Biomedical subjects

J A Robinson

Publications and source records attributed to J A Robinson.

At least 37 records · Page 2Linked to original sources

PubMed1999

On the importance of being aromatic at an antibody-protein antigen interface: mutagenesis of the extracellular interferon gamma receptor and recognition by the neutralizing antibody A6.

A complex formed between the extracellular human interferon gamma receptor alpha-chain (hIFNgammaR) and the Fab fragment of the neutralizing antibody A6 has been studied by site-directed mutagenesis. Five complementarity determining regions of the A6 antibody interact primarily with the CC' surface loop of the receptor, from Lys47 to Trp56, although contact is also made with residues in the neighbouring F strand, in particular with Trp82. The relative contribution that individual side-chains make to complex stabilization was assessed with 21 receptors mutants, whose affinity for A6 was monitored using a surface plasmon resonance biosensor, as well as by solution-phase competition ELISA. The results reveal two lysine side-chains (Lys47 and Lys52), an asparagine side-chain (Asn53), and two aromatic side-chains (Tyr49 and Trp82) in the receptor that are important for recognition by A6. The role of aromatic side-chains in antibody-antigen recognition is of particular interest, not least in this case because 13 aromatic groups (six Tyr, six Trp and one His) are present at the interface (four in VL, six in VH and three in the receptor), and several are proximal to the charged and polar side-chains of Lys47, Lys52 and Asn53 in the receptor. The results highlight the possibility for aromatic rings to participate in networks of co-operative interactions with not only hydrophobic, but also charged and hydrogen bond donor and acceptor groups, properties that are well suited for creating binding sites for protein epitopes, regardless of the distribution of polar and non-polar surface residues. These findings may contribute, therefore, to an understanding of how surface groups on proteins are captured by the often aromatic-rich hypervariable loops of antibodies, and may be of value for the design of molecules with novel recognition properties.

Antibodies, Monoclonal↗

PubMed1999

Plasmapheresis with intravenous immunoglobulin G is effective in patients with elevated panel reactive antibody prior to cardiac transplantation.

BACKGROUND: Patients with a PRA >10% are considered to be at greater risk for the development of not only acute cellular and humoral rejection but also increased mortality when compared to nonsensitized patients following transplantation. All patients with a PRA >10% at our institution are treated with plasmapheresis and intravenous immunoglobulin G immediately prior to cardiac transplantation. METHODS: Sixteen (Group 1) of 118 patients awaiting cardiac transplantation were found to be sensitized. These patients underwent plasmapheresis followed by 20 gm of intravenous immunoglobulin G (IVIG) immediately prior to cardiac transplantation. Group 1 was compared to the remaining 102 patients with a PRA <10% (Group 2). RESULTS: Despite more patients in Group 1 having a positive crossmatch, pulmonary hypertension, and requiring mechanical circulatory support, there was no statistically significant difference in length of stay or mortality at a mean follow-up of 21.6+/-15.0 months. There was no difference in the occurrence of mild, moderate or severe cellular rejection or humoral rejection in these sensitized patients when compared to Group 2. CONCLUSIONS: Pretransplant plasmapheresis followed by intravenous immunoglobulin G may be an effective therapy that obviates the need for a prospective crossmatch and allows sensitized patients to undergo cardiac transplantation. There is no increase in the post transplant length of stay, occurrence of rejection or short term mortality. Long term follow up is necessary to evaluate whether there is a difference in the development of late rejection, transplant vasculopathy and survival.

Adult↗

PubMed1999

Apheresis in thoracic organ transplantation.

The beneficial addition of cyclosporine and tacrolimus to the immunosuppressive armamentarium have unfortunately only partially solved the problems of acute and chronic rejection in thoracic organ transplantation. Apheresis techniques offer creative avenues for modifications of allograft rejection. Plasmapheresis can be used for mechanical reduction of alloantibody burdens in highly sensitized patients and permit transplantation in an otherwise almost hopeless situation and can also be used on a short-term basis for the treatment of acute humoral rejection. Extracorporeal photochemotherapy holds promise as a possibly synergistic adjunct to conventional therapy and may even reduce the severity of graft vasculopathy. The increasing availability of highly specific column immunoadsorption techniques may further increase the applicability of apheresis in transplantation.

