Biomedical subjects

J A Robinson

Publications and source records attributed to J A Robinson.

At least 55 records · Page 3Linked to original sources

PubMed1996

Cloning, sequencing, overexpression in Escherichia coli, and inactivation of the valine dehydrogenase gene in the polyether antibiotic producer Streptomyces cinnamonensis.

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide antibiotic biosynthesis. To determine for the first time the importance of valine dehydrogenase (vdh) in polyether antibiotic biosynthesis, the vdh gene from Streptomyces cinnamonensis has been cloned and sequenced. The enzyme (M(r)37,718 Da) has been produced in large amounts in an active form in the E. coli cytoplasm using a T7 RNA-polymerase expression system. Upon inactivation of the gene in S. cinnamonensis by a double-crossover mechanism, a hyg::vdh mutant was isolated that was devoid of vdh activity. Upon growth in chemically defined media, as well as a complex medium optimised for monensin production, the mutant and wild-type grew equally well and reached the same levels of monensin production. In both strains a valine transaminase activity could be detected that provides an alternative route for converting valine into 2-oxoisovaleric acid. The results show that vdh is not essential for normal growth of S. cinnamonensis, and its inactivation does not significantly affect normal levels of monensin production in this strain.

Amino Acid Oxidoreductases↗

PubMed1996

Production and characterization of anti-human interferon gamma receptor antibody fragments that inhibit cytokine binding to the receptor.

Three single-chain antibody fragments that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R), and inhibit the binding of human IFN gamma, have been produced in Escherichia coli. These fragments are derived from murine anti-receptor monoclonal antibodies, and comprise the variable heavy (VH) domain linked to the variable light (VL) chain through a 15 amino acid linker [(GGGGS)3]. Using surface plasmon resonance technology (BIAcore), the soluble proteins were shown to retain a high affinity for recombinant IFN gamma R, and by radioimmunoassay to possess a high inhibitory activity towards IFN gamma-binding to human Raji cells. The antibody fragments most likely recognize epitopes that overlap the cytokine binding site on the receptor surface. Attempts to dissect further the antibodies to isolated VH- and VL-chains and to synthetic linear and cyclic peptides derived from the individual complementarity determining regions failed to afford fragments with significant IFN gamma R binding affinity. Nevertheless, these native-like variable region fragments and petidomimetics derived from them are of interest in the design of novel IFN gamma R antagonists.

Amino Acid Sequence↗

PubMed1996

Induction of early-phase endotoxin tolerance in horses.

Six, clinically healthy horses, of mixed age and sex, were infused via a jugular venous catheter with 100 ml of pyrogenfree sterile saline (PFSS; 0.9% NaCl). Animals were infused with Escherichia coli O55:B5 endotoxin (total dose = 50 ng/kg bwt), 24 (LPS-1) and 48 h (LPS-2) after PFSS infusion. Blood was collected before, and every 15 min after, each infusion for the first 8 h and then every 2 h for the following 14 h. Clinical responses (rectal temperature, heart rate, respiration rate and blood pressure) were determined before and every 4 h after each infusion for 20 h. Geometric mean anti-endotoxin antibody titres in serum samples, harvested just before each infusion, were unchanged over the course of the experiment. Serum tumour necrosis factor-alpha (TNF alpha) activity was estimated using a cytotoxic bioassay and WEHI 164 clone 13 murine fibrosarcoma cells as targets. Mean clinical parameter values and geometric mean serum TNF alpha activity at given time points were compared across the 3 infusions. Both LPS-1 and LPS-2 resulted in elevated mean rectal temperature at 4 h after infusion. However, duration of mean rectal temperature elevation was greater (P < 0.05) after LPS-1 (through 12 h) than after LPS-2 (through 8 h). More substantial increases in systolic and diastolic blood pressure were observed after LPS-1 than LPS-2 and mean systolic blood pressure after LPS-1 was elevated at 4 h when compared to PFSS (P < 0.05). Decreased systolic and diastolic blood pressures were observed at 16 h after both LPS infusions, when compared to PFSS infusion. Heart rate was increased, compared to PFSS, after both LPS-1 (8-12 h) and LPS-2 (4-12 h) (P < 0.05). No significant elevations in mean respiratory rate were observed after either LPS-1 or LPS-2 when compared to PFSS. However, at 4 h post infusion, mean respiratory rate after LPS-2 was greater (P < 0.05) than that after LPS-1. Serum TNF alpha activity was not detected after infusion of PFSS, but was detected after both LPS-1 and LPS-2. Serum TNF alpha activity was elevated earlier, was present in higher concentrations and persisted longer after LPS-1 than after LPS-2 (P < 0.05). The decreased duration of fever and attenuated serum TNF alpha response subsequent to successive sublethal LPS challenge observed in this study support the conclusion that these horses developed early-phase endotoxin tolerance (EPET) and, therefore, contributes to the understanding of the role of endotoxaemia in a number of clinical conditions in horses.

