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Biomedical subjects

E Lloyd

Publications and source records attributed to E Lloyd.

At least 19 recordsLinked to original sources
PubMed1999

A novel immunohistochemical assay for the detection of Ebola virus in skin: implications for diagnosis, spread, and surveillance of Ebola hemorrhagic fever. Commission de Lutte contre les Epidémies à Kikwit.

Laboratory diagnosis of Ebola hemorrhagic fever (EHF) is currently performed by virus isolation and serology and can be done only in a few high-containment laboratories worldwide. In 1995, during the EHF outbreak in the Democratic Republic of Congo, the possibility of using immunohistochemistry (IHC) testing of formalin-fixed postmortem skin specimens was investigated as an alternative diagnostic method for EHF. Fourteen of 19 cases of suspected EHF met the surveillance definition for EHF and were positive by IHC. IHC, serologic, and virus isolation results were concordant for all EHF and non-EHF cases. IHC and electron microscopic examination showed that endothelial cells, mononuclear phagocytes, and hepatocytes are main targets of infection, and IHC showed an association of cellular damage with viral infection. The finding of abundant viral antigens and particles in the skin of EHF patients suggests an epidemiologic role for contact transmission. IHC testing of formalin-fixed skin specimens is a safe, sensitive, and specific method for laboratory diagnosis of EHF and should be useful for EHF surveillance and prevention.

Adolescent↗
PubMed1998

Bacterial expression and spectroscopic characterization of soybean leghaemoglobin a.

A gene encoding leghaemoglobin a from soybean has been constructed and the soluble recombinant protein expressed in E. coli. The integrity of the recombinant protein has been assessed by a range of spectroscopic techniques. Electrospray mass spectrometry of the protein indicates that the molecular mass of the protein corresponds to the predicted amino acid sequence. Circular dichroism spectra of the ferric derivative and UV-visible spectra of various ferric and ferrous derivatives (pH 6.99, mu = 0.10 M, 25.0 degrees C) are consistent with published data for the wild-type protein. For the ferric derivative, UV-visible (298 and 77 K) and EPR (10 K) spectra indicate the existence of a thermal equilibrium between high- and low-spin forms. Titration of the protein (0.10 M NaCl, mu = 0.10 M, 25.0 degrees C) between pHs 6.68 and 10.35 indicate formation (pKa = 8.3+/-0.03) of a 6-coordinate, hydroxide-bound form of the protein at high pH. All of the above data are consistent with the behaviour of the wild-type protein.

Amino Acid Sequence↗
PubMed1997

Chemical, spectroscopic and structural investigation of the substrate-binding site in ascorbate peroxidase.

The interaction of recombinant ascorbate peroxidase (APX) with its physiological substrate, ascorbate, has been studied by electronic and NMR spectroscopies, and by phenylhydrazine-modification experiments. The binding interaction for the cyanide-bound derivative (APX-CN) is consistent with a 1:1 stoichiometry and is characterised by an equilibrium dissociation binding constant. Kd, of 11.6 +/- 0.4 microM (pH 7.002, mu = 0.10 M, 25.0 degrees C). Individual distances between the non-exchangeable substrate protons of APX-CN and the haem iron were determined by paramagnetic-relaxation NMR measurements, and the data indicate that the ascorbate binds 0.90-1.12 nm from the haem iron. The reaction of ferric APX with the suicide substrate phenylhydrazine yields predominantly (60%) a covalent haem adduct which is modified at the C20 carbon, indicating that substrate binding and oxidation is close to the exposed C20 position of the haem, as observed for other classical peroxidases. Molecular-modelling studies, using the NNM-derived distance restraints in conjunction with the crystal structure of the enzyme [Patterson, W. R. & Poulos, T. L. (1995) Biochemistry 34, 4331-4341], are consistent with binding of the substrate close to the C20 position and a possible functional role for alanine 134 (proline in other class-III peroxidases) is implicated.

Ascorbate Peroxidases↗
PubMed1997

Role of the heme propionates in the interaction of heme with apomyoglobin and apocytochrome b5.

The heme propionate groups of both myoglobin (Mb) and cytochrome b5 form hydrogen bonds with nearby surface amino acids residues that are believed to stabilize the heme-protein complex. To evaluate the magnitude of this stabilization, the kinetics of heme dissociation from variants of horse heart Mb and cytochrome b5 in which these hydrogen bonding interactions have been systematically eliminated were studied by the method of Hargrove and colleagues (1994), and their thermal stability was assessed. Elimination of each hydrogen bond was found to decrease the thermal stability of the proteins and increase the rate constant for heme dissociation in a progressive fashion. For the Mb derivatives, 1H-NMR studies indicate that the elimination of individual hydrogen bonds also affects the rate at which the heme orientational equilibrium is achieved. In both types of kinetics experiment, the effects of decreasing the number of potential hydrogen bonding interactions are found to be cumulative. Despite their kinetic effects, elimination of these hydrogen bonding interactions had no influence on the initial distribution of heme orientational isomers immediately following reconstitution or on the equilibrium constant of heme orientational disorder. The interactions between the heme propionates and nearby protein residues play a partial role in the stabilization of the heme-protein complex and are a major factor in the kinetic "trapping" of the minor heme orientation. Comparisons of the various rate constants determined for the mechanism of heme binding and reorientation suggests that the intramolecular reorientation mechanism is slightly favored over the intermolecular mechanism.