Adult↗

PubMed1999

Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis: influence on polyketide antibiotic biosynthesis.

The coenzyme B(12)-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis. Mutants prepared by inserting a hygromycin resistance gene (hygB) into either icmA or mutB, encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA::hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The mutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. (13)C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.

Bacterial Proteins↗

PubMed1999

A(3) adenosine receptor activation attenuates neutrophil function and neutrophil-mediated reperfusion injury.

This study tested the hypothesis that A(3) adenosine receptors inhibit neutrophil (PMN) function and PMN-mediated reperfusion injury. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), an A(3) agonist, did not attenuate superoxide production or myeloperoxidase release from stimulated PMNs. However, Cl-IB-MECA reduced platelet-activating factor-stimulated PMN adherence to coronary endothelium at low concentrations: 52 +/- 27, 45 +/- 10, and 87 +/- 23 PMNs/mm(2) at 0.1, 1.0, and 10 nM vs. 422 +/- 64 PMNs/mm(2) with platelet-activating factor alone. This inhibition was not blocked by A(1) (5 microM KW-3902) or A(2a) (5 microM KF-21326) antagonists: 44 +/- 3 and 43 +/- 2 PMNs/mm(2), respectively. Endothelial pretreatment with 1 nM Cl-IB-MECA reduced PMN adherence, which was reversed by the A(3) antagonist MRS-1220 (100 nM). PMN-mediated reperfusion injury was initiated in isolated rabbit hearts by infusion of 28 x 10(6) PMNs/min for 10 min early in reperfusion. PMNs caused a significant decrease in recovery of left ventricular developed pressure and positive and negative time derivatives of pressure (23 +/- 3, 25 +/- 3, and 23 +/- 3% of baseline, respectively) vs. buffer-perfused hearts (43 +/- 7, 44 +/- 7, and 45 +/- 6%, respectively). Cl-IB-MECA (10 nM) given at reperfusion attenuated the PMN-mediated loss of contractile recovery (40 +/- 3, 46 +/- 5, and 42 +/- 4% of baseline). Cl-IB-MECA reduced myeloperoxidase release activity (5.3 +/- 0.6 absorbance units/min) and CD18-positive cells (54 +/- 9 cells/slide) compared with the untreated PMN group (17.9 +/- 1.7 absorbance units/min and 183 +/- 68 cells/slide). We conclude that Cl-IB-MECA attenuates reperfusion injury by decreasing PMN-endothelial cell interactions. These results suggest that the A(3) adenosine receptor may be a novel therapeutic target for treatment of myocardial ischemia and reperfusion.

Animals↗

PubMed1999

Vascular damage correlates between heparin-induced thrombocytopenia and the antiphospholipid syndrome.

Antibody-mediated disorders of heparin-induced thrombocytopenia and antiphospholipid antibody syndrome have remarkably similar clinical presentations, both of which can progressively result in severe vascular and thrombotic disorders. We hypothesized that the mechanism of platelet activation as occurs in heparin-induced thrombocytopenia may also occur in antiphospholipid antibody syndrome particularly at the vascular wall, that endothelial injury may be similar in heparin-induced thrombocytopenia and antiphospholipid antibody syndrome, and that these alterations may be caused by related antibodies. Antibody titers and vascular endothelial damage in patients with heparin-induced thrombocytopenia and antiphospholipid antibody syndrome were studied in plasma samples collected from normals (n = 17), heparin-induced thrombocytopenia patients (n = 15), antiphospholipid antibody syndrome patients (n = 30), and patients clinically diagnosed with antiphospholipid antibody syndrome and heparin-induced thrombocytopenia (n = 8). Diagnosis of heparin-induced thrombocytopenia was confirmed by 14C-serotonin release assay or positive antiheparin-platelet factor 4 antibody titer, and antiphospholipid antibody syndrome was confirmed by positive anti-beta 2-glycoprotein (GP) 1/cardiolipin (IgG or IgM) antibody titer. The antiheparin-platelet factor 4 antibody was not detected in any patient with antiphospholipid antibody syndrome. Patients with heparin-induced thrombocytopenia did not have elevated IgG anti-beta 2-GP1 titers, but three (20%) patients had low-positive IgM anti-beta 2-GP1 titers. The endothelial damage markers of soluble thrombomodulin, soluble P-selectin (p < 0.05 vs. normal), plasminogen activator inhibitor-1 and tissue factor were elevated in heparin-induced thrombocytopenia and antiphospholipid antibody syndrome patients. The soluble E-selectin was elevated only in the patients with both heparin-induced thrombocytopenia and antiphospholipid antibody syndrome (p < 0.05 vs. normal). Levels of soluble L-selectin and von Willebrand factor were not different from normals. The pathogenesis of heparin-induced thrombocytopenia and antiphospholipid antibody syndrome appears to be due to two distinct antibodies but associated with similar damage to the vascular endothelium in both diseases.

