Biomedical subjects

R M Smith

Publications and source records attributed to R M Smith.

At least 289 records · Page 16Linked to original sources

PubMed1989

Intranuclear localization of insulin in rat hepatoma cells: insulin/matrix association.

Previous studies have documented nuclear insulin accumulation in a variety of cell types. The present investigation extends these observations by demonstrating that insulin associates with the matrix fraction of H35 rat hepatoma cell nuclei. Nuclei were isolated from [125I]insulin-loaded cells and extracted with DNase I, RNase A and high salt. The resulting matrix fraction was found to contain greater than 75% of the radiolabel initially present. Ultrastructural studies to confirm these findings were carried out using an agarose-encapsulated nuclear matrix preparation. Electron microscopic immunocytochemistry specifically detected insulin in matrices prepared from insulin-treated cells. No reaction was observed in matrices obtained from non-insulin-treated (control) cells. Further biochemical analysis revealed that matrix-associated insulin could be solubilized with 1% sodium dodecyl sulfate (SDS) or in the presence of high urea concentrations. Gel filtration analysis of urea-solubilized matrix material revealed the presence of apparently intact [125I]insulin and a higher molecular weight peak. It is hypothesized that the latter may represent a tightly associated complex of insulin with some matrix protein(s).

Animals↗

PubMed1989

Construct and predictive validities of the Perceptual Ability Test.

The construct and predictive validities of the Perceptual Ability Test (PAT) were examined. The results indicate that the PAT is a multidimensional test of spatial abilities, with each of the PAT subtests exhibiting different predictive validity. Furthermore, a linear combination of the PAT subtest scores was found to be more predictive of first-year dental school technique performance than the total PAT score.

College Admission Test↗

PubMed1988

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-ammonium nitrate eluent. Batch-to-batch reproducibility of the stationary phase.

The effect of changing the brand and batches of the silica stationary phase used with a methanol-aqueous ammonium nitrate eluent for the high-performance liquid chromatographic separation of basic drugs has been studied. Considerable care had to be taken to obtain a reproducible eluent and the effect of small changes in the concentration of the ammonia solution were examined closely. Large differences in both the capacity factor and relative capacity factors were found for separations on columns packed with four different brands of silica. Significant differences were also observed with columns containing eighteen different batches of Spherisorb S5W, which had been manufactured over a period of several years.

Chromatography, High Pressure Liquid↗

PubMed1988

Production and administration to dogs of aerosols of alpha-1-proteinase inhibitor.

The feasibility of aerosol administration of alpha-1-proteinase inhibitor (human) (A1PI) was assessed. Of three different methods of aerosolizing A1PI that were evaluated, an ultrasonic nebulizer was found to be best suited to the present purpose, producing particles of a size that allowed them to reach the distal air spaces of the lung and that retained specific A1PI anti-elastase activity. Administration of 20 mg/kg of A1PI and 150 microCi of 131iodine-A1PI to three dogs was accomplished without complications. Gamma camera scans documented a relatively homogenous distribution throughout the lungs. Bronchial lavage fluid that was recovered from the lungs of the dogs six hours after administration contained large amounts of human A1PI and showed a proportional elevation of anti-elastase activity. There was no evidence of acute toxicity.

Administration, Inhalation↗

PubMed1988

Intravenous administration of alpha-1-proteinase inhibitor in patients of PiZ and PiM phenotype. Preliminary report.

Nine patients with moderate pulmonary emphysema, six of PiZ phenotype and three of PiM phenotype, have received a single intravenous infusion of alpha-1-proteinase inhibitor (human) (A1PI), in a dose of 60 mg/kg over a 30-minute period. They also received a tracer dose (300 microCi) of 131I-labeled A1PI. No active or passive immunization against hepatitis was given. No acute toxicity was observed. Compared with baseline data, significant elevations of serum A1PI (measured both antigenically and as anti-elastase activity) occurred, with a serum half-life approximating 110 hours. Bronchoalveolar lavage fluid, obtained 48 hours after infusion, reflected a significant increase in A1PI concentration versus baseline bronchoalveolar lavage fluid values. Serial gamma camera images of the lungs confirmed persistence of enhanced lung radioactivity for several days. Urinary desmosine excretion did not change following A1PI infusion. During the period of follow-up thus far, no patient has had chronic toxicity, results of liver function tests have been stable, and there has been no development of hepatitis B antigen or antibodies to hepatitis B surface or core antigens.

