Biomedical subjects

G Shearer

Publications and source records attributed to G Shearer.

At least 55 records · Page 3Linked to original sources

PubMed1988

Nitrogen isotopic fractionation and 18O exchange in relation to the mechanism of denitrification of nitrite by Pseudomonas stutzeri.

Two types of mechanisms for the enzymatic reduction of NO2- to N2O have been proposed. In one, two NO2- ions are reduced in parallel, with the nitrogen-nitrogen bond formed from reduced intermediates. In the second, the two NO2- ions enter the reaction sequentially, with the nitrogen of at least one of the two having a valence of 3+ when the nitrogen-nitrogen bond is formed. Our objective was to distinguish between these two types of mechanism. Toward that end, the exchange of 18O from H2O to NO2- and the overall nitrogen isotopic fractionation factor (beta obs) were measured. The rate of exchange of oxygen from H2O to NO2-, resulting from a protonation-dehydration step preceding reductive events in both mechanisms, was less than 10% of the rate of denitrification at both low and high [NO2-]. The value of beta obs was 1.010 +/- 0.001 and 1.020 +/- 0.001 at low and high [NO2-], respectively. Expressions for beta obs, as a function of the measured rate of entry of oxygen from H2O into NO2-, were derived for both types of mechanism. The measured dependence of beta obs on substrate concentration, as constrained by the 18O exchange data, is inconsistent with the first type of mechanism, but consistent with the second type. Thus, by combining nitrogen isotopic fractionation and 18O exchange data, we rule out any mechanism in Pseudomonas stutzeri in which NO2- ions are reduced in parallel, with the nitrogen-nitrogen bond being formed from reduced intermediates.

Mathematics↗

PubMed1988

Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont.

Animals↗

PubMed1988

Cycloheximide efflux in antibiotic-adapted cells of the fungus Mucor racemosus.

Mucor racemosus cells adapted to either cycloheximide or trichodermin were approximately 40-fold more resistant to cycloheximide than nonadapted cells. Ribosomes isolated from adapted and nonadapted cells were equally sensitive to cycloheximide in an in vitro poly(U) translation assay. There was no detectable modification of cycloheximide by adapted cells. Uptake of drug by nonadapted and adapted cells was characterized by a rapid initial accumulation during the first 2 min of incubation with [3H]cycloheximide, followed by a steady-state intracellular drug concentration well below that of the medium. The steady-state drug concentration was approximately 10-fold lower in adapted cells than in nonadapted cells. Treatment of cells with sodium azide or dinitrophenol abolished the difference between uptake of drug by nonadapted and adapted cells and resulted in intracellular drug levels equal to that of the medium. Direct efflux measurements showed that adapted cells loaded with cycloheximide were able to excrete the drug far more rapidly than nonadapted cells. These results suggest that both nonadapted and adapted cells possess an energy-dependent efflux mechanism for transporting cycloheximide and that resistance in adapted cells is due to increased efficiency of transport.

Amino Acids↗

PubMed1987

Mechanical assistance for cardiogenic shock following cardiac surgery, myocardial infarction, and cardiac transplantation.

From January, 1982, to October, 1986, 33 patients were treated with either the Pierce-Donachy prosthetic ventricle or the Bio-Medicus ventricular assist device for cardiogenic shock following a cardiac operation, myocardial infarction, or cardiac transplantation. Twenty-five patients required the assistance for postcardiotomy shock and 8, for a variety of conditions including myocardial infarction shock and myocarditis, and as a bridge to cardiac transplantation. Complications were frequent and usually secondary to prolonged cardiopulmonary bypass. Results were poorest in the group with postcardiotomy shock. Earlier application of an assist device could lead to more frequent survival and avoidance of the detrimental effects of prolonged extracorporeal circulation.

Acute Disease↗

PubMed1987

Heat-shock proteins induced during the mycelial-to-yeast transitions of strains of Histoplasma capsulatum.

