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Biomedical subjects

G Shearer

Publications and source records attributed to G Shearer.

At least 37 records · Page 2Linked to original sources
PubMed1995

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Adenosine Deaminase↗
PubMed1995

Cloning and analysis of cDNA encoding an elongation factor 1 alpha from the dimorphic fungus Histoplasma capsulatum.

The cDNA encoding translation elongation factor 1 alpha (EF-1 alpha) was isolated from the dimorphic fungus, Histoplasma capsulatum (Hc), an important pathogen of man. A cDNA library was probed with the tef1 gene from the fungus Mucor racemosus. Ten independent clones were isolated, all with similar restriction patterns. The longest clone (1.96 kb) was sequenced. Southern blot analysis revealed that the Hc tef1 gene was present as a single copy. A single transcript of approx. 2300 nucleotides was found in total RNA from both the yeast and mold forms of the organism. Comparison of the deduced 460-amino-acid Hc EF-1 alpha protein to EF-1 alpha proteins from other species of fungi revealed the greatest degree of similarity to proteins from the filamentous ascomycetes Podospora anserina and Trichoderma reesei. Phylogenetic tree analysis of fungal tef genes indicated that Hc is most closely related to filamentous ascomycetes and most distantly related to the budding yeast Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1994

Multienzyme nested deletion (MEND) technique for creation of unidirectional deletions in cloned DNA.

An alternative method to construct nested unidirectional deletions of cloned DNA is presented. The multi-enzyme nested deletion technique exploits the fact that the 6-base recognition enzyme ClaI produces termini compatible with at least five restriction enzymes that have different 4-base recognition sequences. A plasmid deletion cloning vector with a ClaI site in the polylinker is linearized with ClaI and then cut with a second enzyme to yield incompatible ends. Directionally cloned insert DNA (in this example SfiI and NotI) is partially digested with one or several of the 4-base recognition enzymes AciI, HinP1I, MaeII, MspI, or TaqI. An aliquot of this partial digest is ligated into SfiI/ClaI cut vector to yield deletions from the NotI end of the insert. Another aliquot is ligated into ClaI/NotI cut vector to yield deletions from the SfiI end of the insert. In general, one of these 4-base cutters will yield deletion clones useful for manual DNA sequencing (i.e., deletions of 300-400 bp or less). It is also simple to perform partial digestion with two enzymes simultaneously if required.

DNA, Complementary↗
PubMed1994

A syringe-based gradient former for linear and exponential gradients.

This report describes a reliable and inexpensive gradient former constructed from disposable plastic syringes. The dual-chamber device produces both linear and exponential gradients which are very reproducible and essentially identical to the theoretical curve. Two identical syringes which serve as a mixing chamber and reservoir chamber are heat sealed at the bottom and glued to a flat plastic support plate. Small holes are drilled into the side walls of the syringes to accommodate an outlet tube and mixing tube. To form linear gradients, equal volumes of solution are placed in both chambers. The solution in the mixing chamber is stirred with a small magnetic stir bar. As starting solution is drawn from the mixing chamber, ending solution is gradually drawn into the mixing chamber to produce a linear gradient. To form exponential gradients a syringe plunger is placed into the mixing chamber to form a fixed volume of starting solution. Ending solution is placed into the reservoir chamber and may be replenished at any time during gradient formation. This allows small gradient formers to produce large volume exponential gradients. These gradient formers, which require only about 10 min to construct, are durable enough to be used many times and inexpensive enough to discard if desired.

Centrifugation, Density Gradient↗
PubMed1993

High-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil in cattle muscle tissue with on-line anion-exchange clean-up.

A high-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil has been developed. The drug was extracted from cattle muscle tissue with 1% triethylamine in acetonitrile. The extract was evaporated to dryness and taken up in 0.1 M ammonium acetate-acetonitrile (50:50, v/v). The extract was then injected onto a polymeric anion-exchange precolumn. After clean-up with 0.1 M ammonium acetate-acetonitrile (50:50, v/v) for 5 min, the precolumn was eluted with 1% aqueous trifluoroacetic acid-acetonitrile (50:50, v/v) onto a PLRP-S polymer column and chromatographed with a mobile phase of 0.01 M phosphate pH 7-acetonitrile (80:20, v/v). Detection was by ultraviolet at 273 nm. Average recoveries at four levels from 0.005 to 1.000 mg kg-1 were > 88%. The limit of determination was 0.005 mg kg-1.

Animals↗
PubMed1993

High-performance liquid chromatographic method for the determination of the dinitrocarbanilide component of nicarbazin in eggs with on-line clean-up.

