisatellited apparently monocentric markers; one of each from chromosomes 13 or 21, 14 or 22, and 15. Two fragment sized small ring markers in one patient and a small ring marker in another were negative with all twenty-two different probes used. In addition, the small ring marker Y chromosome that was found in a boy with karyotype 46,X,-Y,+mar was negative with both pDXZ1 and pDYZ3. This anomaly of negative results with the battery of centromeric alphoid probes can be explained if one breakpoint for some small ring markers is very near to or within the centromere. Only some of the pericentromeric repetitive sequences in the normal chromosome would be represented in the chromosome specific alphoid probes, and presumably those corresponding to the currently available probes are truncated during the formation of the unidentified markers. In three of the small ring markers the FISH signal on the marker was much stronger than on the normal homologues in various proportions of cells, and this may indicate that some of the fragment sized small rings were multicentric. The literature was reviewed for Distamycin A/DAPI negative small ring markers that were present as extra chromosomes. There were only single published cases of most small rings but there were three r(8) cases, two r(1) cases, two r(12) cases, and two r(20) cases, uncomplicated by the presence of other chromosome abnormalities. Most cases with similar small rings were quite dissimilar phenotypically and syndrome identification was not possible, but in pooled data, 18/23 (about 80%) were developmentally and/or phenotypically abnormal. Some patients (5/23, about 20%) with small rings were dysmorphic without intellectual handicap. Of 28 such patients with small ring markers (Distamycin/Dapi negative) in pooled data there are 6 (about 20%) with multiple markers mostly derived from different chromosomes. This is a very high figure and would suggest that the ring formation events, although involving different chromosomes, must be related and must be an indicator of the mechanism of origin of this group of markers.

Adolescent↗
PubMed1994

The three-dimensional structure in solution of the paramagnetic high-potential iron-sulfur protein I from Ectothiorhodospira halophila through nuclear magnetic resonance.

The three-dimensional structure in solution of reduced recombinant high-potential iron-sulfur protein iso-I from Ectothiorhodospira halophila was determined using 948 relevant interproton NOEs out of the 1246 observed NOEs. The determination was accomplished using the XEASY program for spectral analysis and the distance geometry (DG) program DIANA for generation of the structure as described by Wüthrich [Wüthrich, K. (1989) Acc. Chem. Res. 22, 36-44]. The FeS cluster was simulated using an amino acid residue constructed for the present work from a cysteinyl residue with an iron and a sulfur atom attached to the terminal thiol. The family of structures obtained from distance geometry were subjected to energy minimization and molecular dynamics simulations using previously defined force field parameters. The quality of these structures at each stage of the refinement process is discussed with respect to the dihedral angle order parameter and the root-mean-square deviation of the atomic coordinates. The latter values for the backbone atoms vary from 67 pm for the distance-geometry structures to 60 pm for the energy-minimized structures to 51 pm for the structures subjected to restrained molecular dynamics. Finally, the structure in best agreement with the NOE constraints has been further treated with extensive restrained molecular dynamics in water. The solution structure is well defined and is very similar to the available X-ray structure. We do not know of any previous determination of the structure of a paramagnetic protein in solution by NMR. The effect of paramagnetism on the quality of the structure determination is discussed.

Bacteria↗
PubMed1994

Mefloquine distribution in postmortem cases.

Mefloquine is currently the drug-of-choice for malaria prophylaxis among military personnel. Four active duty military personnel receiving 250 mg mefloquine per week were killed in the line of duty under combat conditions. Samples of blood, bile, liver, kidney, muscle, brain, spleen and lung were submitted to the Division of Forensic Toxicology, Office of the Armed Forces Medical Examiner, for routine toxicologic analysis. Qualitative screening revealed only the presence of ethanol (< 25 mg/dl, probably attributable to postmortem formation) and mefloquine. Quantitation of mefloquine was performed using an HP 5880 gas chromatograph equipped with a nitrogen/phosphorus detector. The column was an HP-5 cross-linked 5% phenyl methyl silicone fused silica capillary column (15 m x 0.25 mm i.d. x 0.25 microns film thickness). The temperature program began at 110 degrees C, was held for 1 min and ramped at 20 degrees C/min to 200 degrees C, held for 1 min and then ramped at 10 degrees C/min to 280 degrees C and held for 10 min. Mefloquine elutes with a relative retention time similar to that of the tricyclic antidepressants. No postmortem data concerning mefloquine concentrations or tissue distribution was available. Quantitated blood concentrations in the presented cases were greater than the expected therapeutic values indicating the possibility of postmortem redistribution of this drug. No mefloquine overdoses were identified in the literature making comparison to the postmortem therapeutic concentrations impossible at this time.