Heat-shock proteins (hsp) were elicited when mycelia of the Downs strain and the more virulent G184A and G222B strains of Histoplasma capsulatum were shifted up to temperatures which induced the mycelial-to-yeast transition (34-40 degrees C). The classes of the major hsp which increased in synthesis in each strain were similar. However, the pattern of synthesis of these proteins at the different temperatures in Downs differed from those in the G184A and G222B strains: hsp synthesis in Downs peaked at 34 degrees C, whereas in G184A and G222B it was highest at 37 degrees C.

Heat-Shock Proteins↗

PubMed1987

[Combined effect of cytomegalovirus infection and allo-immunization on the cytotoxic T lymphocyte response in the mouse].

We have investigated the reciprocal influence of CMV infection and allo-immunisation on the CTL response of mice against allogeneic cells and TNP modified syngeneic cells. The data show that allo-immunisation regulates the immunological modifications brought about by CMV infection; allo-immunisation impedes the increased reactivity induced by CMV and counters the suppression induced by CMV when the mice are tested against a non specific target, whereas CMV induced immunosuppression persists against the specific immunizing antigen. Reciprocally CMV infection modulates the effect of all-immunisation: whatever may be the effect of CMV infection, the allo-suppression persists against the specific target, whereas it is abolished against a non specific one. We conclude that there is an immune interaction between CMV infection and allo-immunisation which might have clinical implications.

Animals↗

PubMed1986

Cytotoxic T cell response and thymic hormonal dysfunction in graft-vs-host mice.

As an approach to dissect complex mechanisms that lead to graft-vs-host (GvH)-associated immune disorders, we have compared the splenic cytotoxic T lymphocyte (CTL) response and thymic hormonal function in nonirradiated F1 hybrid mice injected with parental spleen cells. Thymic secretory function was studied by the determination of serum thymulin levels, and the number of thymic epithelial cells containing thymulin as assessed by indirect immunofluorescence with the use of an anti-thymulin monoclonal antibody. In addition, the epithelial cell network was analyzed with an anti-keratin serum, and the general histology pattern was studied by conventional histologic methods. An initial analysis was performed on day 15, which was characterized by CTL suppression mediated by parental suppressor T cells. No thymic abnormalities were detected at this time. By day 45 after GvH induction, active CTL suppression had decreased, and GvH was associated with a progressive decline in thymic hormonal function. Finally, by day 60 and thereafter, F1 GvH mice recovered normal in vitro CTL responsiveness, which contrasted with profound alterations of the epithelial cell network and severely reduced serum thymulin levels. This hormonal dysfunction was shown to be directly associated with a reduction in the number of thymulin-containing cells. Moreover, no anti-thymulin auto-antibodies could be detected. The results are discussed with respect to the role of thymic hormonal dysfunction in the modulation of F1 CTL responses observed during the course of a GvH reaction, and the additional analogy of this GvH model with human immunodeficiency.

Animals↗

PubMed1985

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis.

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 degrees C; pH optimum was 7-7.5; Mg+2 optimum was 5-10 mM. The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.

Blastomyces↗

PubMed1985

Mechanical assistance for biventricular failure following coronary bypass operation and heart transplantation.

From September 1983 to March 1985, five patients who could not be weaned from extracorporeal circulation or who deteriorated in the recovery room have been treated with biventricular mechanical support using two vortex pumps, standard cannulas and tubing. One patient was supported for six hours following heart transplantation and acute graft failure until another donor heart could be found. Intraaortic balloon pumping was utilized in each patient to augment the circulation and produce pulsatile flow. Four of the five patients were weaned from the device. Only one patient is currently a long-term survivor. Although long-term survival has been low, the ability of the myocardium to recover was impressive and warrants the efforts at assisting the heart in biventricular failure.

Adult↗

PubMed1984

Increase of natural N enrichment of soybean nodules with mean nodule mass.

The (15)N abundance of soybean (Glycine max L. Merrill var Harosoy) nodules is usually greater than it is for other tissues or for atmospheric N(2). Results of experiments in which nodules were separated by size show that the magnitude of the (15)N enrichment is correlated with nodule mass. The results support the hypothesis that (15)N enrichment of nodules results from differential N isotopic fractionation for synthesis of nodule tissue versus synthesis of compounds for export from the nodule. The physiological significance of this hypothesis is that it requires that a substantial fraction of the N for nodule tissue synthesis in (15)N-enriched nodules be N recently fixed within the same nodule.