Antiphospholipid Syndrome↗

PubMed1999

Plasmapheresis in the management of heparin-induced thrombocytopenia.

This study investigated the role of plasmapheresis in the treatment of severe heparin-induced thrombocytopenia (HIT). Patients diagnosed with HIT were divided into three experimental groups. Sixteen patients did not receive plasmapheresis (control). Twenty-one patients received plasmapheresis within 4 days of onset of thrombocytopenia (early group). Seven patients received plasmapheresis 4 days or later after onset (late group). Most patients underwent a second plasmapheresis 24 to 48 hours after the first, when clinically indicated, and platelet aggregation tests became negative in 75% of these patients. Heparin administration was discontinued after 1.4 days in the early group of patients and 4.2 days in the late group, as compared with 2.4 days in the control group. The 30-day mortality rate was 4.8% among patients in the early group and 57% in the late group, as compared with 32% in the control group. Platelet recovery time, incidence of thrombotic events, and length of hospital stay were similar in the early group and controls, but were somewhat higher in the late group. Thus, plasmapheresis within 4 days of the onset of thrombocytopenia reduced mortality in HIT patients, whereas plasmapheresis after 4 days was not beneficial. There were no adverse events related to plasmapheresis. These findings suggest that plasmapheresis may be useful in the treatment of HIT when initiated within 4 days of onset of thrombocytopenia.

Aged↗

PubMed1998

Cloning, sequencing, expression, and insertional inactivation of the gene for the large subunit of the coenzyme B12-dependent isobutyryl-CoA mutase from Streptomyces cinnamonensis.

Purification of the coenzyme B12-dependent isobutyryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of approximately 65 kDa by SDS-polyacrylamide gel electrophoresis, whose gene icmA was cloned using sequences derived from tryptic peptide fragments. The gene encodes a protein of 566 residues (62, 487 Da), with 43-44% sequence identity to the large subunit of methylmalonyl-CoA mutase (MCM) from S. cinnamonensis and Propionibacterium shermanii. Targeted disruption of the icmA gene yielded an S. cinnamonensis mutant devoid of ICM activity. The IcmA protein is approximately 160 residues shorter than the large subunit of the bacterial MCMs, corresponding to a loss of the entire C-terminal coenzyme B12 binding domain. The sequence of the (beta/alpha)8-barrel comprising residues A1-A400 in P. shermanii MCM is highly conserved in IcmA. The protein was produced in Streptomyces lividans and Escherichia coli with an N-terminal His6 tag (His6-IcmA), but after purification His6-IcmA showed no ICM activity. In the presence of coenzyme B12, protein from S. lividans and S. cinnamonensis of approximately 17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His6-IcmA from a Ni2+ affinity column. After purification, this small subunit showed no ICM activity but gave active enzyme when recombined with coenzyme B12 and IcmA or His6-IcmA.

Amino Acid Sequence↗

PubMed1998

Effective oral ganciclovir prophylaxis against cytomegalovirus disease in heart transplant recipients.

The presented data show the combined sequential use of i.v. G for 14 days followed by PO G for 90 days is a much more effective prophylaxis for CMVD after heart transplantation than use of i.v. G for 14 days followed by PO A for 90 days. A need for hospitalization due to CMVD is significantly reduced by this new strategy. The follow-up in group II is shorter than in group I but is now at least 6 months in group II, without any new cases in the first 6 months after cardiac transplantation. Some currently unknown adverse effect of prolonged PO G, which may be present, is not identified in this analysis.