A high-performance liquid chromatographic method for the determination of the dinitrocarbanilide component of the anti-coccidial drug nicarbazin has been developed. The drug was extracted from egg with acetonitrile. The extract was evaporated to dryness and taken up in water-acetonitrile (80:20, v/v). The extract was then injected onto a reversed-phase precolumn. After clean-up with 20% aqueous acetonitrile for 5 min, the precolumn was eluted onto a Chromspher C18 cartridge column with 0.01 M potassium dihydrogen-phosphate pH 4.0-acetonitrile (50:50, v/v). Detection was by ultraviolet at 343 nm. Average recoveries at five levels from 0.005 to 0.500 mg kg-1 were > 80%. The limit of determination was 0.005 mg kg-1.

Animals↗
PubMed1993

Alloactivated cytotoxic T cells recognize the carboxy-terminal domain of human immunodeficiency virus-1 gp120 envelope glycoprotein.

Infection with the human immunodeficiency virus (HIV) virus leads to clinical disease in humans but not in chimpanzees. Progression to disease is associated with activation of the immune system followed by loss of T helper cell function and a slow decline in CD4-positive lymphocytes. The presence of autoreactive and cytotoxic cells in humans but not chimpanzees suggests that mechanisms other than, or in addition to, direct virus-induced cell killing, are required for disease to develop. The observed changes are similar to those seen in chronic allogeneic disease. Here we show that a peptide from the carboxy terminus of gp120, predicted to have a structure similar to the major alpha-helices of major histocompatibility complex (MHC) class I and class II, acts as a cytolytic target when presented on syngeneic cells for alloactivated cytotoxic T effector cells. These data add further evidence to the hypothesis that HIV can act as an allostimulant due to its dual properties of CD4 binding and MHC mimicry. The ability to signal nonspecifically through the T cell receptor could explain the initially paradoxical responses of proliferation, anergy and apoptosis.

Amino Acid Sequence↗
PubMed1992

Determination of tranquilisers and carazolol residues in animal tissue using high-performance liquid chromatography with electrochemical detection.

A multi-residue method for the determination of tranquiliser residues in animal tissue is described. The procedure may be used to determine residues of the tranquilisers acepromazine, azaperone, chlorpromazine, haloperidol, propionylpromazine, xylazine, the metabolite of azaperone, azaperol, and the beta-adrenoreceptor blocking agent carazolol. Existing methods of analysis for tranquilisers are based on ultraviolet and fluorescence detection and have been used for pig kidney analysis. Determination in this method was by high-performance liquid chromatography with electrochemical detection in the screen mode. The enhanced selectivity offered by the electrochemical detector allowed determination in liver extracts, which often give rise to more interferences on chromatographic traces when using conventional methods of detection. The method offers up to a ten-fold improvement in limits of determination over methods using ultraviolet and fluorescence detection. Recoveries and coefficients of variation have been determined in the range 2-25 micrograms/kg in pig kidney and liver. This electrochemical detection method has been used to measure residues in routine surveillance programmes.

Adrenergic beta-Antagonists↗
PubMed1992

Improved high-performance liquid chromatographic procedure for the determination of lasalocid in chicken tissues and egg using polymeric and porous graphitic carbon columns.

A high-performance liquid chromatographic (HPLC) method for the determination of the ionophore coccidiostat lasalocid in poultry muscle and eggs was developed. The drug was extracted from tissue with acetonitrile. The extract was partitioned between saturated salt and carbon tetrachloride and the organic layer evaporated to dryness. Clean-up was by solid-phase extraction on a silica column. HPLC analysis was carried out on either a polymeric PLRP-S or a porous graphitic carbon Hypercarb column with a basic mobile phase and fluorescence detection with excitation at 310 nm and emission at 420-430 nm. Average recoveries from poultry muscle at the 0.002, 0.010 and 0.050 mg kg-1 levels were 65.7, 72.0 and 77.9%, respectively. Average recoveries from egg at the 0.010 and 0.100 mg kg-1 levels were 76.2 and 76.4%, respectively.

Animals↗
PubMed1992

High-performance liquid chromatographic determination of imidocarb in cattle kidney with cation-exchange clean-up.

A high-performance liquid chromatographic (HPLC) method for the determination of the antiprotozoal agent imidocarb in cattle kidney is developed. The drug is extracted from tissue with acetone in the presence of base. The extract is partitioned between saturated salt and chloroform and the organic layer evaporated to dryness. Clean-up is by cation-exchange solid-phase extraction on a carboxylic acid column. HPLC analysis is carried out on a Spherisorb S3W-PC18 column with ultraviolet detection at 260 nm. Average recoveries at the 0.05 and 0.01 mg kg-1 levels are 77.5 and 76.3%, respectively. The limit of detection is 0.001 mg kg-1.