Journal Article↗

PubMed1983

Variable expression of the nitrogen isotope effect associated with denitrification of nitrite.

The overall isotopic fractionation factor observed for denitrification of NO-2 by resting cultures and cell-free extracts of Pseudomonas stutzeri varied widely with the concentration of NO-2 and reductant. The observed isotope effect (beta obs) increased linearly with velocity when the concentration of nitrite was varied and decreased with velocity when reductant concentration was varied. At any given ratio of velocity to maximum velocity, beta obs was approximately the same in intact cells and cell free extracts. These results indicate the following: (a) neither uptake (whole cells) nor enzyme-substrate association (cell-free extracts) is the sole cause of saturation of the overall rate as [NO-2] is increased; (b) a reductive step lies beyond the initial step and at or before the first unidirectional step; and (c) in intact cells, uptake of NO-2 by the cell and egress of NO-2 from it are very rapid compared to reduction at all concentrations of NO-2. A corollary of the last conclusion is that variation of beta obs in intact cells is due entirely to variation in the relative rates of intracellular steps of the denitrification process. The linear relation between beta obs and velocity imposes constraints on any proposed mechanism of denitrification.

Isotope Labeling↗

PubMed1983

Site of natural N enrichment of soybean nodules.

Soybean (Glycine max L. Merrill) nodules are usually more enriched in (15)N than other tissues. We show that both bacteroids and nodule cortex are considerably more enriched in (15)N than nodule cytosol, with bacteroids being slightly more enriched than the cortex. Hence, (15)N enrichment occurs in cells of both plant and bacterial origin.

Journal Article↗

PubMed1983

Relationship between N(2)-Fixing Efficiency and Natural N Enrichment of Soybean Nodules.

Non-nodular tissue of soybean (Glycine max L. Merrill) plants grown hydroponically in the absence of added N have a (15)N abundance close to that of atmospheric N(2). In contrast, nodules are usually enriched in (15)N. In this paper, we report measurements of the (15)N abundance of foliar tissue and nodules of soybeans inoculated with 11 variably efficient strains of Rhizobum japonicum and grown hydroponically with no added N. The efficiency of the 11 symbioses varied over a wide range as judged by a 16-fold difference in N content. The degree of (15)N enrichment of nodules was closely correlated with N(2)-fixing efficiency (milligrams N fixed per milligram N in the nodules).These results confirm prior preliminary data based on six variably efficient R. japonicum strains. The strong correlation between (N)N enrichment of soybean nodules and N(2)-fixing efficiency is consistent with the hypothesis that new nodule tissue is synthesized from a pool of recently fixed N within the same nodule.

Journal Article↗

PubMed1982

N abundance of nodules as an indicator of N metabolism in n(2)-fixing plants.

This paper expands upon previous reports of (15)N elevation in nodules (compared to other tissues) of N(2)-fixing plants. N(2)-Fixing nodules of Glycine max (soybeans), Vigna unguiculata (cowpea), Phaseolus vulgaris (common bean), Phaseolus coccineus (scarlet runner bean), Prosopis glandulosa (mesquite), and Olneya tesota (desert ironwood) were enriched in (15)N. Nodules of Vicia faba (fava beans), Arachis hypogaea (peanut), Trifolium pratense (red clover), Pisum sativum (pea), Lathyrus sativus (grass pea), Medicago sativa (alfalfa), and Lupinus mutabilis (South American lupine) were not; nor were the nodules of nine species of N(2)-fixing nonlegumes. The nitrogen of ineffective nodules of soybeans and cowpeas was not enriched in (15)N. Thus, (15)N elevation in nodules of these plants depends on active N(2)-fixation. Results obtained so far on the generality of (15)N enrichment in N(2)-fixing nodules suggest that only the nodules of plants which actively fix N(2) and which transport allantoin or allantoic acid exhibit (15)N enrichment.

Journal Article↗

PubMed1980

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum.

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5% of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 micrograms/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.

Animals↗

PubMed1980

Isotopic Fractionation Associated With Symbiotic N(2) Fixation and Uptake of NO(3) by Plants.