Animals↗
PubMed1996

Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant.

The structural and functional consequences of the introduction of a negatively charged amino acid into the active site of horse heart myoglobin have been investigated by replacement of the proximal Ser92 residue (F7) with an aspartyl residue (Ser92Asp). UV-visible absorption maxima of various ferrous and ferric derivatives and low-temperature EPR spectra of the metaquo (metMb) derivative indicate that the active site coordination geometry has not been perturbed significantly in the variant. 1H-NMR spectroscopy provides direct evidence for the existence of a distal water molecule as the sixth ligand in the oxidized form of the variant at pD 5.7. Spectrophotometric pH titration of the Ser92Asp variant is consistent with this finding and with a pKa = 8.90 +/- 0.02 [25.0 degrees C, mu = 0.10 M (NaCl)] for titration of the distal water molecule, identical to the value reported for the wild-type protein. X-ray crystallography of the metMb derivative indicates that the heme substituents conserve their orientations in the variant protein, except for a slight reorientation of the pyrrole A propionate group to which Ser92 normally hydrogen bonds and reorientation of the carboxyl end of the pyrrole D propionate group. No change is observed in conformation of the proximal (His93) or distal (Wat156) heme ligands. 1H-NMR spectroscopy of the metMbCN form of the protein indicates that a slight rotation of the proximal His93 ligand has occurred in this derivative. Resonance Raman experiments indicate increased conformational heterogeneity in the proximal pocket of the variant. Failure to detect electron density for the Asp residue in the X-ray diffraction map of the variant protein and high average thermal factors for the pyrrole A propionate substituent are consistent with this observation. The variant exhibits novel pH-dependent behavior in the metMb form, as shown by 1H-NMR spectroscopy, and provides evidence for a heme-linked titratable group with a pKa of 5.4 in this derivative. The metMbCN and deoxyMb derivatives also exhibit pH-dependent behavior, with pKas of 5.60 +/- 0.07 and 6.60 +/- 0.07, respectively, compared to the wild-type values of 5.4 +/- 0.04 and 5.8 +/- 0.1. The heme-linked ionizable group is proposed to be His97 in all three derivatives. The reduction potential of the variant is 72 +/- 2 mV vs SHE [25.0 degrees C, mu = 0.10 M (phosphate), pH 6.0], an increase of 8 mV over the wild-type value. The possible influence of a number of variables on the magnitude of the reduction potential in myoglobin and other heme proteins is discussed.

Animals↗
PubMed1996

A blood group-related polymorphism of CD44 abolishes a hyaluronan-binding consensus sequence without preventing hyaluronan binding.

CD44 is a widely expressed integral membrane protein that acts as a receptor for hyaluronan (HA) and is proposed to be important to cell-extracellular matrix interaction. The Indian (In) blood group antigens reside on CD44, and most individuals express the Inb antigen. Homozygosity for the Ina allele occurs as a rare event and is associated with production of alloantibody to the common Inb antigen after transfusion or pregnancy. The present study demonstrates that a single point mutation (G252 --> C) causes an Arg46 --> Pro substitution, which is responsible for the Inb/Ina polymorphism. Additional mutations were found in In(a+b-) cDNA but were not necessary to the antigenic phenotype as determined in site-directed mutagenesis studies. In studies using CD44 chimeric constructs, Arg46 has previously been shown to be crucial for maintenance of HA-binding ability to a CD44 peptide. However, the present study demonstrates that the Arg46 --> Pro substitution does not reduce HA binding to the intact CD44 protein, which contains two proposed extracellular HA-binding motifs. Down-regulation of HA binding to In(a+b-) CD44 by anti-CD44 monoclonal antibody (mAb) ligands, however, was weakened, although all mAbs tested bound In(a+b-) and In(a-b+) CD44 equally well. Competitive inhibition studies using human anti-Inb also showed that some mAbs that inhibit HA binding to CD44 may do so by interacting with a domain separate from, but affecting the structure of, the Inb epitope.

Amino Acid Sequence↗
PubMed1995

Arthritis and other rheumatic conditions: who is affected now, who will be affected later? National Arthritis Data Workgroup.

OBJECTIVE: We present national and state estimates for 1990 and projections for the year 2020 of the prevalence of self-reported arthritis and other rheumatic diseases and related activity limitations. We further suggest a research and policy agenda to address this important and growing public health problem. METHODS: Estimates and projections were derived from household interviews conducted for the 1989-1991 National Health Interview Survey, and were applied to United States census population estimates for 1990 and projections for 2020. RESULTS: The prevalence rate of self-reported arthritis and other rheumatic conditions in the United States is projected to increase from 15.0% (37.9 million) of the 1990 population to 18.2% (59.4 million) of the estimated 2020 population. Activity limitation attributed to these conditions is projected to increase from 2.8% (7.0 million) of the 1990 population to 3.6% (11.6 million) of the 2020 population. Prevalence rates were higher for older persons, women, residents of nonmetropolitan areas, and those with less education or lower income. CONCLUSIONS: Arthritis and other rheumatic conditions are frequent and disabling public health problems now, and are projected to become even more so by 2020. Implementing the suggested research and public health agenda could reduce the occurrence and impact of these conditions.

