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Biomedical subjects

R D Allen

Publications and source records attributed to R D Allen.

At least 55 records · Page 3Linked to original sources
PubMed1997

Use of transgenic plants to study antioxidant defenses.

The abundance of O2 and the highly energetic electron transfer reactions associated with thylakoid membranes make chloroplasts a major source of reactive oxygen intermediates (ROI) in photosynthetic tissues of plants. Attempts to reduce oxidative damage in chloroplasts have included the manipulation of ROI-scavenging enzymes by gene transfer technology. Much of this work has focused on chloroplast-localized superoxide dismutase (SOD), both chloroplast-targeted and cytosolic ascorbate peroxidase (APX) and glutathione reductase (GR). Increased activity of SOD in chloroplasts of transgenic tobacco plants generally leads to increased protection from membrane damage caused by exposure to methyl viologen (MV). In addition, overexpression of chloroplastic Cu/Zn SOD can lead to increased protection from photooxidative damage caused by high light intensity and low temperatures. Transgenic tobacco plants that overexpress APX either in the cytosol or chloroplastic compartments also show reduced damage following either MV exposure or photooxidative treatment and transgenic plants that express increased levels of GR have elevated pools of ascorbate and GSH. Though still preliminary, these results clearly indicate that alterations in the expression of enzymes involved in ROI-scavenging can have significant metabolic effects and provide the hope that this strategy can be used to develop crop plants with increased stress tolerance.

Antioxidants↗
PubMed1997

Cloning and expression of an Arabidopsis gene encoding a putative peroxisomal ascorbate peroxidase.

An Arabidopsis 14-3-3 protein, AFT1, was used as a 'bait' in the two-hybrid system to identify its interacting proteins. One of the candidate proteins, APX3, was identified as a putative peroxisomal membrane-bound ascorbate peroxidase. Ascorbate peroxidases are important defense enzymes that protect plant cells from oxidative stress damage. DNA blot analysis indicates that APX3 is encoded by a single-copy gene in the Arabidopsis genome. RNA blot analyses show that APX3 transcript levels increase slightly in response to cold, UV light, and treatments with hydrogen peroxide and paraquat. The activity of APX3 in Arabidopsis may be controlled in two ways: its enzymatic activity through protein-protein interactions and its transcription by transcriptional or posttranscriptional regulation.

14-3-3 Proteins↗
PubMed1997

Overexpression of glutathione S-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress.

Transgenic tobacco seedlings that overexpress a cDNA encoding an enzyme with both glutathione S-transferase (GST) and glutathione peroxidase (GPX) activity had GST- and GPX-specific activities approximately twofold higher than wild-type seedlings. These GST/GPX overexpressing seedlings grew significantly faster than control seedlings when exposed to chilling or salt stress. During chilling stress, levels of oxidized glutathione (GSSG) were significantly higher in transgenic seedlings than in wild-types. Growth of wild-type seedlings was accelerated by treatment with GSSG, while treatment with reduced glutathione or other sulfhydryl-reducing agents inhibited growth. Therefore, overexpression of GST/GPX can stimulate seedling growth under chilling and salt stress, and this effect could be caused by oxidation of the glutathione pool.

Agriculture↗
PubMed1996

The effect of venous drainage on glucose homeostasis after experimental pancreas transplantation.

In this canine study, glucose homeostasis after clinical pancreas transplantation is complex, with the relative effect of systemic versus portal delivery of insulin remaining unresolved. Thirty-two pancreatectomized dogs received either systemic venous drainage (SVD) with bladder exocrine drainage (n = 16), or portal venous drainage (PVD) with gastric exocrine drainage (n = 16). Cyclosporine (CsA) based immunosuppression was commenced on day -7. The effect of immunosuppression was a significant increase in fasting blood glucose (FBGL) (P = 0.002), fasting insulin (P = 0.024), AUC for insulin (P = 0.009), and K values decreased (P = 0.009). FBGL and K values remained abnormal after transplantation with no significant difference seen between SVD and PVD. However, fasting insulin became significantly lower after PVD and AUC insulin fell in both groups. CsA levels fell in both groups after transplantation, mirroring the fall in AUC insulin, and implicating CsA as a major cause of peripheral resistance to insulin. In conclusion, PVD did not demonstrate a significant advantage over SVD in handling an intravenous glucose challenge. The need for pancreatectomy in large animals may make them an unsatisfactory experimental model to evaluate the glucoregulatory effects of pancreas allotransplantation.

