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Biomedical subjects

R Heath

Publications and source records attributed to R Heath.

At least 37 records · Page 2Linked to original sources
PubMed1988

Human erythrocyte protein 4.1 is a phosphatidylserine binding protein.

The aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS) are the major phospholipids contained in the cytoplasmic leaflet of the human erythrocyte (RBC) plasma membrane and are largely confined to that leaflet over the entire RBC lifespan. In particular, PS, which comprises approximately 13% of total RBC membrane phospholipids, is normally restricted entirely to the cytoplasmic leaflet. However, molecular mechanisms that regulate this asymmetric distribution of phospholipids are largely unknown. We examined elliptocytic RBCs that completely lacked protein 4.1 (HE [4.1 degrees]), but contained normal amounts of all other peripheral membrane proteins, and found approximately 10% of total membrane PS was accessible in the exoplasmic leaflet of these membranes. Inside out vesicles (IOVs) derived from HE [4.1 degrees] RBCs bound fewer PS liposomes than did IOVs derived from normal RBCs. Normal IOVs that were depleted of proteins 2.1 (ankyrin), 4.1, and 4.2 bound fewer PS liposomes similar to HE [4.1 degrees] IOVs, and repletion with protein 4.1 restored PS liposome binding to control levels. Addition of purified protein 4.1 to PS liposomes resulted in saturable binding with the extent of binding being proportional to the liposome PS content. Our data suggests that human RBC protein 4.1 is a PS binding protein and may be involved in the molecular mechanisms that stabilize PS in the cytoplasmic leaflet of the human RBC plasma membrane.

Blood Proteins↗
PubMed1988

Deficiency of protein 4.2 in erythrocytes from a patient with a Coombs negative hemolytic anemia. Evidence for a role of protein 4.2 in stabilizing ankyrin on the membrane.

A patient with a mild hemolytic anemia and osmotically fragile, spherocytic erythrocytes was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of protein 4.2 (less than 0.10% of normal). The protein 4.2-deficient erythrocytes contained normal amounts of all other membrane proteins, although the amount of band 3 was slightly reduced and the amount of band 6 (G3PD) was slightly elevated. The spectrin content of these cells was normal, as measured by both SDS-PAGE and radioimmunoassay. Erythrocytes from the patient's biologic parents were hematologically normal and contained normal amounts of protein 4.2. Immunological analysis using affinity purified antibodies revealed that the patient's protein 4.2 was composed of equal amounts of a 74-kD and 72-kD protein doublet, whereas the normal protein was composed primarily of a 72-kD monomer. Proteolytic digestion studies using trypsin, alpha-chymotrypsin and papain demonstrated that the patient's protein 4.2 was similar but not identical to the normal protein. Binding studies showed that the protein 4.2-deficient membranes bound purified protein 4.2 to the same extent as normal membranes, suggesting that the membrane binding site(s) for the protein were normal. Depleting the protein 4.2-deficient membranes of spectrin and actin resulted in a loss of nearly two-thirds of the membrane ankyrin, whereas similar depletion of normal membranes resulted in no loss of ankyrin. Repletion of the protein 4.2-deficient membranes with purified protein 4.2 before spectrin-actin extraction partially prevented the loss of ankyrin. These results suggest that protein 4.2 may function to stabilize ankyrin on the erythrocyte membrane.

Adult↗
PubMed1988

Changes in proteinase/proteinase inhibitor levels in rat skeletal muscle tissue during diabetes and fasting.

The skeletal muscle content of three rat proteinase inhibitors, a 1-proteinase inhibitor, contrapsin and a 1-cysteine proteinase inhibitor was measured by immunochemical techniques following streptozotocin-induced diabetes. When compared with normal rats, a 1-cysteine proteinase inhibitor and a 1-proteinase inhibitor levels remained essentially unchanged, whereas the content of rat contrapsin was reduced by nearly 80% after the onset of diabetes. Similarly, fasting of rats for three days resulted in a lowering of the levels of contrapsin in skeletal muscles. Under these conditions, levels of chymotrypsin-like activity (chymase) were increased by 150%, whereas the content of the trypsin-like, neutral proteinase was unchanged. Kinetic studies in vitro with Tosyl-Gly-Pro-Arg-4-nitroanilide as substrate showed no inhibition of the trypsin-like proteinase by a 1-proteinase inhibitor, while contrapsin inhibited the enzyme with a Ki value of 40nM. The changing pattern of these proteinases and their potential inhibitors (chymase/a 1-proteinase inhibitor and trypsin-like proteinase/contrapsin) may be a factor contributing to muscle wasting as observed in diabetes and fasting.

Animals↗
PubMed1987

Physicochemical stability and inactivation of human and simian rotaviruses.

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.

Animals↗
PubMed1987

Human milk and preterm formula compared for effects on growth and metabolism.