Administration, Oral↗

PubMed1998

Assessment of reductive acetogenesis with indigenous ruminal bacterium populations and Acetitomaculum ruminis.

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H2 disposal mechanism in the rumen. H2/CO2-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 x 10(5) cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH13CO3, and 100% H2 gas phase since [U-13C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10(7) CFU/ml). To assess the relative importance of population density and/or H2 concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N2 gas phase. Both selective inhibition of methanogenesis and A. ruminis 190A4 fortification (>10(5) CFU/ml) were necessary for the detection of reductive acetogenesis under H2-limiting conditions. Under these conditions, H2 accumulated to 4, 800 ppm. In contrast, H2 accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H2 concentrations correlated well with the pure culture H2 threshold concentrations determined for A. ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H2 partial pressure below the level necessary for H2 utilization by A. ruminis 190A4.

Acetic Acid↗

PubMed1998

Estrogen regulation of a transforming growth factor-beta inducible early gene that inhibits deoxyribonucleic acid synthesis in human osteoblasts.

This laboratory reported the identification and characterization of a unique three zinc finger, transcription factor-like, transforming growth factor-beta inducible early gene (TIEG) (see Ref. 35). TIEG expression has been shown to be tissue- and cell type specific, enhanced by specific growth factors, and to decrease with advancing stages of breast cancer. Recent studies involving TIEG overexpression in pancreatic carcinoma cells indicate that TIEG expression inhibits DNA synthesis, similar to a tumor suppressor-like gene, and plays a role in apoptosis (see Ref. 37). This paper describes the rapid, but transient, induction of TIEG steady-state messenger RNA (mRNA) levels by 17beta-estradiol (E2) in estrogen receptor (ER)-positive, human fetal osteoblastic (hFOB/ER) cells. This rapid induction is shown to be ER- and steroid dose-dependent but protein synthesis independent. An antagonism between E2 and PTH, which occurs in skeletal metabolism, is shown to concur rapidly with TIEG mRNA expression. Scanning confocal microscopy (using polarized, laser-based immunofluorescence) shows that TIEG protein is localized in the nucleus of hFOB/ER cells, with the levels rapidly increasing after E2 treatment. The rapid E2-induced increase in TIEG expression is followed by an E2-induced inhibition of DNA synthesis in the hFOB/ER cells. Antiestrogens block not only the induction of TIEG mRNA levels but also the inhibition of cell proliferation. Lastly, hFOB cells, stably transfected with a TIEG expression vector, display markedly reduced DNA synthesis/cell proliferation, compared with nontransfected cells. These results support the finding that TIEG is an early responding regulatory gene for E2 in human osteoblast cells that inhibits DNA synthesis. It is speculated that TIEG may play a role in the signaling pathway for E2 in inhibiting cell proliferation.

Cell Division↗

PubMed1997

Neutralizing epitopes on the extracellular interferon gamma receptor (IFNgammaR) alpha-chain characterized by homolog scanning mutagenesis and X-ray crystal structure of the A6 fab-IFNgammaR1-108 complex.

The extracellular interferon gamma receptor alpha-chain comprises two immunoglobulin-like domains, each with fibronectin type-III topology, which are responsible for binding interferon gamma at the cell surface. The epitopes on the human receptor recognized by three neutralizing antibodies, A6, gammaR38 and gammaR99, have been mapped by homolog scanning mutagenesis. In this way, a loop connecting beta-strands C and C' in the N-terminal domain was identified as a key component of the epitopes bound by A6 and gammaR38, whereas gammaR99 binds to the C-terminal domain in a region including strands A and B and part of the large C'E loop. The epitope for A6 was confirmed in a crystal structure of a complex between a recombinant N-terminal receptor domain and the Fab fragment from A6, determined by X-ray diffraction to 2.8 A resolution. The antibody-antigen interface buries 1662 A2 of protein surface, including 22 antibody residues from five complementarity determining regions, primarily through interactions with the CC' surface loop of the receptor. The floor of the antigen binding cavity is formed mainly by residues from CDR L3 and CDR H3 while a surrounding ridge is formed by residues from all other CDRs except L2. Many potential polar interactions, as well as 13 aromatic side-chains, four in VL, six in VH and three in the receptor, are situated at the interface. The surface of the receptor contacted by A6 overlaps to a large extent with that contacted by interferon-gamma, in the ligand-receptor complex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a surface loop on this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites.