Animals↗
PubMed1996

Experimental hyperacute rejection in pancreas allotransplants.

A model of sensitization by intraperitoneal lymph node inoculation was developed to test the hypothesis that hyperacute rejection (HAR) could occur in sensitized recipients of vascularized pancreas allografts. Ten pairs of outbred mongrel dogs that were lymphocytotoxic cross-match assay negative were inoculated with homogenized lymph nodes on either three or four occasions at fortnightly intervals before renal transplantation. A renal allograft from the same donor was used to test the HAR response and to further enhance sensitization by rejection of a vascularized organ. Pancreas transplants were performed 2 weeks later, with biopsies of the graft and blood samples taken at 0, 10, 20, and 30 min and then at 30-min intervals until the grafts were no longer viable. All renal and pancreas grafts were rejected in a classical hyperacute pattern. Within 4 min of revascularization of the pancreas, central lobular hemorrhage and vascular congestion appeared, followed by general edema. Histology demonstrated parallel changes of edema, vascular congestion, necrosis, hemorrhage, and leukocytic infiltrate, which all preceded graft infarction. A sharp decline in both arterial and venous white blood cell count and platelets occurred within 10 min of revascularization with initial sequestration and subsequent release of platelets from the graft (P=0.02). In summary, HAR of the allografted pancreas can be observed by the surgeon within minutes of revascularization, with predictable macroscopic and microscopic changes. This study supports the use of routine lymphocytotoxic cross-match tests for all recipients of pancreas transplants and implies that particular care is warranted in regraft pancreas allograft recipients.

Acute Disease↗
PubMed1996

Reversal of cyclosporine malabsorption in diabetic recipients of simultaneous pancreas and kidney transplant using a microemulsion formulation.

In view of the known problems that diabetic recipients have with drug absorption and of the potential advantages of the microemulsion formulation of cyclosporine (Neoral), 17 previously diabetic recipients of simultaneous pancreas and kidney allografts were studied before and after conversion from Sandimmune (SIM) to Neoral (NEO) formulations of cyclosporine a mean of 4.75 +/- 2.0 years after transplantation. The aims of this study were to determine whether there were changes in the absorption profiles using the 2 formulations and to analyze the safety of conversion from SIM to NEO on a 1:1 ratio. Pharmacokinetic, clinical, and renal function parameters were measured 2 weeks before, and 2 and 4 weeks after conversion. Pharmacokinetics demonstrated that, when taking SIM, only 5 patients had "good absorption" (maximal concentration >500 ng/ml and time to maximal concentration < / = 2 hr), but after conversion to NEO, 14 at week +2, and 13 at week +4 were good absorbers (P<0.02 and <0.01, respectively). There was a 50% reduction in the time to maximal blood concentration (from 3.2 +/- 1.9 hr with SIM, to 1.8 +/- 1.3 hr on NEO [P<0.007]) and a 36% increase in trough and a 143% increase (P<0.01) in maximal blood concentrations, although the 9-hr cyclosporine AUC showed an 89% increase (P<0.01). There was an 18% mean reduction in dose per kilogram prescribed 4 weeks after conversion. Renal function, measured by serum creatinine and technecium-99m diethylenetriamine pentaacetic acid glomerular filtration rate, was not altered and no adverse events were attributed to NEO. This study confirmed that previously diabetic recipients of simultaneous renal pancreas allografts had variable and poor absorption of cyclosporine using conventional Sandimmune and that this absorption was improved by conversion to Neoral.

Absorption↗
PubMed1996

Clinical determinants of glucose homeostasis after pancreas transplantation.