Adult↗

PubMed1988

Application of packed column supercritical fluid chromatography to the analysis of barbiturates.

The analysis of barbiturates using packed column supercritical fluid chromatography (SFC) has been investigated. The separations were carried out on either an ODS bonded silica or polystyrene-divinylbenzene polymer column with carbon dioxide as the mobile phase and flame ionisation detection. All the barbiturates were strongly adsorbed on the ODS-silica column, but reasonable separations were obtained by using the polymer column.

Journal Article↗

PubMed1988

Preparation and characterization of a colloidal gold-insulin complex with binding and biological activities identical to native insulin.

We studied the binding and biological activities of gold-insulin complexes to develop a complex with properties identical to native insulin. Stabilizing amounts of insulin absorbed to 5-, 10-, or 15-nm gold particles resulted in complexes with 40-327 insulin molecules per gold particle and 4-111 times the biological activity of unlabeled insulin, based on the molar concentration of gold complex. These data suggested that these complexes behaved as multivalent ligands. Gold-insulin complexes were prepared with 5% of the stabilizing insulin concentration and were stabilized with bovine serum albumin. This resulted in a complex with 5-7 insulin molecules per 10-nm gold particle, which stimulated glucose oxidation in rat adipocytes and competed with [125I]-insulin for binding to the insulin receptor identically to unlabeled insulin on an equimolar basis. The organization and distribution of insulin receptors occupied by this monovalent-behaving gold-insulin complex were virtually identical to previous observations using monomeric ferritin-insulin. Since multivalent ligands may affect receptor binding, re-distribution, and intracellular processing, the use of electron-dense probes that resemble the unlabeled ligand in biological and binding properties is appropriate when studying receptor dynamics of in vivo or in vitro biological systems. The gold-insulin complex developed in this study should serve this function.

Adipose Tissue↗

PubMed1988

Effect of growth hormone on growth and myelination in the neonatal hypothyroid rat.

The possible involvement of a deficit of GH and insulin-like growth factor-I (somatomedin C) (IGF-I/SMC) in mediating the effects of propylthiouracil (PTU)-induced hypothyroidism on body and skeletal growth and myelination was studied in the neonatal rat. Myelination (as assessed by 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity), skeletal growth (as assessed by tail length) and body weight of pups from PTU-treated mothers were significantly retarded compared with normal animals or euthyroid controls. At 20 days after birth, plasma GH in hypothyroid animals was undetectable (less than 10 micrograms/l), pituitary GH content was 1.2% of control, and plasma, liver and kidney IGF-I/SMC concentrations were 63, 68 and 50% of control values respectively. CNP activity in hypothyroid brain was 52% of normal controls but the concentration of IGF-I/SMC was 113-154% of control. Treatment of hypothyroid animals from day 1 with GH (10 mg/kg body weight per day) restored liver and plasma IGF-I/SMC concentrations at 20 days to values above those of normal animals and euthyroid controls. The concentration of IGF-I/SMC was also significantly (P less than 0.001) restored in hypothyroid kidney (79% of normal), but the concentration in brain was unaffected. These observations provide evidence that the GH treatment employed in the present experiments was adequate to restore the deficit. GH treatment had no significant effect on tail length or CNP activity, and only a small (4-24%) effect on body weight at 20 days. Only thyroxine was able fully to restore body weight and substantially restore tail length and CNP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Autoradiographic evidence for insulin and insulin-like growth factor binding to early mouse embryos.

