Biomedical subjects
Publications and source records attributed to A Davies.
Four Asian babies were investigated because they failed to thrive. In all four cases the failure to thrive was a result of the mother's social isolation and inability to communicate, and to the father's refusal to accept that there was a problem in the family.
Details are given of the sexual assaults dealt with by this laboratory, with particular mention of the increasing number of rape cases submitted each year. The contribution of a forensic biologist to the investigation of alleged rapes is summarized. The laboratory's sexual assault index is explained using examples of three series of linked rapes recognized by this method. The uses and limitations of the ABO grouping of semen are briefly mentioned and an example given showing how the male's ABO group may be ascertained even in the absence of semen.
Our previously described immunoadsorption method for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters from 3T3-L1 adipocytes has been improved in two ways. First, the minimal number of g minutes required to sediment the plasma membranes from the cell homogenate has been determined and, as a result, the supernatant used for immunoadsorption in the new procedure contained twice as much of the intracellular transporters. Second, the immunoadsorption has been performed with affinity-purified antibodies directed against the carboxy terminal peptide of the transporter, rather than against the entire protein. 10(7) cells (10 mg protein) yielded about 12 micrograms of vesicular protein and 11 micrograms of vesicular phospholipid. The transporter constituted 3% of the protein in the vesicles; this amount equates to approx. eight copies of the transporter per 50 nm vesicle. The polypeptide composition of the vesicles was determined by gel electrophoresis and protein staining. Major components, other than the glucose transporter, are polypeptides of Mr 270,000, 245,000, 165,000 and 115,000. The vesicles contained several phosphoproteins; the major ones have a Mr of 245,000, 190,000, 115,000 and 25,000. Insulin treatment of adipocytes did not significantly change the phosphoprotein composition of the vesicles. The vesicles were not enriched in the Golgi marker enzyme, galactosyltransferase. The cellular content of the marker for the trans-Golgi reticulum, sialyltransferase, was too low to detect.
The nucleoside transporter has been purified by passage of a preparation of human erythrocyte-membrane band-4.5 proteins through a column of immobilized antibodies specific for the glucose transporter. This procedure removed greater than 99.8% of the glucose transporters and achieved an approx. 18-fold purification of the nucleoside transporter, constituting a 478-fold purification from erythrocyte membranes. The isolated protein migrated as a single broad band of average apparent Mr 55,000 on SDS/polyacrylamide gels and bound approx. 0.6 mol of nitrobenzylthioinosine/mol of polypeptide, with a Kd of 1.1 +/- 0.14 (S.E.M.) nM. Upon reconstitution into large unilamellar phospholipid vesicles it catalysed the uptake of uridine with an apparent specific activity 6-fold greater than that of the unfractionated band-4.5 proteins. Furthermore, the purified nucleoside transporter was not labelled on Western blots by monoclonal antibody raised against the glucose transporter. It is concluded that the nucleoside transporter has been purified to near homogeneity.
The purpose of the present study was to explore the effects of protein kinase C (PKC) stimulation on two cell surface receptors that regulate T cell growth: the T cell antigen receptor/CD3 complex and the CD2 antigen. The data show that PKC differentially regulates the expression and functions of CD2 and CD3 molecules. Thus, activation of PKC induced a decrease in cell surface levels of CD3 molecules but an increase in the expression of CD2 antigens. Additionally, prolonged stimulation of PKC inhibited subsequent T cell activation via CD3 but promoted activation via CD2 molecules. These results suggest that the CD2 cellular activation pathway would be preferred in T cells which have been exposed to stimulators of PKC. The molecular basis for the regulatory effects of PKC on CD3 and CD2 molecules and its physiological significance are discussed.