Although successful simultaneous pancreas and kidney transplantation (SPK) achieves normoglycemia in the majority of diabetic recipients with end-stage renal disease, little is known about the factors that influence long-term endocrine function. In this prospective study of 48 bladder-drained SPK patients, 209 oral glucose tolerance tests were performed between 3 months and 6 years after transplantation. Normal fasting glucose levels and systemic hyperinsulinemia were stable for up to 6 years after SPK. Multivariate analysis revealed that increased area-under-curve (AUC) levels of C-peptide 3 months after transplantation were predicted by short surgical pancreas anastomosis time, greater recipient body weight, and total HLA mismatch score. Episodes of acute pancreas rejection were not associated with reduced allograft insulin output in the long term. Insulin output, stimulated by oral glucose tolerance tests and assessed by the ratio of AUC insulin to AUC glucose, fell gradually after transplantation and was decreased by an elevated serum calcium level and high cyclosporine dose. The ratio of fasting insulin to glucose, which acts as a marker of peripheral insulin resistance, fell with time after transplantation and was increased by greater body weight, higher prednisolone dose, and lower cyclosporine dose. The inhibitory effect of cyclosporine on both fasting and postprandial insulin output was, however, minor when quantified by multivariate analysis. Endocrine function of the transplanted pancreas was not correlated with its exocrine function measured by urinary amylase excretion, nor was there a correlation with change in renal function measured by isotopic glomerular filtration rate. In summary, simultaneous pancreas and kidney transplantation leads to excellent long-term glucose homeostasis maintained at the expense of systemic hyperinsulinemia. The key factors adversely affecting peripheral resistance in SPK were corticosteroid therapy, body weight, and time after transplantation. The susceptibility of islets to ischemia-reperfusion injury, as quantitated by surgical anastomosis time, may have implications for islet transplantation programs, as may the relative resistance of islets to allograft rejection. Glucose homeostasis after SPK, while remaining abnormal, may be used as the standard against which islet transplantation must be measured.

Adult↗
PubMed1996

Restriction endonuclease mapping and molecular cloning of the human herpesvirus 6 variant B strain Z29 genome.

Human herpesvirus 6(HHV-6) variants A and B differ in cell tropism, reactivity with monoclonal antibodies, restriction endonuclease profiles, and epidemiology. Nonetheless, comparative nucleotide and amino acid sequences from several genes indicate that the viruses are very highly conserved genetically, The B variant is the major etiologic agent of exanthem subitum and is frequently isolated from children with febrile illness; no disease has been etiologically associated with HHV-6A. One HHV-6A strain has been cloned and sequenced, but similar information and reagents are not available for HHV-6B. We report here the determination of maps of the restriction endonuclease cleavage sites for BamHI, C1aI, HindIII, KpnI, and Sa1I, and the cloning in plasmids and bacteriophages of fragments representing over 95% of the HHV-6B strain Z29 [HHV-6B(Z29)] genome. Hybridization experiments and orientation of several blocks of nucleotide sequence information onto the genomic map indicate that HHV-6A and HHV-6B genomes are colinear.

Antibodies, Monoclonal↗
PubMed1996

Australian multicentre evaluation of a new polyurethane vascular access graft.

BACKGROUND: A new three-layered cast polyurethane vascular access graft (Thoratec VAG) is the most recent addition to the list of materials used in the search for the perfect prosthetic graft material for haemodialysis vascular access. Despite its use in 23 countries, a clinical assessment has not been published. METHODS: An independent retrospective evaluation by questionnaire was obtained for 145 implantation procedures performed by 30 surgeons. RESULTS: Ninety-two per cent of procedures were performed for an acute need for vascular access and 73% had prior failed vascular access surgery. Patients were hospitalized for a median of 4 days and the graft was initially used at a median of 3 days. Median follow-up was 306 days. Thoratec VAG had a problem-free (primary) patency of 44.9% and a functional (secondary) patency 64.5% at 1 year. Major causes of graft loss were thrombosis (17%) and infection (11%). Intra-operative thrombosis was the only significant risk factor for subsequent thrombosis (P = 0.013). Infection was less in patients with antibiotic prophylaxis that included vancomycin (P = 0.02). CONCLUSION: The ability to use the graft soon after surgery is a major advance for patients who urgently need reliable medium to long-term haemodialysis access.