Metabolic tolerance to a 'premature formula' feed was studied in a group of small immature infants, mean (SD) gestation 27.8 (1.4) weeks. Ten infants weighing 880-1295 g at the time of the study were fed on SMA low birthweight formula for a mean (SD) of 23.5 (5.5) days and were compared with 10 who were fed on expressed breast milk for 25.8 (6.1) days. The infants were well matched for weight, gestation, and postnatal age at the time of the study and were receiving full enteral feeds. They were investigated by balance techniques and plasma sampling on at least two occasions. Ten larger infants weighing 1330-1740 g and being fed on the same formula feed were also studied as an additional control group. Formula fed infants retained more nitrogen and gained weight faster. Plasma phosphorus concentrations were higher in the group fed on the formula feed, and alkaline phosphatase activity was lower. There were no significant differences in plasma concentrations of urea, electrolytes, or albumin or in acid base status. Taurine and arginine concentrations were higher in the group being breast fed, but there were no other significant differences in plasma amino acids, and no toxic concentrations occurred after either feed. The results of this study show that this formula (and presumably other feeds of similar composition) seem to be metabolically safe for the smallest infants.

Anthropometry↗
PubMed1987

Acquired immune deficiency syndrome without the recognized risk factors.

We report two cases of acquired immune deficiency syndrome (AIDS), apparently without the usual exposure factors, in whom a temporal association was detected after detailed epidemiological investigation. The index case, a 45 year old housewife, had provided terminal home-nursing care for a 33 year old African man, who died from an undiagnosed encephalitis. At that time she had fissures of the skin of both her hands. Review of post-mortem pathology specimens of the African man allowed a retrospective diagnosis of AIDS with cerebral toxoplasmosis to be made. The type of home-nursing care given by the index case was quite different from that normally provided by health care workers with the training and facilities to prevent the spread of infection.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Alkaline proteinase localization in myoblasts.

Recent interest in elucidating the role of non-lysosomal proteases in intracellular protein catabolism in muscle has led to various investigations with three alkaline proteases: a trypsin-like, a chymotrypsin-like, and a high molecular weight cysteine proteinase. Although in vitro biochemical assays have revealed the catabolic potential of at least two of these proteases, confirmation of their presence in muscle cells has been difficult. In this study immunohistochemical techniques were employed to localize each of these proteases in rat myoblasts. Antisera against the trypsin-like and chymotrypsin-like proteinase (both serine proteinases) showed strong localization in the cytoplasm immediately around the nucleus. Both also stained chromatin material in the nucleus of these cells. Fluorescent localization of the high molecular weight cysteine proteinase (Proteinase I) also appeared to be cell-associated in the myoblasts. The use of myoblasts in cell culture sections of whole muscle was advantageous, since localization of the proteases could be assessed in the absence of other cell types.

Animals↗
PubMed1987

Slow moving proteinase. Isolation, characterization, and immunohistochemical localization in gastric mucosa.

Human gastric mucosa contains three immunochemically distinguishable aspartic proteinases, pepsinogen I (pepsinogen A), pepsinogen II (pepsinogen C, progastricsin), and a nonpepsinogen proteinase also termed slow moving proteinase (SMP). The properties of SMP, and in particular its relationship to another aspartic proteinase, cathepsin D, were examined in this study. Slow moving proteinase and cathepsin D were isolated, respectively, from gastric mucosa and human spleen. Antiserum specific to each proteinase was prepared in rabbits. Rabbit anti-SMP did not recognize cathepsin D, and conversely, anticathepsin D did not react with SMP. Immunohistochemical studies localized SMP to surface epithelial cells in both the fundic and pyloric gland areas of the stomach. In contrast, cathepsin D was found mainly in mononuclear cells in the lamina propria and in parietal cells. Slow moving proteinase exhibited considerably lower Km values for its interaction with two chromogenic substrates than did cathepsin D. An even greater distinction between the two enzymes was found with the protein inhibitor from Ascaris lumbricoides; the activity of SMP was inhibited very strongly, whereas that of cathepsin D was not affected. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions, SMP consisted of two subunits with apparent molecular weights of 42,500 and 41,000. The last two properties characterize a less-well-known aspartic proteinase, cathepsin E. We conclude that SMP is not cathepsin D, but that it may be cathepsin E.

Cathepsin D↗
PubMed1986

Immunogold electron-microscopic localisation of calpain I in skeletal muscle of rats.

By using a double-affinity-purified first antibody and colloidal gold-conjugated second antibody, it is shown that calpain I (a cysteine proteinase activated by micromolar concentrations of Ca2+) has a predominant intracellular location in the I-band region of the extensor digitorum longus (EDL) muscle of the rat, but is not exclusively associated with the Z-line.

Animals↗
PubMed1986

Antigenic analysis of rotavirus isolates using monoclonal antibodies specific for human serotypes 1, 2, 3 and 4, and SA11.