Animals↗

PubMed1996

Interspecies acetate transfer influences the extent of anaerobic benzoate degradation by syntrophic consortia.

Benzoate degradation by an anaerobic, syntrophic bacterium, strain SB, in coculture with Desulfovibrio sp. strain G-11 reached a threshold value which depended on the amount of acetate added and ranged from about 2.5 to 29.9 (mu)M. Increasing acetate concentrations also uncompetitively inhibited benzoate degradation. The apparent V(infmax) and apparent K(infm) for benzoate degradation decreased with increasing acetate concentration, but the benzoate degradation capacities (V(infmax)/K(infm)) of cell suspensions remained comparable. The addition of an acetate-using bacterium to cocultures after the threshold was reached resulted in the degradation of benzoate to below the detection limit. Mathematical simulations showed that the benzoate threshold was not predicted by the inhibitory effect of acetate on benzoate degradation kinetics. With nitrate instead of sulfate as the terminal electron acceptor, no benzoate threshold was observed in the presence of 20 mM acetate even though the kinetics of benzoate degradation were slower with nitrate rather than sulfate as the electron acceptor. When strain SB was grown with Desulfovibrio sp. strain DG2 that had a fourfold-lower V(infmax) for hydrogen use than strain G-11, the V(infmax) for benzoate degradation was 37-fold lower than that of strain SB-G-11 cocultures. The Gibb's free energy for benzoate degradation was less negative in cell suspensions with a threshold than in suspensions without a threshold. These studies showed that the threshold was not a function of the inhibition of benzoate degradation by acetate or the toxicity of the undissociated form of acetate. Rather, a critical or minimal Gibb's free energy may exist where thermodynamic constraints preclude further benzoate degradation.

Journal Article↗

PubMed1996

The importance of hydrogen in landfill fermentations.

Forty-two samples taken from two landfills were monitored for CH(inf4) production and apparent steady-state H(inf2) concentration. The rates of methanogenesis in these samples ranged from below the detection limit to 1,900 (mu)mol kg (dry weight)(sup-1) day(sup-1), and the median steady-state hydrogen concentration was 1.4 (mu)M in one landfill and 5.2 (mu)M in the other. To further investigate the relationship between hydrogen concentration and methanogenesis, a subset of seven landfill samples was selected on basis of their rates of CH(inf4) production, H(inf2) concentrations, sample pHs, and moisture contents. Samples with H(inf2) concentrations of <20 nM had relatively small amounts of volatile fatty acids (VFAs) (undetectable to 18.6 mmol of VFA kg [dry weight](sup-1)), while samples with H(inf2) concentrations of >100 nM had relatively high VFA levels (133 to 389 mmol of VFA kg [dry weight](sup-1)). Samples with high H(inf2) and VFA contents had relatively low pH values (<=6.3). However, methanogenic and syntrophic bacteria were present in all samples, so the lack of methanogenesis in some samples was not due to a lack of suitable inocula. The low rates of methanogenesis in these samples were probably due to inhibitory effects of low pH and VFA accumulation, resulting from a thermodynamic uncoupling of fatty acid oxidation. As in other anaerobic ecosystems, H(inf2) is a critical intermediate that may be used to monitor the status of landfill fermentations.

Journal Article↗

PubMed1996

Osteoclasts and transforming growth factor-beta: estrogen-mediated isoform-specific regulation of production.