Arteriovenous Fistula↗
PubMed1996

Hyperosmotic stress leads to reversible dissociation of the proton pump-bearing tubules from the contractile vacuole complex in Paramecium.

To study the effect of hyperosmotic stress on the structure and function of the contractile vacuole complex of Paramecium multimicronucleatum, we employed two different monoclonal antibody markers: one to a decorated spongiome antigen (A4) and a second to an antigen found on all other membranes of the contractile vacuole complex (G4). A hyperosmotic condition was produced by adding sorbitol to the axenic culture medium which induced both dose- and time-dependent decreases in the vacuole's expulsion rate. The addition of 150 mM sorbitol to the medium (making a final osmolarity of 230 mOsmol) was sufficient to completely stop the expulsion of the contractile vacuole. Immunofluorescence demonstrated that the blocking of fluid output was accompanied by the disappearance of most fluorescence labeling from the decorated spongiome (the A4 antigen). Electron microscopy revealed that the disappearance of the labeling was accompanied by the disappearance of the decorated tubules from around the collecting canals. These tubules vesiculate. The other membranes of the contractile vacuole complex remained unaffected which was demonstrated by both electron microscopy and indirect immunolabeling using the mAb against the G4 antigen. These results show that the decorated spongiome is formed from a distinct membrane pool separate from that of the smooth spongiome, collecting canals and the contractile vacuole. Recovery of the decorated spongiome rapidly followed the return of the cell to an isotonic environment and was completed within 3 hours. Decorated tubule recovery paralleled the recovery of the function of the contractile vacuole. Recovery was also observed during continuous hyperosmotic treatment with the reappearance of the contractile vacuole activity starting at 3 hours and stabilizing at around 10 hours of incubation. Functional recovery under these conditions was accompanied by a reappearance of the decorated tubules but the total fluid output was always lower than for cells in an isotonic environment. Thus, cells were shown to be capable of adapting to high hyperosmotic conditions. We conclude that the dissociation and reassociation of the decorated spongiome is an important regulatory feature controlling the activity of the contractile vacuole complex and of intracellular osmoregulation in Paramecium.

Adaptation, Physiological↗
PubMed1996

High resolution view of the true cytosolic membrane surface of phagosomes of known ages purified from Paramecium.

Techniques were used for viewing the true cytosolic surfaces of the membranes of intracellular organelles by field emission scanning electron microscopy (SEM). Cells of Paramecium multimicronucleatum were fed briefly with magnetic beads followed by a chase which advanced the newly formed digestive vacuoles (DVs) to predetermined ages. These bead-containing phagosomes were isolated from the whole cell homogenates with a magnet and were determined to be intact by fluorescence microscopy. Antigenically, these DVs were similar to those in situ. The DVs prepared for transmission electron microscopy or SEM showed extensive adherence of cellular debris. The use of 0.2 M KCl in the wash buffer eliminated much of this debris and exposed the true vacuolar surfaces. Three populations of tightly bound vesicles and numerous globular particles of 10 to 20 nm became visible on the DV surfaces. The attached vesicles, having diameters of approximately 300 and approximately 200 nm each, corresponded to the acidosomes and lysosomes that are known to be associated with the DV-I and DV-II, respectively. High resolution SEM also revealed a third set of small vesicles (50-150 nm), which were previously not known to be associated with DVs. The 10 to 20 nm globular particles were judged to be the cytosolic extensions of transmembrane protein complexes as their patterns of distribution on DVs of various ages corresponded to the transmembrane particles previously seen in these membranes in freeze-fracture studies.

Acid Phosphatase↗
PubMed1995

Renal dysfunction in acute rejection. Effect of HLA typing, therapy, and histology.