Activities of Daily Living↗
PubMed1994

Recombinant human erythrocyte cytochrome b5.

The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.

Amino Acid Sequence↗
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

Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli.

Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (approximately 16-17% total) of two sulphmyoglobin isomers. The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer. Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX. The possible relationship of this observation to recombinant expression of other heme proteins is discussed.

Animals↗
PubMed1993

Pulse radiolysis and related studies on the Ru-modified His 16 derivative of Anabaena variabilis [2Fe-2S] ferredoxin.

The preparation and characterisation of a Ru-modified derivative of the [2Fe-2S] ferredoxin FdI component of A. variabilis by attachment of Ru(NH3)5 to a surface histidine has been carried out. Metal analyses by ICP gave an Fe/Ru ratio of 1.97:1. From NMR the attachment is confirmed as a modification at His-16 and not His-92. No Ru-modification of the [2Fe-2S] FdI component of spinach, which has His-92 but no His-16, was observed. Histidine pKa values have been determined and anomalously low values for A. variabilis (5.3) and spinach (< 5.0) His-92 residues are noted, whereas His-16 gives a pKa of 6.95. Pulse radiolysis techniques with e-aq as reductant have been used to generate the metastable Fe(II)Fe(III)Ru(III) state from Fe(III)2Ru(III), k = 8.8 x 10(10) M-1 s-1. Intermolecular, k(inter) = 4.3 x 10(6) M-1 s-1 at approx. 19 degrees C, but no intramolecular electron-transfer (ET) Fe(II)Fe(III) to Ru(III) contribution was observed with Fe(III)2Ru(III) in the range 7-30 microM. The driving force for ET is approx. 500 mV, and the edge to edge distance from the C gamma of His-16 to the nearest cysteinyl S-atom attached to the Fe2S2 cluster is 16.1 A. Using the Beratan-Onuchic pathway approach, the most favourable ET intramolecular route consists of 20 covalent and 3 H-bonds, with a total distance from the S of Cys-49 to the C gamma of His-16 of 37.1 A. We note that the reduced Fe is on the remote side of the [2Fe-2S] cluster from the site of Ru-attachment.

Amino Acid Sequence↗
PubMed1990

Type of childcare at 18 months--I. Differences in interactional experience.

A longitudinal study has followed two-parent families and their first-born child. The families were chosen so that there were three groups of dual-earner families using relatives, childminders and nurseries for day care and one group of single-earner families. At 18 mths of age children in the study were observed in the four types of childcare setting. The data from the detailed observations were used to compare the children's interactional experience. The results indicate marked variation in the quality of children's experiences between different childcare settings. Possible reasons for such variation are discussed.

Attention↗
PubMed1990

Type of childcare at 18 months--II. Relations with cognitive and language development.

In a longitudinal study of women and their first-born children the relationship between type of day care experience and cognitive and language development at 18 mths of age was considered. There was a strong association between socio-economic characteristics and type of day care and analyses allowed for this. The results for cognitive development indicate a relationship with mother's education but not with type of day care. For language development the results indicate that children who experience group care were less likely to show much production of different word combinations, but that this was related to the children's language environments.

Child Care↗
PubMed1988

Filling defects of the kidney pelvis.

Filling defects in the renal pelvis should alert radiologists to the possibility of underlying tuberculosis. A case is presented with the major criteria used in differentiating tuberculosis from a neoplasm.

Adult↗
PubMed1983

Sexual experience and plasma testosterone levels in male veterans after spinal cord injury.

Fifty men with spinal cord injuries (SCI) were asked to complete a questionnaire concerning their sexuality before and after injury. Medical examination confirmed the location and completeness of the injury and extracted information about genitourologic status. The respondents rated sexuality highly as a concern in living, and a wide variety of sexual techniques were reported. A marked decrease in sexual activity, satisfaction, and feelings of sexual adequacy was reported after injury, as compared to retrospective "before injury" responses, lack of opportunity being reported as causative by 66% of the subjects and insufficient personal satisfaction by 59%. Seventy-five percent of the subjects experienced sexual arousal from genital stimulation, and several methods of eliciting erection were cited. Orgasm was described by a variety of terms. Significant differences were found between quadriplegic and paraplegic patients in answers to several items, though there was generally no difference between cervical and thoracic groups, which were more specifically broken down with respect to motor or sensory/complete or incomplete lesions. Plasma testosterone levels were found to fall well within the normal adult male range, as were levels of free testosterone and serum sex binding protein. The resulting information demonstrated more sexual concern among men with SCI than the literature previously indicated.

Adult↗