Estrogen deficiency induced by menopause leads to an increase in bone resorption that is not compensated for by a comparable increase in bone formation, resulting in excessive bone loss. Clinically, estrogen replacement reverses these processes, but the mechanisms by which this takes place are not completely understood. Both osteoclasts and osteoblasts contain functional estrogen receptors and, therefore, may be directly involved in these responses. Because both osteoclasts and osteoblasts secrete transforming growth factor-beta (TGF beta), and because 17 beta-estradiol (E2) treatment increases TGF beta production by osteoblast-like cells in vitro, we have investigated the possibility that E2 also may increase the production of TGF beta by isolated osteoclasts in vitro. Highly purified avian osteoclasts were treated with either vehicle or E2, and TGF beta protein accumulation in culture was measured by bioassay. Although an E2 dose-dependent increase in TGF beta protein accumulation in osteoclast-conditioned medium was measured at 4 h of treatment, a steroid dose-dependent decrease in the accumulation of active TGF beta was measured after 18 h of estrogen treatment. The steroid specificity of the increased TGF beta accumulation was confirmed by demonstrating that the E2-induced increase in TGF beta protein levels in the medium was inhibited by cotreatment with a specific E2 antagonist. Interestingly, E2 treatment induced a TGF beta isoform change from TGF beta 2 to predominantly TGF beta 3. Thus, the data suggest that a direct action of E2 on osteoclasts to lower resorption activity may be mediated by autocrine/paracrine production and activation of TGF beta, perhaps including modulation of specific isoform production.

Animals↗

PubMed1996

Potential mechanism of estrogen-mediated decrease in bone formation: estrogen increases production of inhibitory insulin-like growth factor-binding protein-4.

Using a recently developed human osteoblastic cell line (hFOB/ER9) with high levels (approximately 4,000 per nucleus) of estrogen receptors and the characteristic phenotype of the mature osteoblast, we tested the hypothesis that estrogen decreases bone formation by inhibiting the action of the insulin-like growth factor (IGF) paracrine/autocrine system. IGF-II, the predominant IGF produced by osteoblastic cells, was measurable in hFOB/ER9-conditioned medium (approximately 10 ng/mL) and its level did not change significantly after treatment with 17 beta-estradiol (E2) or anti-estrogens. Treatment with E2 at 0.1-100 nM decreased [3H]thymidine uptake to 53% of control (p < 0.001) in a dose-dependent fashion. The predominant IGF-binding proteins (IGFBPs) produced by hFOB/ER9 and by normal trabecular osteoblasts are IGFBP-3 and IGFBP-4, of which IGFBP-4 is consistently inhibitory of IGF action. Treatment with E2 at 0.01-10 nM for 48 h increased IGFBP-4 mRNA to 346% +/- 90% (mean +/- SE) of control (p < 0.05) and IGFBP-4 protein to 278% +/- 75% of control (p < 0.01) in a dose-dependent fashion but did not alter IGFBP-3 mRNA or protein. E2 treatment also attenuated IGF-dependent, IGFBP-4 specific proteolysis to approximately 50% of control. ICI 182,780, a pure anti-estrogen, completely blocked E2-mediated decreases in cell proliferation and increases in levels of IGFBP-4 mRNA and protein. Treatment of the hFOB/ER9 cells with recombinant human IGFBP-4 (200 ng/mL) decreased cell proliferation to 55% of control (p < 0.01). Thus, E2 acts on osteoblastic cells to increase availability of inhibitory IGFBP-4, by both increasing its production and decreasing its degradation, which may oppose the mitogenic effect of the IGFs on osteoblastic cells. This action may mediate, at least in part, the decreases in bone formation that are observed after estrogen treatment in vivo.

Antibodies↗

PubMed1996

Humoral (antibody-mediated) rejection in lung transplantation.