Animals↗
PubMed1992

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.

Animals↗
PubMed1992

Proline fed to intact soybean plants influences acetylene reducing activity and content and metabolism of proline in bacteroids.

Supplying l-proline to the root system of intact soybean (Glycine max [L.] Merr.) plants stimulated acetylene reducing activity to the same extent as did supplying succinate. Feeding l-proline also caused an increase in bacteroid proline dehydrogenase activity that was highly correlated with the increase in acetylene-reducing activity. Twenty-four hours after irrigating with l-proline, endogenous proline content had increased in host cell cytoplasm and bacteroids, about three- and eightfold, respectively. In bacteroids, proline concentration was calculated to be at least 3.5 millimolar. In experiments in which [U-(14)C]l-proline was supplied to uprooted, intact plants incubated in aerated solution, (14)C-labeled products of proline metabolism, as well as [(14)C]proline itself, accumulated in both host cells and bacteroids. When plants were incubated in aerated solutions containing [5-(3)H]l-proline, (3)H-labeled proline was found in host cells and bacteroids. [(3)H] Pyrroline-5-carboxylate was found in bacteroids, but not host cells, after a 2-hour incubation in [5-(3)H]l-proline. When [U-(14)C]l-proline was supplied for 24 hours, a significant amount of [(14)C] pyrroline-5-carboxylate was found in the host cells, in contrast with the results from the shorter incubation in [5-(3)H]proline, although the amount in the host cells was only about half the quantity found in the bacteroids. Taken as a whole, these results indicate that proline crosses both plant and bacterial membranes under the in vivo experimental conditions utilized and are consistent with a significant role for proline as an energy source in support of bacteroid functioning. In spite of the increase in acetylene-reducing activity when proline was supplied to the root system of intact plants, proline application did not rescue stemgirdled plants from loss of acetylene-reducing activity, although succinate application did. This suggests a nonphloem route for succinate, but not proline, from roots to nodules.

Journal Article↗
PubMed1991

Comparison of spinal fluid beta 2-microglobulin levels with CD4+ T cell count, in vitro T helper cell function, and spinal fluid IgG parameters in 163 neurologically normal adults infected with the human immunodeficiency virus type 1.

Beta 2-microglobulin levels were measured in the cerebrospinal fluid (CSF) and serum of 163 human immunodeficiency virus-positive (HIV+) persons with normal neurologic physical examinations. None were on antiretroviral therapy. Only 3% had a positive CSF HIV p24 antigen test. The CSF beta 2-microglobulin levels increased as the CD4+ T cell count decreased. Intrathecal production of beta 2-microglobulin was suggested by finding CSF concentrations greater than serum concentrations in 15% of patients. The CSF beta 2-microglobulin levels rose as in vitro T helper cell function deteriorated, independent of CD4+ T cell count. CSF beta 2-microglobulin levels paralleled CSF IgG, IgG index, and IgG synthesis. Higher CSF beta 2-microglobulin levels were found in persons with positive CSF oligoclonal bands. CSF beta 2-microglobulin concentration may serve as a marker for subclinical neurologic damage due to HIV. If this is established, defining the effect of anti-HIV interventions on CSF beta 2-microglobulin would be warranted.

Albumins↗
PubMed1990

Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules.

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N(2) fixation, and production of NADP(+) to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N(2) is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N(2)-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N(2) fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.

Journal Article↗
PubMed1989

Distribution of N within Pea, Lupin, and Soybean Nodules.

The (15)N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in (15)N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring (15)N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in (15)N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest (15)N enrichment. In soybean nodules, the (15)N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in (15)N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in (15)N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual (15)N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in (15)N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in (15)N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess (15)N of lupin and soybean bacteriods is offered.

Journal Article↗
PubMed1988

Development of disease and virus recovery in transgenic mice containing HIV proviral DNA.

Transgenic mice containing intact copies of the human immunodeficiency virus (HIV) proviral DNA were constructed. Founder animals were not viremic for HIV and remained healthy during a 9-month observation period. After being mated with nontransgenic animals, one founder mouse (No. 13) gave rise to F1 progeny that developed a disease syndrome characterized by marked epidermal hyperplasia, lymphadenopathy, splenomegaly, pulmonary lymphoid infiltrates, growth retardation, and death by day 25 of life. Infectious HIV, indistinguishable from parental virus by immunoblot analysis, was recovered from the spleen, lymph nodes, and skin of five of five affected animals.

Acquired Immunodeficiency Syndrome↗