Adult↗
PubMed1994

Active site coordination chemistry of the cytochrome c peroxidase Asp235Ala variant: spectroscopic and functional characterization.

Asp235 in yeast cytochrome c peroxidase forms a hydrogen bond with His175, the proximal histidyl residue, that has been suggested to be important in determining the electronic properties of the heme iron and that may be involved in stabilizing the higher oxidation states of the peroxidase that form transiently during catalysis. The current study employs 1H and 15N-NMR spectroscopy to study the electronic properties of and the effects of pH on the active site of the Asp235Ala variant. This variant exhibits three spectroscopic species between pH 5 and 9: a high-spin species that forms at low pH and two low-spin species that form successively at higher pH. Nevertheless, the activity of the variant exhibits a pH dependence virtually identical to that of the wild-type protein, though the activity of the variant is 3 orders of magnitude lower at all values of pH between pH 5 and 8.5. These findings suggest that the spin state and coordination environment of the heme iron in cytochrome c peroxidase do not dictate the rate of substrate (cytochrome c) oxidation. Binding of cyanide to the variant enzyme results in formation of a single species as detected by NMR spectroscopy. Analysis of high-resolution 1D and 2D 1H-NMR and 15N-NMR spectra of the cyanide adduct has permitted characterization of the properties of this derivative and the strength of the proximal ligand bond to the heme iron. Disruption of the hydrogen bond between the proximal histidine and Asp235 that exists in the wild-type enzyme dramatically reduces the strength of the interaction between the proximal ligand and the iron; this effect combined with concurrent changes in the distal heme-binding pocket accounts for the increase in reduction potential reported for the Fe3+/Fe2+ couple. The catalytic consequences of the structural and electronic properties of the variant elucidated in this study are discussed.

Alanine↗
PubMed1994

Primary sequence and immunological characterization of beta-subunit of high conductance Ca(2+)-activated K+ channel from smooth muscle.

The charybdotoxin receptor, purified from bovine tracheal smooth muscle, consists of two subunits (alpha and beta) and, when reconstituted into planar lipid bilayers, forms functional high conductance Ca(2+)-activated K+ channels. Amino acid sequence, obtained from proteolytic fragments of the beta-subunit, was used to design oligonucleotide probes with which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative transmembrane) domains and bears little sequence homology to subunits of other known ion channels. Site-directed antisera, raised against putative extracellular epitopes of this protein, specifically immunoprecipitated 125I-labeled Bolton-Hunter beta-subunit as well as [125I]charybdotoxin-cross-linked beta-subunit. Under nondenaturing conditions, however, these anti-beta sera immunoprecipitated a complex consisting of both the alpha- and beta-subunits. The data demonstrate that, in vivo, the high conductance Ca(2+)-activated K+ channel exists as a multimer containing both alpha- and beta-subunits, and this cDNA represents the first beta-subunit of a potassium channel cloned to date. Furthermore, we demonstrate that the cloned protein is the subunit to which charybdotoxin is specifically and covalently incorporated when cross-linked to the channel.

Amino Acid Sequence↗
PubMed1994

Regulation of iron uptake and transport by transferrin in Caco-2 cells, an intestinal cell line.