To confirm the existence and characterize the pathologic features of humoral (antibody-mediated) lung rejection, we prospectively studied 55 lung transplant recipients (24 male [44%] and 31 female [56%], age range 14 to 69 years [mean 45]). The time between transplantation and biopsy ranged from 2 to 1546 days (mean 274). We performed direct immunofluorescence with C3, immunoglobulin M, and immunoglobulin G antibodies on frozen sections of 106 transbronchial biopsies and one wedge biopsy and compared the results with 13 explanted lungs, one donor lung, and two controls. The histologic diagnoses of these 107 biopsies included acute cellular rejection (62, 58% [minimal 23, mild 33, moderate 5, and severe 1]), chronic rejection (eight, 7%), chronic vascular rejection (two, 2%), acute vasculitis (five, 5%), cytomegalovirus pneumonitis (two, 2%), acute pneumonia (two, 2%), acute organizing pneumonia (two, 2%), diffuse alveolar damage (one, 1%), no evidence of rejection or infection (30, 28%), lipoid pneumonia (one, 1%), and inadequate for histologic diagnosis (one, 1%). Eighty-nine of 106 (84%) transbronchial biopsies, the wedge biopsy, and control lungs were satisfactory for direct immunofluorescence, because each contained alveolate lung parenchyma and arterioles or venules. There was no demonstrable immunofluorescence in the wall of the blood vessels or in the lung parenchyma in any case. We conclude that (1) transbronchial biopsies and wedge biopsies provide adequate material to evaluate humoral rejection, and (2) in spite of the large population studied, the satisfactory material obtained, and the wide range of histologic diagnoses, we could not demonstrate the occurrence of humoral rejection in the lung.

Adolescent↗

PubMed1996

Anticipatory reaching by infants and adults: the effect of object features and apertures in opaque and transparent screens.

Perception of the manner in which objects may be grasped was examined in a series of experiments with adults and 10-month-old infants; visuomotor adjustment in hand orientation before making contact with objects served as the index throughout. In the first experiment, infants modified their hand orientation to match more closely the long axis of an object. They did so even though the target object could have been grasped by either end with hands oriented orthogonally to the long axis of the object. In two subsequent experiments involving reaching through narrow apertures to grasp the target, anticipatory adjustments in hand posture were evident for adults but not for infants. However, anticipatory adjustments by infants, sometimes appropriate and sometimes inappropriate, were made when the object was of such a size that it could neither be grasped nor retrieved through the aperture. In the final experiment, infants directed their hands first toward a handle, the only graspable part of an object, and oriented their hands so as to be nearly parallel with it prior to contact. This was the only orientation in which a grasp was possible. It was concluded that infants at 10 months respond more appropriately to object properties than to surface apertures that place limitations on how an object may be grasped.

Adult↗

PubMed1995

Mutagenesis of immunoglobulin-like domains from the extracellular human interferon-gamma receptor alpha chain and their recognition by neutralizing antibodies monitored by surface plasmon resonance technology.

The extracellular portion of the human interferon-gamma receptor (IFN-gamma R) is predicted to adopt two Ig-like domains each with Ig superfamily type C2 topology. Surface plasmon resonance technology has been used here to compare the equilibrium and kinetic constants for binding reactions between these and several mutant domains, and neutralising anti-IFN-gamma R monoclonal antibodies. The biosensor measurements provide a sensitive method for monitoring the effects of mutations on the functional epitopes recognized by the neutralising antibodies. Thus, using recombinant native-like proteins made in E. coli, the ten N-terminal residues of the receptor were found not to be essential for domain folding, nor for recognition by the antibodies A6, D2 and gamma R38. In a similar way, residues in the interdomain region were found to play an important functional role in the epitope recognized by the antibody gamma R99.

Amino Acid Sequence↗

PubMed1995

Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.

The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., & Kossiakoff, A. (1992) Science 255, 306-312], a distant relative in the cytokine receptor superfamily. In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold. The N-terminal domain (residues 1-108), with a Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli. Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding. Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells. Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure. The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.

Amino Acid Sequence↗

PubMed1995

Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

Amino Acid Sequence↗

PubMed1995

Polyethers.

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Anti-Bacterial Agents↗

PubMed1995

Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors.