Five neutralizing monoclonal antibodies produced against human rotavirus (HRV) serotypes 1, 2, 3 and 4 and the simian rotavirus (SA11) were used to study 59 rotavirus isolates of human, simian and feline origin previously serotyped using polyclonal antisera. In neutralization tests, 19 of 26 HRV serotype 1 isolates, both strains of HRV serotype 2, 14 of 24 HRV serotype 3 isolates and all of seven serotype 4 isolates were neutralized by the homologous serotype-specific monoclonal antibodies. Use of the panel of monoclonal antibodies revealed antigenic differences between strains within serotypes 1 and 3 and, in the case of the serotype 3 strains, each variant had a unique RNA electropherotype. An enzyme immunoassay (EIA) which utilized the monoclonal antibodies essentially confirmed the neutralization results. Preliminary results show that direct serotyping in faecal extracts by EIA using these monoclonal antibodies is specific but lacks sensitivity.

Antibodies, Monoclonal↗
PubMed1985

Fetal plasma carbonic anhydrase III in prenatal diagnosis of Duchenne muscular dystrophy.

Carbonic anhydrase III (CAIII), a skeletal-muscle-specific enzyme which is elevated in the plasma of Duchenne muscular dystrophy (DMD) patients, was measured by radioimmunoassay in fetal plasma in order to evaluate its application to prenatal diagnosis of DMD. Using fetoscopy, pure fetal blood samples were taken at 17-24 weeks gestation from 25 fetuses at risk for DMD and from 78 control fetuses. Care was taken in the handling and storage of all samples. Normal sons were born in eight cases at risk for DMD. The CAIII levels in the infants were not significantly different from those of the control infants. Pregnancies were terminated in the remaining 17 at-risk cases. The CAIII levels in the fetuses were significantly different (p = 0.0034) from those of the control fetuses, although the distributions overlapped. Based on prior maternal risk, seven affected fetuses were expected in the terminated group; five had CAIII levels at or above the 95th centile of the control range. It is suggested that measurement of CAIII achieves partial discrimination between affected fetuses and their normal at-risk brethren.

Carbonic Anhydrases↗
PubMed1985

Evaluation of carrier detection of Duchenne muscular dystrophy using carbonic anhydrase III and creatine kinase.

Carbonic anhydrase III (CAIII) and creatine kinase (CK) were measured in plasma samples from a series of females at-risk as carriers of Duchenne muscular dystrophy and compared with control groups. Both plasma CAIII and CK levels were raised in a proportion of carriers. Although measurement of CAIII and CK was no more successful in identifying carriers than CK alone, CAIII could fulfill a useful confirmatory role, particularly for cases with a marginally elevated CK or where the sample is poorly preserved. The difference between the CK and CAIII results could indicate biochemical heterogeneity in the expression of Duchenne muscular dystrophy.

Carbonic Anhydrases↗
PubMed1984

Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking.

Dimethyl adipimidate (DMA), an effective antisickling agent in vitro, reacts with free amino groups producing chemically modified and cross-linked molecules. In this report, we have investigated the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization. Since the extent of cross-linking is pH-dependent, we first compared the solubilities of deoxygenated hemolysates prepared from sickle cells previously treated with dimethyl adipimidate at either pH 7.4 or 8.4. The solubility of the hemolysate increased from 18.6 +/- 0.8 g/dl in the untreated sample to 20.9 +/- 1.5 g/dl (pH 7.4) and to 25.4 +/- 3.0 g/dl (pH 8.4) after dimethyl adipimidate treatment. Removal of cross-linked hemoglobin tetramers from hemolysate obtained from dimethyl adipimidate-treated cells abolished part of this effect; at pH 7.4, the solubility decreased from 20.9 +/- 1.5 to 19.4 +/- 0.2 and at pH 8.4 from 25.4 +/- 3.0 to 21.0 +/- 1.5. However, the ratio of [14C]DMA-labelled hemoglobin in the sol phase to that in the gel phase in the unfractionated hemolysate was 1.17 at pH 7.4 and 1.25 at pH 8.4, suggesting that part of the cross-linked hemoglobin tetramers was incorporated into the gel. In order to further investigate the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization, we separated cross-linked hemoglobin tetramers on a gel-filtration column, prepared mixtures of untreated sickle hemoglobin and cross-linked hemoglobin tetramers and studied the polymerization of these mixtures. The Csat of the untreated hemolysate increased progressively from 18.6 +/- 0.8 to 22.5 +/- 0.8 g/dl with 33% cross-linked hemoglobin tetramers. The hemoglobin concentration in the gel decreased from 43 +/- 1.0 to 33.8 +/- 1.0 g/dl with 33% cross-linked hemoglobin tetramers, while the pellet volume fraction, phi p, increased with and almost approached 1 at 50% cross-linked hemoglobin tetramers. In addition, the sol phase contained a higher molecular weight distribution of cross-linked hemoglobin tetramers than the gel phase. These observations suggest that a loose polymer was formed in the gel phase with a hemoglobin concentration much lower than that of the control. Thus, polymerization of sickle hemoglobin is inhibited by: (1) exclusion of higher molecular weight cross-linked hemoglobin tetramers from the gel, and (2) loose incorporation of cross-linked hemoglobin tetramers into the gel, perhaps preventing lateral packing and formation of tightly ordered fibers.

Chromatography, Gel↗