Acute rejection is a frequent cause of early graft dysfunction in renal transplantation, and serum creatinine is the most important measure of clinical response. However, there is little information on serum creatinine during rejection episodes. In this study, the determinants that might influence the clinical response of serum creatinine during acute rejection were evaluated in 96 renal transplant recipients with 100 episodes of biopsy-proven rejection, by univariate and multivariate analysis. The factors assessed for their influence on serum creatinine in acute rejection included: presence and severity of vascular or cellular rejection on biopsy, time taken to institute therapy, HLA mismatch, and the use of OKT3. The presence of vascular rejection on biopsy (n = 28) was a major determinant of impaired reciprocal area under the curve (AUC) of serum creatinine (P < 0.01), and was correlated with HLA-A and -B mismatch (r = 0.21, P < 0.05). Acute rejection associated with HLA-DR mismatch resulted in a more rapid increase of serum creatinine, a higher maximal creatinine, and a greater AUC creatinine (all P < 0.05). Treatment of acute rejection with OKT3 had a beneficial effect on AUC creatinine (P < 0.05) when compared with intravenous corticosteroids, by multivariate analysis. However, vascular rejection and HLA-DR mismatch had no effect on serum creatinine 1 year after transplantation. The biopsy cellular rejection score had no effect on AUC creatinine, although there was a modest effect on gradient of creatinine prior to biopsy. A minor adverse effect of delay in therapy of acute rejection could be demonstrated in the methylprednisolone-treated subgroup. In summary, HLA-DR mismatch and the presence of vascular rejection were the most important predictors of the severity of rejection assessed by the response of serum creatinine to treatment. Appropriate antirejection therapy should be instituted promptly to optimize clinical response during acute rejection.

Adult↗
PubMed1995

Predicting glomerular filtration rate after kidney transplantation.

Serum creatinine is an important clinical measure of impairment of glomerular filtration rate (GFR) after kidney transplantation. The use of formulas that predict GFR (such as the Cockcroft-Gault) derived from patients with chronic renal failure and standardized against measured creatinine clearance may not be accurate when applied to kidney transplant recipients. The purpose of this study, was to investigate the level of inaccuracy and its causes and then to derive predictive GFR formulas that are appropriate to renal transplantation. Determinants of isotopic GFR, serum creatinine, and muscle mass were evaluated in consecutive kidney recipients (n = 146) using 99mTc DTPA GFR (n = 751) as a reference method. Factors that predicted GFR apart from serum creatinine included sex, height, body weight, serum urea, years on dialysis, numbers of rejections and infective episodes, and prednisolone dose. The relationship between serum creatinine and GFR was highly variable and dependent on factors that alter muscle mass and muscle catabolic rate. The relationship was further altered by ATN and chronic rejection when tubular secretion of creatinine was reduced. Three alternative GFR formulas (which can be applied to renal transplant patients according to the availability of clinical parameters) were derived and tested against six published methods of GFR estimation. Our derived formulas had the highest correlation, no overall bias, least scatter of sum of squares, and least error at low levels of GFR. They represent a better estimation of GFR in kidney transplantation than published formulas, and would allow a standardized approach to the study of long-term renal dysfunction.

Adult↗
PubMed1995

Repression of motility and flagellin production at 37 degrees C is stronger in Listeria monocytogenes than in the nonpathogenic species Listeria innocua.

Listeria monocytogenes and Listeria innocua differ markedly in virulence but are indistinguishable by classical taxonomic criteria. Both species are actively motile and produce abundant flagellin at 22 degrees C. We have found, however, noticeable differences between L. monocytogenes and L. innocua in motility and flagellin production at 37 degrees C. At this temperature, L. monocytogenes strains were virtually nonmotile and produced little or no detectable flagellin, whereas strains of L. innocua were frequently motile and produced substantial amounts of flagellin. This flagellin was recognized by a Listeria genus-specific monoclonal antibody that also recognized flagellin produced at 22 degrees C. These results suggest differential regulation of flagellin production between L. monocytogenes and L. innocua at 37 degrees C.

Antibodies, Bacterial↗
PubMed1995

The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps.

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm 'pegs' protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