Human bone marrow contains a distinct cell population that expresses bone proteins and responds to transforming growth factor beta 1 (TGF-beta), but not to hematopoietic growth factors (Long, M. W., J. L. Williams, and K. G. Mann. 1990. J. Clin. Invest. 86:1387-1395). We now report the isolation, characterization, and growth factor responsiveness of these precursors to human osteoblasts and the identification of a human osteoprogenitor cell. Immunological separation of human bone marrow nonadherent low-density (NALD) cells results in a marked enrichment of cells that express osteocalcin, osteonectin, and bone alkaline phosphatase. Flow cytometric analyses show that distinct cell subpopulations exist among these isolated cells. The majority of the bone antigen-positive cells are approximately the size of a lymphocyte, whereas other, less frequent antibody-separated subpopulations consist of osteoblast-like cells and osteoprogenitor cells. In serum-free cultures, TGF-beta stimulates the small, antigen-positive cells to become osteoblast-like, as these cells both increase in size, and express increased levels of osteocalcin and alkaline phosphatase. Antibody-separated cells also contain a separate population of clonal progenitor cells that form colonies of osteoblast-like cells when cultured in serum-free, semi-solid media. Two types of human osteoprogenitor cells are observed: a colony-forming cell (CFC) that generates several hundred bone antigen-positive cells, and a more mature cluster-forming cell that has a lesser proliferative potential and thus generates clusters of 20-50 antigen-positive cells. Osteopoietic colony-forming cells and cluster-forming cells have an obligate but differential requirement for osteogenic growth factors. The CFCs respond to TGF-beta, basic fibroblast growth factor (bFGF), bone morphogenic protein-2 (BMP-2), and 1, 25-dihydroxy vitamin D3 (1,25-OH D3). In contrast to the colony-forming cells, cluster-forming cells are regulated predominantly by 1,25-OH D3 and TGF-beta, but fail to respond to bFGF. We conclude that human bone marrow contains a nonhematogenous, heterogeneous population of bone precursor cells among which exists a population of proliferating osteoprogenitor cells. Further characterization of these bone precursor cell populations should yield important information on their role in osteogenesis in both health and disease.

Alkaline Phosphatase↗

PubMed1995

The effect of slaframine on salivary output and subacute and acute acidosis in growing beef steers.

Experiments were conducted to determine 1) the effect of injecting slaframine (SF) on salivary output in growing beef steers and 2) whether increased salivary output after SF injection would inhibit the decrease in ruminal pH that occurs after experimentally induced subacute and acute ruminal acidosis. In Exp. 1 and 2, we measured ruminal pH and salivary output in ruminally and esophageally cannulated beef steers fed an 88% concentrate diet. Injections of 66 or 100 micrograms of SF/kg BW increased salivary flow approximately 50% compared with controls. Those doses were tested in subacute and acute acidosis models using ruminally cannulated beef steers in Exp. 3 and 4, respectively. In these experiments, salivation was assessed indirectly using a visual scoring system. In the subacute acidosis model, SF reduced (P < .10) the decrease in ruminal pH (1.1, .7, and .6 pH units for control, 66, and 100 micrograms of SF/kg BW doses, respectively), and excessive salivation was observed in all SF-injected steers. In the acute acidosis model, there were no differences (P > .10) in ruminal pH at 12 h after injection between control and SF-treated steers. Mean ruminal lactate concentrations for all treatment groups were between 87 and 112 mM. Although treatment with 66 micrograms of SF/kg BW reduced (P < .10) ruminal lactate concentrations, all ruminal lactate concentrations were indicative of acute acidosis. These results indicate that SF will reduce the decrease in ruminal pH associated with subacute acidosis in growing beef steers, but SF does not attenuate acute ruminal acidosis.

Acidosis↗

PubMed1995

The effect of daily slaframine injection of salivary score, feed intake, ruminal pH, and circulating concentrations of somatotropin and insulin-like growth factor I.