The glucose transporter of the facilitated diffusion type has been localized in sections of innervated rat diaphragm muscle and sciatic nerve by immunofluorescence, using affinity-purified antibodies against both the entire transporter and the carboxy-terminal peptide. In both tissues the transporter was very abundant in the perineurial sheath of cells surrounding the nerve fibres. The transporter also appeared to be abundant in the endoneurial blood vessels of the sciatic nerve. The identity of the antigen as the glucose transporter was established by extracting sciatic nerve with sodium dodecylsulphate and immunoblotting the extract. A single reactive polypeptide with the expected molecular weight of 55,000 was found. The high concentration of glucose transporter in the cells of the blood-nerve barrier presumably ensures an adequate supply of glucose to the nerve fibres.
The ABO grouping results from approx. 1000 seminal stains have been collected and analysed. Most of the stains came from rape cases where the ABO and secretor status of both complainant and suspect were known. The results of the survey provided information concerning the usefulness of elution and inhibition as methods of body fluid grouping, the relative strengths of reaction of the A, B and H antigens in body fluids and the interpretation of the ABH reactions of body fluid stains.
The complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase. Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.
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The lungs of anaesthetized rabbits were ventilated with a frequency of 30 Hz and a displacement of 5 ml. High-frequency ventilation (HFV) was superimposed on static inflation or deflation pressures of 2.5 and 5 cmH2O and was maintained for 10 s. Changes in pattern of breathing in response to this procedure were recorded before and during block of pulmonary stretch receptors by SO2. With lung stretch receptors intact apnoea or extended duration of expiration demonstrated the predominant role of pulmonary stretch receptors in the response to HFV. Block of stretch receptors exposed effects of other pulmonary afferents, presumably rapidly adapting receptors, during HFV, and demonstrated the influence of their sustained stimulation on pattern of breathing as augmented breaths and the reduction of duration of expiration. High-frequency ventilation may represent a useful experimental method of stimulating rapidly adapting receptors.
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We have measured upper airways resistance from the trachea and from the pharynx to the atmosphere, EMG of genioglossus muscle, and the sound of snoring, in anaesthetized greyhounds breathing spontaneously through the upper airways. Using extra-corporeally produced continuous flow we determined flow/pressure curves for the upper airways in an expiratory direction and analysed them in terms of resistances from the trachea and from the pharynx. Resistances and other variables were determined with the nose open and the nostrils blocked. About one-third of the dogs snored spontaneously and most of the remainder did so when the nose was blocked. During snoring with nasal blockage the upper airways resistance increased considerably, and the sound of snoring and genioglossus EMG were also enhanced. The results show that the anaesthetized greyhound is a suitable model for studying snoring with simultaneous measurements of upper airways resistance and the activity of pharyngeal dilator muscles.
We measured upper airways resistance from the trachea and from the pharynx to the atmosphere, EMG of the genioglossus muscle and the sound of snoring, in anaesthetized greyhounds, breathing spontaneously through the upper airways. Using extra-corporeally produced continuous flow we determined flow/pressure curves for the upper airways and resistances from the trachea and from the pharynx. We tested the effects of 0.9% saline and of Sonarex (a proprietary mixture containing sodium chloride, glycerol, polysorbate 80 and benzalkonium chloride). Both saline and Sonarex decreased upper airways resistance, but the latter did so more consistently. With Sonarex, genioglossus activity increased and the sound of snoring decreased. Flow/pressure curves 5-20 min after Sonarex showed a decrease in upper airways resistance and a smoother curve, whereas those with saline showed an increase in resistance. The sound produced by continuous flow through the upper airways was decreased by Sonarex but increased by saline. Thus, both Sonarex and saline decrease upper airways resistance, but Sonarex also reduces the sound of snoring and the resistance and sound of continuous airflow through the upper airways.