Isotopic fractionation associated with N(2) fixation and NO(3) (-) uptake by plants are relevant to the accuracy of estimates of N(2) fixation based on differences in the natural abundance of (15)N between N(2) fixing and nonfixing plants. The isotope effect on N(2) fixation by soybeans (Glycine max [L] Merrill, variety Harosoy) and red clover (Trifolium pratense [L]) was determined from the difference in (15)N abundance between atmospheric N(2) and the total N of plants grown hydroponically with N-free nutrient solution. In soybeans the isotope effect was found to be +0.98 +/- 0.18 per thousand (beta = 0.99902). In clover the isotope effect was +1.88 +/- 0.14 per thousand (beta = 0.99812). The magnitude of these inverse isotope effects is small. However, they would lead to an underestimation of the amount of N(2) fixed, since the N of atmospheric origin which finally appears in the plant is made richer in (15)N by the inverse isotope effects than is atmospheric N(2), and, to that degree, is attributed to soil-derived N in the calculation.Isotopic fractionation associated with NO(3) (-) uptake by plants does not have a critical effect on estimates of N(2) fixation which are based on natural abundance of (15)N since the (15)N abundance of soil-derived N in plants is measured directly (i.e. after the N has undergone fractionation). Nevertheless, such fractionation is of some interest from the point of view of deciding upon the most appropriate sampling time. The isotope effect on NO(3) (-) uptake by a nonnodulating isoline of soybeans (variety Harosoy), marigold (Tagetes erecta [L]) and ryegrass (Lolium perenne [L]) was estimated from the difference between the (15)N abundance of the total N of plants grown hydroponically and that of NO(3) (-) supplied in the medium. It was found to be about -5 per thousand (beta = approximately 1.005).

Journal Article↗

PubMed1980

Distribution of N among plant parts of nodulating and nonnodulating isolines of soybeans.

Differences among plant parts in the natural abundance of (15)N are of interest from the point of view of developing a sampling strategy for using (15)N measurements to estimate the contribution of symbiotically fixed N to N(2) fixing plants, and because they reflect isotopic fractionation associated with degradation, transport, and resynthesis of N-bearing molecules. This paper reports such differences in nodulating and nonnodulating isolines of soybeans (Glycine max [L] (Merrill, variety Harosoy)) grown under several different conditions.Nodules were strikingly enriched in (15)N compared to other plant parts (by an average of 8.3 per thousand excess (15)N), and the enrichment increased with time during the growing season. (15)N was much more uniformly distributed among other plant parts. Although there were significant differences among other plant parts, the maximum deviation of the (15)N abundance of any plant part from that of the entire plant was about 2 per thousand (15)N excess. The (15)N abundance of the seed N was most representative of the whole plant.There were significant differences between isolines in the distribution of (15)N. The distribution of (15)N within plants also varied with experimental conditions. The implications of these results for estimation of N(2) fixation from measurements of the natural abundance of (15)N are discussed.

Journal Article↗

PubMed1980

Estimates of n(2) fixation based on differences in the natural abundance of N in nodulating and nonnodulating isolines of soybeans.

Estimates of the contribution of biologically fixed N to the total N of nodulating soybeans (Glycine max (L) Merrill, variety Harosoy) grown under a variety of conditions were made from: (a) differences in N yield between nodulating and nonnodulating isolines; and (b) differences in (15)N abundance between the two isolines. For plants grown in a greenhouse in nutrient-poor soil, both estimates showed a high level of N(2) fixation; from 58 to 89% N fixed by differences in N yield and from 51 to 95% by differences in (15)N abundance. Decreasing contributions of fixed N were estimated by both methods with increasing levels of added NO(3) (-). Results of field experiments carried out over two years on an unamended highly fertile midwestern soil showed a modest level of N(2) fixation by both methods (7.3 to 51% by differences in N yield, and 5.4 to 46% by differences in (15)N abundance). When the soil was amended with ground corn cobs, both methods showed higher contributions of fixed N. The two methods of estimating N(2) fixation gave similar results. Both appear to be semiquantitative and the standard errors of the estimates were about the same (6% on the average).

Journal Article↗