In a randomized complete block design with repeated measures, ruminally cannulated crossbred beef steers (n = 24; 279 +/- 3.2 kg) had ad libitum access to a 90% concentrate diet and were injected daily with slaframine dichloride (SF) at doses of 0, 30, or 60 micrograms SF free-base/kg BW. On d 1, 2, 7, 14, and 21, salivary scores were determined hourly from -2 to 8 h relative to injection (0 h), and ruminal pH was measured at 0, 4, and 8 h. Feed intake was measured daily. Serum concentrations of somatotropin (ST) were measured in samples collected at 20-min intervals from -40 min to 8 h on d 2, 7, 14, and 21, and IGF-I was measured at 8 h. Mean salivary score increased with SF injection (P < .01; .4, 3.3, and 3.7 for 0, 30, and 60 micrograms of SF/kg BW, respectively), although there was a dose x day interaction (P < .01). Injection of SF reduced DMI (P < or = .01; 6.39, 4.95, and 2.64 kg of DM/d for 0, 30, and 60 micrograms of SF/kg BW, respectively). Mean ruminal pH was increased (P < .01); 6.03, 6.42, and 6.72 for 0, 30, and 60 micrograms of SF/kg BW, respectively). Steers administered 60 micrograms of SF/kg BW lost weight (P = .02; 15.1 and -20.3 kg for 0 and 60 micrograms of SF/kg BW, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1994

Diminished cytotoxic gene expression in rat cardiac transplants with low-dose cyclosporine/methotrexate combination therapy.

We have previously shown that administration of a combination low-dose cyclosporine (CsA) (1.0 mg/kg/day)/methotrexate (MTX) (450 micrograms/kg/wk) treatment significantly increases the survival of rat cardiac allografts, and may therefore potentially serve as an alternative immunosuppressive therapy designed to promote transplant survival while minimizing high-dose CsA side effects. In contrast to high-dose CsA, low-dose CsA/MTX treatment does not appear to alter IL-2 gene expression, since similar patterns of IL-2 gene transcripts were found in both low-dose CsA/MTX-treated and untreated control allografts on days 1 through 8 posttransplantation (post-tx). The mechanism(s) by which low dose CsA/MTX therapy increases the time of allograft survival remains to be elucidated. The aim of the present study was to determine the effects of low-dose CsA/MTX on the expression of the cytotoxic cytokines, TNF alpha, TNF beta, or lymphotoxin (LT), and the serine proteases HF and C11 (granzymes A and B, respectively) in rat cardiac allografts during rejection. RNA blot analysis showed significant suppression of TNF alpha, LT, HF, and C11 gene expression on days 1 through 8 post-tx in cardiac allografts from low-dose CsA/MTX-treated recipients compared with untreated allograft controls. TNF protein levels in cardiac allografts from low-dose CsA/MTX-treated recipients were also found to be significantly reduced on days 1 through 8 post-tx when compared with time-matched untreated allograft controls (P < or = 0.001). We conclude that low-dose CsA/MTX treatment, while effective in prolonging cardiac transplant survival, appears to act at the mRNA level to downregulate cytotoxic cytokine gene expression. Such trials aimed at evaluating low-dose combination therapy may afford new insight into mechanisms underlying improvement in immunosuppressive treatment.

Animals↗

PubMed1994

Spatial variability in biodegradation rates as evidenced by methane production from an aquifer.

Accurate predictions of carbon and energy cycling rates in the environment depend on sampling frequencies and on the spatial variability associated with biological activities. We examined the variability associated with anaerobic biodegradation rates at two sites in an alluvial sand aquifer polluted by municipal landfill leachate. In situ rates of methane production were measured for almost a year, using anaerobic wells installed at two sites. Methane production ranged from 0 to 560 mumol . m . day at one site (A), while a range of 0 to 120,000 mumol . m . day was measured at site B. The mean and standard deviations associated with methane production at site A were 17 and 57 mumol . m . day, respectively. The comparable summary statistics for site B were 2,000 and 9,900 mumol . m . day. The coefficients of variation at sites A and B were 340 and 490%, respectively. Despite these differences, the two sites had similar seasonal trends, with the maximal rate of methane production occurring in summer. However, the relative variability associated with the seasonal rates changed very little. Our results suggest that (i) two spatially distinct sites exist in the aquifer, (ii) methanogenesis is a highly variable process, (iii) the coefficient of variation varied little with the rate of methane production, and (iv) in situ anaerobic biodegradation rates are lognormally distributed.

Journal Article↗