The glucose-transport protein from bovine cerebral-cortex microvessels has been identified and characterized by virtue of its ability to bind the ligand [4-3H]cytochalasin B. Microvessel membranes were found to contain a single set of glucose-inhibitable high-affinity cytochalasin B-binding sites [113 +/- 16 (S.E.M.) pmol/mg of membrane protein], with an association constant of 6.8 +/- 1.8 (S.E.M.) micron-1. D-Glucose inhibited the binding to these sites with a Ki of 31 mM. The transport protein was identified by photoaffinity labelling with [4-3H]cytochalasin B and was found to migrate as a broad band of apparent Mr 55,000 on SDS/polyacrylamide gels. Labelling was inhibited by D-glucose, but not by L-glucose. Treatment with endoglycosidase F yielded a sharper band of apparent Mr 46,000, indicating that the transport protein is glycosylated. However, in contrast with the human erythrocyte glucose transporter, digestion with endo-beta-galactosidase had little effect on the electrophoretic mobility of the microvessel protein. Tryptic digestion of the photolabelled protein yielded a radioactive fragment of apparent Mr 18,000, similar to that of the fragment produced by digestion of the labelled human erythrocyte glucose transporter. In addition, a protein of Mr identical with that of the photolabelled transporter was labelled on Western blots of microvessel membranes by antisera raised against the intact erythrocyte transporter and against synthetic peptides corresponding to its N- and C-terminal regions. It is concluded that the glucose-transport protein of bovine cerebral-cortex microvessel endothelial cells shows structural homology with the human erythrocyte glucose transporter.
Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.
Based on the Rieder and Gershon (1978) biological genetic study paradigm we compared the prevalence of a putative vulnerability factor (rapid cholinergic REM sleep induction) between 35 affectively ill and 31 well first-degree relatives of 34 probands chosen on the basis of presence of primary affective illness and positive marker status. A significantly higher percentage of ill relatives (63%, 22 out of 35 subjects) had a supersensitive REM-induction response as compared to well relatives (22%, 7 out of 31 subjects). Since the current study does not rule out the effects of prior depressive episodes or treatment on cholinergic sensitivity, caution must be exercised in the interpretation of our findings.
Immune responses of BALB/c mice to immunopurified tetanus toxoid entrapped in dehydration-rehydration vesicles composed of equimolar egg phosphatidylcholine and cholesterol were compared to those of free toxoid. Animals were injected intramuscularly with the free or liposomal toxoid and identical injections were repeated 4 weeks and, in some experiments, 24 weeks later. Analysis of IgG1, IgG2a, IgG2b, IgG3 and IgM in the sera by an enzyme-linked immunosorbent assay suggested that adjuvanticity of liposomes is reflected in most antibody subclasses and that there is no shift in subclasses compared to the response obtained with the free antigen, thus establishing liposomes as a type I adjuvant. In other, appropriately designed experiments, the relative importance of events following the first and second injections in determining the adjuvant effect of liposomes was investigated. It was found that liposome adjuvanticity is the outcome of events following primary immunization.
Tetanus toxoid was incorporated into liposomes composed of equimolar phospholipid and cholesterol. The toxoid was either passively entrapped into multilamellar vesicles prepared by the dehydration-rehydration procedure (DRV) or covalently coupled by diazotization to the surface of multilamellar vesicles (MLV) prepared by the classical procedure. Up to 82.3% of the antigen used was entrapped in neutral, negatively and positively charged DRV composed of a variety of unsaturated and saturated phospholipids and 63.1% was coupled to MLV composed of egg phosphatidylcholine. After freeze-drying of toxoid-incorporating DRV and MLV and subsequent rehydration, up to 93.5% of the antigen was recovered with liposomes and, in the case of MLV, retained its external localization. Upon freeze-drying in the presence of 0.25 M trehalose, up to 96.1% of the antigen was recovered with the DRV liposomes. In immunization studies using Balb/c mice, DRV composed of equimolar egg phosphatidylcholine and cholesterol were shown to act as immunological adjuvants to the entrapped tetanus toxoid. In addition, there was no difference in immune responses between DRV and MLV of identical composition but bearing the toxoid on their surface. Comparison of immune responses to the toxoid entrapped in DRV made of phospholipids with varying gel to liquid crystalline transition temperature (Tc) revealed a reduction in responses to very low or nil values for DRV made of distearoyl phosphatidylcholine (Tc 54 degrees C).