Amino Acid Sequence↗

PubMed1997

Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene.

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5 alpha-dihydrotestosterone (5 alpha-DHT) (10(-8) M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 +/- 73 (mean +/- SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 +/- 395 functional hARs/nucleus in hFOB/AR-2 cells, and 3,987 +/- 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5 alpha-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5 alpha-DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

Androgen Antagonists↗

PubMed1997

Dynamical studies of peptide motifs in the Plasmodium falciparum circumsporozoite surface protein by restrained and unrestrained MD simulations.

The immunodominant region on the circumsporozoite surface (CS) protein of the malaria parasite Plasmodium falciparum contains 37 repeated copies of a asparagine-alanine-asparagine-proline (NANP) motif NMR studies of linear synthetic peptides containing one, two or three repeat units provided evidence for nascent type I beta-turns within the NPNA cadence in aqueous solution. The beta-turns could be stabilised upon substituting proline for alpha-methylproline (p(Me)) in the dodecamer (NP(Me)NA)3, without loss of the ability to elicit antibodies cross-reactive with P. falciparum sporozoites. In this work, four 4 ns MD simulations of the dodecapeptide Acetyl-(NP(Me)NA)3, in water, using NOE distance restraints, using 3J-coupling constant restraints, using both these restraints and without restraints, were carried out to determine the conformations of this peptide in aqueous solution. An unrestrained MD simulation of the unmethylated Ac-(NPNA)3 peptide in water was also carried out to investigate the effect of the additional methyl groups on the structure and dynamics of the peptide. The application of NOE distance restraints and 3J-coupling constant restraints leads to contradictory results, probably due to different averaging time scales inherent to the measurement of these data, which exceed the 100 ps averaging applied in the simulations. The additional methyl groups lead to more compact structures, which display enhanced local fluctuations. The central tetrapeptide adopts a type I beta-turn, while the outer motifs display more conformational variability. The three motifs in the methylated dodecamer peptide, however, adopt frequently in the distance restrained MD simulation a compact structure such that the outer motifs appear to form a hydrophobic core by stacking of their two proline rings. This arrangement also suggests how a peptide containing multiple tandemly linked copies of a stable beta-turn NPNA motif might adopt a folded stem-like structure, which conceivably may be of biological relevance in the native CS protein.

Animals↗

PubMed1997

Plasmapheresis followed by intravenous immunoglobulin in presensitized patients awaiting thoracic organ transplantation.

Four highly sensitized patients awaiting thoracic organ transplantation were treated immediately preceding transplantation with 1 plasmapheresis and infusion of high dose intravenous immunoglobulin (IVIG). All 4 underwent successful surgery and have had minor to no rejection episodes over a range from 5 1/2 to 12 months. All panel reactive antibodies (PRA) were dithiotheitol (DTT) resistant, and 1 patient had IgG specific alloantibodies to a donor alloantigen. All 4 patients had positive donor cross matches prior to transplantation, and 3 of the 4 patients remained PRA negative for up to 9 months after allografting. Possible mechanisms of this therapy include inhibition of proliferating alloreactive B cells or suppression by antiidiotypic antibodies. Further study is warranted.

Female↗

PubMed1997

Statistical modeling of methane production from landfill samples.