Insulin binding to mouse oocytes and preimplantation embryos was assessed by light-microscopic autoradiography. Significant insulin binding was present on the cells of morulae and increased twofold at the blastocyst stage of development. Insulin binding was markedly decreased by native insulin and to a lesser extent by insulin-like growth factors (IGFs). No specific insulin binding was detected on oocytes or embryos throughout the eight-cell stage. Specific binding of IGF-I and IGF-II was also observed on the cells of blastocyst outgrowths. The findings demonstrate that specific binding of insulin and IGF is temporally expressed on the cells of pre- and peri-implantation mouse embryos. These results confirm and extend our previous immunofluorescence study.

Animals↗

PubMed1988

Protection against hyperoxia by serum from endotoxin treated rats: absence of superoxide dismutase induction.

Endotoxin greatly reduces lung injury and pleural effusions in adult rats exposed to normobaric hyperoxia (greater than 98% oxygen for 60 hours). This study reports that serum from endotoxin treated donor rats protects serum recipients against hyperoxic lung injury without altering lung superoxide dismutase (SOD) activity. Rats pretreated with endotoxin alone were protected and exhibited an increase in lung SOD activity as previously reported by others. Protection by serum was not due to the transfer of residual endotoxin or SOD. These results show that protection from oxygen toxicity can occur in rats without an increase in lung SOD and suggest that a serum factor may be involved.

Animals↗

PubMed1988

Receptor-mediated endocytosis and intracellular processing of insulin: ultrastructural and biochemical evidence for cell-specific heterogeneity and distinction from nonhormonal ligands.

This review has presented some of the data which indicate that there are substantial differences in the mechanisms of receptor-mediated uptake and intracellular processing of ligands. Data from various laboratories indicate that receptor-mediated endocytosis is not necessarily synonymous with the concentrative, coated-pit-mediated internalization of ligand-receptor complexes. That differences exist both between the internalization of different types of ligands on the same cell type and between different types of cells for same ligand has clearly been documented. The differences in mechanisms or structures involved in receptor-mediated endocytosis have been demonstrated both biochemically and ultrastructurally. The reasons for these two diverse internalization pathways utilizing coated and non-coated membrane domains are not known and require further investigation to substantiate or disprove hypotheses presented here and elsewhere. Similarly, while differences in intracellular routing of hormonal and non-hormonal ligands and their receptors certainly exist, the factors controlling the itinerary of the ligand or its receptor need to be identified. The differences in ligand-receptor processing already demonstrated should indicate that interpretation of the results of future studies must be made with a great deal of caution and conservatism particularly when attempting to apply knowledge of one ligand and cell system to pharmacologically, ultrastructurally, or physiologically dissimilar systems.

Adipose Tissue↗

PubMed1988

Pulmonary oxygen toxicity in rats: prevention by pyrogenic diphosphoryl lipid A and potentiation by nontoxic monophosphoryl lipid A and lipid X.

Pretreatment with gram-negative bacterial endotoxin protects rats from the lethal intrathoracic edema produced by continuous exposure to greater than 95% O2 for 3 days. To help elucidate structure-activity relationships of endotoxin, we administered lipid part-structures of endotoxin intraperitoneally (at dosages up to 5.0 mg/kg) to male Sprague-Dawley rats prior to exposure to greater than 99% O2 (at 760 mm Hg) for 72 hr. We found that Salmonella minnesota mutant Re595 diphosphoryl lipid A protected rats from O2 toxicity as effectively as the parent S. minnesota endotoxin molecule at equimolar concentration. Diphosphoryl lipid A exhibited less acute toxicity (2 hr post-treatment hypothermia) than did endotoxin, although both produced prolonged fevers (greater than 24 hr) and similar patterns of protection in O2. In contrast, nontoxic lipid X (2,3-diacylglucosamine 1-phosphate) and monophosphoryl (1-dephospho) lipid A potentiated pulmonary O2 toxicity. We conclude that within the range of dosages investigated, diphosphoryl lipid A is the minimal essential structure responsible for the protection of rats from O2 toxicity by endotoxin.

Animals↗

PubMed1988

Revision of Dental Admission Test standard score scale.