Caco-2 cells grown in bicameral chambers, a model of intestinal epithelial iron transport (Biochim. Biophys. Acta (1991) 1070, 205-208), were used to study the effect of apo-transferrin (apo-Tf) in the basal chamber on 59Fe uptake from the apical surface, intracellular 59Fe distribution, and 59Fe transport into the basal chamber. Caco-2 cells were grown with varying amounts of iron to achieve cells that were either iron-deficient (FeD), or normal iron status (FeN), or iron-loaded (FeH). The effect of apo-Tf was most marked in FeD cells with the transport of 59Fe from 1 microM 59Fe-ascorbate on the apical side to the basal chamber measured as (22.2 +/- 3.0) x 10(4), (8.2 +/- 0.6) x 10(4), and (2.7 +/- 0.4) x 10(4) atoms 59Fe/cell/min in the presence of apo-Tf, BSA, and no added protein, respectively. Unexpectedly in FeD cells total 59Fe uptake (i.e., both 59Fe in the cells and that transported into the basal chamber) was decreased by basolateral apo-Tf with total uptake of (2.6 +/- 0.3) x 10(5), (4.8 +/- 0.6) x 10(5), and (4.8 +/- 0.7) x 10(5) atoms/cell/min with apo-Tf, BSA, and no additions, respectively. Analysis of intracellular 59Fe by isoelectrofocusing in polyacrylamide gels demonstrated 59Fe migrating both with a basic pI and with the pI values of ferritin (Ft) at a ratio of 200:1 (basic pI moiety: ferritin) in FeD cells. The presence of Tf further decreased the small amount of 59Fe in Ft. These studies demonstrate that basolateral Tf affects the apical uptake of 59Fe, the intracellular distribution of 59Fe, and the transport of 59Fe across intestinal epithelium, the latter effect occurring even when cellular content of ferritin is high.

Biological Transport↗
PubMed1994

FTIR analysis of the interaction of azide with horse heart myoglobin variants.

The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhibit a high-spin/low-spin equilibrium. Introduction of positively charged amino acid residues shifts nu max for the low-spin form to higher energy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and His64Ile variants is accompanied by a shift of the nu max for the low-spin infrared band to lower energy and by a significant increase in the corresponding half-bandwidths. This observation indicates greater mobility of the bound azide ligand in these variants. The His64Lys variant exhibits two infrared bands attributable to low-spin forms that are assigned to two different conformations of the lysyl residue. In one conformation, the lysine is proposed to form a hydrogen bond with the bound azide similar to that proposed to occur between the distal histidine and bound azide, and in the other conformation no interaction occurs.

Animals↗
PubMed1994

Spectroscopic, electrochemical, and ligand binding properties of the horse heart metmyoglobin His64-Tyr variant.

The distal histidine (E7) of horse heart myoglobin (Mb) has been replaced by tyrosine using site-specific mutagenesis. The resulting green Mb variant (His64-Tyr) was expressed in Escherichia coli JM101, isolated and purified to homogeneity. Spectrophotometric pH titrations of the variant exhibit a change in spectrum that occurs with a pKa of 4.7 (25 degrees C). The midpoint reduction potential of the variant is 20 mV (vs. SHE at pH 7, 25 degrees C). Cyanide and azide binding measurements indicate that the oxidized variant binds these anionic ligands with much greater affinity at pH 4.0 than at neutral pH. Extended X-ray absorption fine structure (EXAFS) spectroscopy establishes that the variant is six coordinate at pH 7.0 and pH 4.2. Higher shell contributions to the iron EXAFS observed at pH 7.0 are attributed to tyrosine. These contributions are absent at pH 4.2. Thus, the sixth heme iron ligand of the oxidized variant Mb at pH 7.0 is attributed to oxygen from the hydroxyl group of tyrosine and the sixth ligand present at pH 4.2 is attributed to the oxygen atom of a coordinated water. The EXAFS spectra, electronic absorption spectra, and ligand binding properties of the His64-Tyr Mb variant are consistent with the binding of Tyr-64 as the sixth heme iron ligand between pH 5 and 12 and with the replacement of Tyr-64 by a water molecule at low pH with a pKa of 4.7.

Animals↗
PubMed1994

Structural characterization of heme ligation in the His64-->Tyr variant of myoglobin.

A site-specific mutant of horse heart myoglobin has been prepared in which the distal heme pocket residue, His64, is replaced by tyrosine. The structure of this myoglobin variant has been determined to 2.0-A resolution using x-ray diffraction techniques and refined to a final crystallographic R-factor of 16.9%. The polypeptide backbone conformation of the His64-->Tyr variant of myoglobin is very similar to that of the wild-type protein. However, in the variant the water normally found coordinated to the heme iron atom and hydrogen-bonded to His64 has been displaced by the hydroxyl oxygen of the Tyr64 side chain. The tyrosine oxygen atom is directly coordinated to the heme iron atom with a bond length of 2.18 A. Distortion of heme planarity and changes in the packing of the Leu29 and Leu104 side chains are related to this mutation. The ligand environment of the ferric iron has been studied by electron paramagnetic resonance (EPR) spectroscopy using crystalline material and protein in solution. The protein in solution exhibits a rhombically split ferric high spin EPR spectrum with g values of 6.64, 5.34, and 1.98. The EPR spectrum of the crystalline sample consists of two different ferric high spin signals. The main signal is similar to the signal observed in solution and is assigned to His93-Fe(III)-Tyr64 coordination. The relatively high rhombicity of this signal can be explained as arising from distortions of the heme plane seen in the crystal structure. The second, more axial high spin signal found in the crystalline state can be tentatively assigned to another form of iron ligation with a different iron-tyrosine bond length and a less distorted heme plane.