Multiple-regression analysis was conducted to evaluate the simultaneous effects of 10 environmental factors on the rate of methane production (MR) from 38 municipal solid-waste (MSW) samples collected from the Fresh Kills landfill, which is the world's largest landfill. The analyses showed that volatile solids (VS), moisture content (MO), sulfate (SO(inf4)(sup2-)), and the cellulose-to-lignin ratio (CLR) were significantly associated with MR from refuse. The remaining six factors did not show any significant effect on MR in the presence of the four significant factors. With the consideration of all possible linear, square, and cross-product terms of the four significant variables, a second-order statistical model was developed. This model incorporated linear terms of MO, VS, SO(inf4)(sup2-), and CLR, a square term of VS (VS(sup2)), and two cross-product terms, MO x CLR and VS x CLR. This model explained 95.85% of the total variability in MR as indicated by the coefficient of determination (R(sup2) value) and predicted 87% of the observed MR. Furthermore, the t statistics and their P values of least-squares parameter estimates and the coefficients of partial determination (R values) indicated that MO contributed the most (R = 0.7832, t = 7.60, and P = 0.0001), followed by VS, SO(inf4)(sup2-), VS(sup2), MO x CLR, and VS x CLR in that order, and that CLR contributed the least (R = 0.4050, t = -3.30, and P = 0.0045) to MR. The SO(inf4)(sup2-), VS(sup2), MO x CLR, and CLR showed an inhibitory effect on MR. The final fitted model captured the trends in the data by explaining vast majority of variation in MR and successfully predicted most of the observed MR. However, more analyses with data from other landfills around the world are needed to develop a generalized model to accurately predict MSW methanogenesis.

Journal Article↗

PubMed1997

Immunotoxicological assessment of cyclosporin A by conventional pathological techniques and immune function testing in the rat.

1. Groups of male rats were given different doses of cyclosporin A, ranging from the maximum tolerated dose (20 mg/kg/day) downwards, 7 days a week for 28 days using a protocol derived from OECD test guideline 407. 2. At the end of the test, one set of animals underwent a detailed necropsy and histopathological examination of lymphoid tissues. Immune function was assessed using the lymphoproliferative response and natural killer cell activity of their spleen cells. Another set of animals was immunised with sheep erythrocytes on day 25 and used to evaluate the ability to produce specific anti-sheep red blood cell antibody. 3. Cyclosporin A produced detectable effects on the immune system at all doses and at doses lower than other toxic effects. Both histopathological techniques and one of the immune function tests were able to identify changes at the lowest dose, 1.25 mg/kg/day. 4. The results of this investigation suggest that conventional histopathological techniques, if applied to a range of lymphoid organs, are sufficient to identify potential immunotoxicants without recourse to immune function tests.

Animals↗

PubMed1997

Estrogen regulation of human osteoblastic cell proliferation and differentiation.

Estrogen (E2) has been shown to prevent bone loss among postmenopausal women. The molecular mechanism(s) by which this is accomplished is not clear. The discovery of E2 receptor (ER) in osteoblasts and osteoclasts has implicated these cells as direct targets for E2. Previous studies on the effects of E2 on osteoblastic cells in vitro or in organ culture present conflicting results, possibly due to heterogeneity in cell types, stage of differentiation, ER levels, and/or species differences. The effects of E2 on gene expression during various stages of human osteoblast cell differentiation has not been investigated extensively. In this study we employed a newly developed human fetal osteoblastic cell line (hFOB/ER9) that contains high levels of ER to examine the effects of E2 on osteoblast proliferation and differentiation. The basal levels and E2 effects on the expression of various extracellular matrix proteins were also characterized throughout different stages of differentiation. These stages include a proliferative/relatively undifferentiated stage (day 6), a matrix maturation stage (days 10-14), and a mineralization/calcified nodule stage (day 18). During the stage of rapid cell proliferation, E2 treatment of hFOB/ER9 cells resulted in a dose-dependent decrease in [3H]thymidine incorporation to a maximum of 72% compared to the vehicle control value. Treatment of hFOB/ER9 cells with 10(-9) M E2 for 48 h resulted in an increase in alkaline phosphatase (AP) activity throughout cell differentiation. The magnitude of AP induction varied from approximately 200-500%. In contrast, E2 decreased osteocalcin protein levels to a minimum of 54% compared to the vehicle control value. The steady state messenger RNA levels for AP increased and osteocalcin decreased after E2 treatment, similar to the responses observed at the protein level. At all stages, there was little or no effect of E2 on type I collagen protein levels or osteonectin steady state messenger RNA levels. The E2 responses on hFOB/ER9 cell matrix protein expression and cell proliferation were mediated through the ER, as cultures cotreated with a 100-fold molar excess of a type II anti-E2 (ICI 182,780) abrogated these effects. These results support the hypothesis that E2 does have an effect on osteoblastic differentiation by decreasing hFOB/ER9 cell proliferation and differentially regulating extracellular matrix expression.

Bone and Bones↗