In October 1988 the Dental Admission Testing Program will begin reporting DAT scores on a new standard score scale. This scale will be based on the underlying ability metric rather than on the normal distribution that is the basis of the current-1 to 9 scale. A comparison of the two standard score scales is included, as well as a discussion of the advantages offered by the ability based metric.

American Dental Association↗

PubMed1987

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-ammonium nitrate eluent. The effect of the mobile phase and the operating conditions.

The reproducibility of capacity factors and relative capacity factors are compared as methods for recording retentions for the high-performance liquid chromatography of basic drugs on a silica column with methanol-aqueous ammonium nitrate as the eluent. The effects of changing the column temperature and the ionic strength, pH and proportion of organic modifier in the eluent on the retentions and selectivity have been studied. The results suggest that the mobile phase and operating conditions must be closely defined in order to obtain results of adequate reproducibility to develop a data base of retention values for interlaboratory comparisons and/or for the identification of basic drugs.

Chemical Phenomena↗

PubMed1987

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects.

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.

Affinity Labels↗

PubMed1987

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-ammonium nitrate eluent. Interlaboratory collaborative study.

The reproducibility of the separation of basic drugs on silica columns has been tested in a collaborative study between nine laboratories. The results from the different laboratories were very similar. Various methods of recording the retention properties of the drugs were compared with reference to their reproducibility and ability to discriminate between different compounds. Relative retention times, relative capacity factors, and corrected capacity factors were much better than retention times and capacity factors, although all the methods suffered from large errors with weakly retained compounds. All the work was carried out using a single batch of silica to avoid variations due to the stationary phase.

Ammonia↗

PubMed1987

Retention reproducibility of thiazide diuretics and related drugs in reversed-phase high-performance liquid chromatography.

A method has been developed for the separation of thiazide diuretics and a number of related drugs by high-performance liquid chromatography on an ODS-Hypersil column with acetonitrile-1% aqueous acetic acid as the eluent. The effects caused by changes in the separation conditions on the reproducibility and robustness of alternative methods for recording retentions (including capacity factors, retention indices based on the alkyl aryl ketone scale, and relative capacity factors compared to a thiazide standard) have been examined. The results confirm that good interlaboratory reproducibility will only be achieved when operators control the temperature of the column and use the same brand of column packing material. The retentions should be recorded using a relative method, as these were found to be virtually independent of minor variations in the eluent composition.

Acetates↗

PubMed1987

Insulin binding and processing by H4IIEC3 hepatoma cells: ultrastructural and biochemical evidence for a unique route of internalization and processing.

Biochemical and ultrastructural studies of insulin binding and cellular processing by cultured H4IIEC3 hepatoma cells were performed. Insulin binding and intracellular accumulation were rapid and after 30 min at 37 degrees C, 65% of the total cell-associated 125I-insulin was in an acid-stable compartment. Chloroquine had no significant effect on the amount of total cell-associated insulin or the percentage of insulin in the acid-stable compartment or cell-associated insulin degradation under those conditions, but after 60-min incubations, it slightly decreased the rate of dissociation of internalized hormone. Ultrastructural analysis revealed that monomeric ferritin-insulin (Fm-I) initially bound to single or paired receptors on microvilli. Within 5 min occupied insulin receptors microaggregated and migrated to the intervillous cell surface. During the next 5-10 min occupied receptors aggregated into large clusters on the plasma membrane. Large amounts of insulin were internalized by macropinocytosis and the majority of internalized Fm-I was found in phagosomes. Less than 10% of the membrane-bound insulin was associated with pinocytotic invaginations or coated pits and less than 5% of the total cell-associated insulin was found in lysosomes. Chloroquine had no detectable effect on the amount of Fm-I or its distribution among the intracellular organelles. These studies demonstrated that, compared to previous studies with rat adipocytes or 3T3-L1 adipocytes, insulin interalization and intracellular processing in this hepatoma cell were unique. These differences provide further evidence that insulin binding and processing may be controlled by cell-specific mechanisms and that substantial heterogeneity exists in pathways previously presumed to be similar for all cell types.

Animals↗