Animals↗
PubMed1994

Heterodimerization of the yeast MATa1 and MAT alpha 2 proteins is mediated by two leucine zipper-like coiled-coil motifs.

The yeast Saccharomyces cerevisiae has three cell types distinguished by the proteins encoded in their mating type (MAT) loci: the a and alpha haploids, which express the DNA binding proteins a1 and alpha 1 and alpha 2, respectively, and the a/alpha diploid which expresses both a1 and alpha 2 proteins. In a/alpha cells, a1-alpha 2 heterodimers repress haploid-specific genes, while alpha 2 homodimers repress a-specific genes, indicating a dual regulatory function for alpha 2 in mating type control. a1 does not form homodimers. We have identified two sequences in the alpha 2 N-terminal domain which contain the 3,4-hydrophobic heptad repeat pattern characteristic of coiled-coils. Mutational analyses show that both sequences are important to a1-alpha 2 heterodimerization. We propose that these two sequences associate in a coiled-coil-like manner with a sequence within a1 which bears two adjacent, overlapping 3,4-hydrophobic heptad repeats. This model, which describes a novel dimerization motif for homeodomain proteins, also provides a mechanism by which a1-a1 homodimerization is prevented.

Amino Acid Sequence↗
PubMed1994

The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c.

High resolution three-dimensional structures for the N52I and N52I-Y67F yeast iso-1-cytochrome c variants have been completed in both oxidation states. The most prominent structural difference observed in both mutant proteins is the displacement of a conserved, internally bound water molecule (Wat166) from the protein matrix. In wild-type yeast iso-1-cytochrome c the position and orientation of this water molecule is found to be dependent on the oxidation state of the heme iron atom. Overall our results suggest the function of Wat166 and its associated hydrogen bond network is threefold. First, the presence of Wat166 provides a convenient mechanism to modify the hydrogen bond network involving several residues near the Met80 ligand in an oxidation state dependent manner. Second, Wat166 is necessary for the maintenance of the spatial relationships between nearby side-chains and the hydrogen bond interactions formed between these groups in this region of the protein. An essential part of this role is ensuring the proper conformation of the side-chain of Tyr67 so that it forms a hydrogen bond interaction with the heme ligand Met80. This hydrogen bond influences the electron withdrawing power of the Met80 ligand and is therefore a factor in controlling the midpoint reduction potential of cytochrome c. Elimination of this interaction in the N52I-Y67F mutant protein or elimination of Wat166 in the N52I protein with the subsequent disruption in the position and interactions of the Tyr67 side-chain, leads to a drop of approximately 56 mV in the observed midpoint reduction potential of the heme group. Third, Wat166 also appears to mediate increases in the mobility of three nearby segments of polypeptide chain when cytochrome c is in the oxidized state. Previous studies have proposed these changes may be related to oxidation state dependent interactions between cytochrome c and its redox partners. Coincident with the absence of Wat166, such mobility changes are not observed in the N52I and N52I-Y67F mutant proteins. It is possible that much of the increased protein stability observed for both mutant proteins may be due to this factor. Finally, our results show that neither heme iron charge nor heme plane distortion are responsible for oxidation state dependent conformational changes in the pyrrole A propionate region. Instead, the changes observed appear to be driven by the change in conformation that the side-chain of Asn52 experiences as the result of oxidation state dependent movement of Wat166.

Amino Acid Sequence↗
PubMed1994

Mutation of tyrosine-67 to phenylalanine in cytochrome c significantly alters the local heme environment.

The high resolution three-dimensional atomic structures of the reduced and oxidized states of the Y67F variant of yeast iso-1-cytochrome c have been completed. The conformational differences observed are localized directly in the mutation site and in the region about the pyrrole A propionate. Shifts in atomic positions are largely restricted to nearby amino acid side-chains, whereas little perturbation of the polypeptide chain backbone is observed. One prominent difference between the variant and wild-type structures involves a substantial increase in the size of an already existing internal cavity adjacent to residue 67. This same cavity contains an internally bound water molecule (Wat166), which is found in all eukaryotic cytochromes c for which structures are available. In the reduced Y67F mutant protein a second water molecule (Wat300) is observed to reside in this enlarged internal cavity, assuming a position approximately equivalent to that of the hydroxyl group of Tyr67 in the wild-type protein. A further consequence of this mutation is the alteration of the hydrogen bond network between Tyr67, Wat166 and other nearby residues. This appears to be responsible for the absence of oxidation state dependent changes in polypeptide chain flexibility observed in the wild-type protein. Furthermore, loss of the normally resident Tyr67 OH to Met80 SD hydrogen bond leads to a significantly lower midpoint reduction potential. These results reaffirm proposals that both Tyr67 and Wat166 play a central role in stabilizing the alternative oxidation states of cytochrome c.

Cytochrome c Group↗
PubMed1994

National survey on hospital-based privileges in family practice obstetrics.

PURPOSES: To document the content and level of obstetrical hospital-based privileges for members of the American Academy of Family Physicians and to describe variations between regions, rural vs urban practices, and various physician characteristics. METHODS: About 12% of the active members of the American Academy of Family Physicians listed as offering obstetrical care by the Academy as of March 1991 were randomly sampled by mailed questionnaire. Samples were drawn from three national regions. Privileges were grouped by degree of restriction, based on whether consultation or transfer was required. RESULTS: Of 1464 surveys mailed, 1026 physicians (70%) responded. Only 740 (72%) stated that they still practiced obstetrics. Privileges ranged from least restricted (100% provided vaginal vertex delivery, with no consultation required) to most restricted (79% provided amniocentesis, with consultation or transfer required). A surprisingly large proportion of physicians reported having fewer routine and more advanced privileges without consultations being required, such as ultrasonography (53%), vaginal breech delivery (41%), and cesarean section (25%). Physicians having more advanced privileges tended to be located in the West or mountain-plains region; be trained in the Midwest, mountain-plains region, or the West; work in middle-sized, nonteaching hospitals in more rural countries; have completed advanced obstetrical training (> or = 6 months); and deliver more than 40 infants per year. CONCLUSIONS: Overall, a considerable number of hospital-based obstetrical privileges are granted to family physicians. No uniformity in privileges prevails, owing to significant regional and practice variations. Teaching hospitals reportedly restrict obstetrical care by family physicians more than other hospitals. The variations in restrictions could not be explained by degree of training.

Adult↗
PubMed1994

Modulation of angiogenesis in vitro by laminin-entactin complex.

Laminin is a cross-shaped glycoprotein whose inner cross-region binds to the glycoprotein entactin (nidogen) forming a stable complex extractable from basement membrane matrices with chelating agents. In this study we evaluated the effect of the laminin-entactin complex on angiogenesis in serum-free collagen gel culture of rat aorta. Laminin-entactin stimulated or inhibited angiogenesis depending on its concentration in the gel. Stimulatory concentrations of laminin-entactin (30 to 300 micrograms/ml) promoted an increase in the number and length of microvessels. A similar effect was observed with purified laminin. Elongation of microvessels was also obtained with the laminin fragments E1' and E8 and with entactin, all of which have binding sites for endothelial cells. By contrast the E4 fragment of laminin, which has no cellular binding sites, failed to promote microvascular elongation. Inhibitory concentrations of laminin-entactin (3000 micrograms/ml) allowed formation of only a few stubby endothelial sprouts. Laminin-entactin promoted dose-dependent stabilization of microvessels preventing their regression. Microvessels stabilized by laminin-entactin were surrounded by thick patches of basement membrane-like material, whereas untreated microvessels prone to regression had a highly attenuated basement membrane. The angiogenic effect of laminin-entactin was enhanced by exogenous bFGF. However, bFGF was unable to stimulate angiogenesis over control values when incorporated in gels containing a high concentration of laminin-entactin. Our results indicate that laminin and its supramolecular complex laminin-entactin play an important modulatory role in angiogenesis. The dose-dependent effects of the laminin-entactin complex suggest that the basement membrane is a dynamic regulator of angiogenesis whose function varies depending on the concentration of its molecular components.

Animals↗