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Biomedical subjects

R Dixon

Publications and source records attributed to R Dixon.

At least 19 recordsLinked to original sources
PubMed2001

A novel purification method for histidine-tagged proteins containing a thrombin cleavage site.

A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified protein. The advantage of this method is that thrombin is used instead of imidazole in the final purification step. Imidazole can influence NMR experiments, competition studies, or crystallographic trials, and the presence of imidazole often results in protein aggregates. Removal of the His-tag results in a form of the protein of interest in which no additional tags are present, resembling the native form of the protein, with only three additional amino acids at the N-terminal side. Our method is compared with a more conventional method for the purification of the Azotobacter vinelandii NIFL PAS domain, overexpressed in Escherichia coli. It also proves to be successful for three different His-tagged proteins, the Klebsiella pneumoniae NTRC protein, and the A. vinelandii NIFA and NIFL proteins, and therefore it is a general method for the purification of His-tagged proteins.

Azotobacter vinelandii↗
PubMed2001

Coal face and stockpile ash analyser for the coal mining industry.

A portable nucleonic instrument was developed for the determination of coal ash on the coal face or the surface of coal stockpiles. The instrument employs the backscattered gamma-gamma technique. There are two gamma-ray sources used in this instrument: a 1.1 MBq 133Ba source as the primary source of radiation and a 37 kBq 137Cs for gain stabilization. The instrument is commercially available.

Journal Article↗
PubMed2001

Concerted inhibition of the transcriptional activation functions of the enhancer-binding protein NIFA by the anti-activator NIFL.

The Azotobacter vinelandii NIFL regulatory flavoprotein responds to the redox, energy and nitrogen status of the cell to inhibit transcriptional activation by the sigmaN-dependent enhancer binding protein, NIFA, via the formation of a NIFL-NIFA protein complex. The NIFA protein contains three domains: an N-terminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the sigmaN-RNA polymerase holoenzyme; and a C-terminal DNA-binding domain. We report that truncated NIFA proteins that either lack the amino-terminal domain or contain only the isolated central domain remain responsive to inhibition by NIFL but, in contrast to native NIFA, continue to hydrolyse nucleotides when NIFL is present. We also report that NIFL is competent to inhibit the DNA-binding function of NIFA. Taken together, these results suggest that NIFL inhibits NIFA via a concerted mechanism in which DNA binding, catalytic activity and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environmental conditions.

Azotobacter vinelandii↗
PubMed2001

Role of Escherichia coli nitrogen regulatory genes in the nitrogen response of the Azotobacter vinelandii NifL-NifA complex.

The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (nif)-specific transcriptional activator NifA in Azotobacter vinelandii in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the A. vinelandii NifL-NifA system responds to fixed nitrogen in vivo is unknown, the glnK gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of A. vinelandii glnK in this response is difficult to establish because of the essential nature of this gene. We have shown previously that A. vinelandii NifL is able to respond to fixed nitrogen to control NifA activity when expressed in Escherichia coli. In this study, we investigated the role of the E. coli PII-like signal transduction proteins in nitrogen control of the A. vinelandii NifL-NifA regulatory system in vivo. In contrast to recent findings with Klebsiella pneumoniae NifL, our results indicate that neither the E. coli PII nor GlnK protein is required to relieve inhibition by A. vinelandii NifL under nitrogen-limiting conditions. Moreover, disruption of both the E. coli glnB and ntrC genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that glnB ntrC and glnB glnK ntrC mutant strains accumulate high levels of intracellular 2-oxoglutarate under conditions of nitrogen excess. These findings are in accord with our recent in vitro observations (R. Little, F. Reyes-Ramirez, Y. Zhang, W. Van Heeswijk, and R. Dixon, EMBO J. 19:6041-6050, 2000) and suggest a model in which nitrogen control of the A. vinelandii NifL-NifA system is achieved through the response to the level of 2-oxoglutarate and an interaction with PII-like proteins under conditions of nitrogen excess.

Azotobacter vinelandii↗
PubMed2001

Protein-protein interactions in the complex between the enhancer binding protein NIFA and the sensor NIFL from Azotobacter vinelandii.

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.

Adenosine Triphosphate↗
PubMed2000

Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein.

PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs. The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction. Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins. We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate. We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation. Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.

Adenine Nucleotides↗
PubMed2000

Responses of cloned rainbow trout Oncorhynchus mykiss to an attenuated strain of infectious hematopoietic necrosis virus.

The objective of this work was to examine the response of homozygous clones of rainbow trout to vaccination by an attenuated strain (Nan Scott Lake; NSL) of infectious hematopoietic necrosis virus (IHNV). Adult rainbow trout of the Hot Creek Strain (YY males maintained in a recirculating system at 12 degrees C) were injected 3 times with 10(5) to 10(7) plaque forming units (pfu) of NSL. Intraperitoneal injections were given at Day 0 and at 2 and 4 mo post-infection. All fish were nonlethally bled at monthly intervals for 18 mo. Serum from each fish was analyzed by the complement-dependent neutralization assay and by western blot against purified NSL virus. The highest virus neutralization titers were detected 4 mo after the first injection, and peaked at 1280. When sera were analyzed by western blot, the predominating responses of the serum from immunized fish on the reduced western blot were against M1, a matrix protein of the virus and to a 90 kDa stress protein. The 90 kDa protein was identified by a monoclonal antibody as a stress protein derived from the CHSE-214 cells in which the purified IHN virus was grown and which associates with the virus during purification.

Animals↗
PubMed2000

Exclusion limits on the WIMP-nucleon cross section from the cryogenic dark matter search.

The Cryogenic Dark Matter Search (CDMS) employs Ge and Si detectors to search for weakly interacting massive particles (WIMPs) via their elastic-scattering interactions with nuclei while discriminating against interactions of background particles. CDMS data, accounting for the neutron background, give limits on the spin-independent WIMP-nucleon elastic-scattering cross section that exclude unexplored parameter space above 10 GeV/c2 WIMP mass and, at >75% C.L., the entire 3sigma allowed region for the WIMP signal reported by the DAMA experiment.

Journal Article↗
PubMed2000

Discovery and characterization of OC144-093, a novel inhibitor of P-glycoprotein-mediated multidrug resistance.

OC144-093 is a novel substituted diarylimidazole (Mr 495) generated using the OntoBLOCK system, a solid-phase combinatorial chemistry technology, in combination with high-throughput cell-based screening. OC144-093 reversed multidrug resistance (MDR) to doxorubicin, paclitaxel, and vinblastine in human lymphoma, breast, ovarian, uterine, and colorectal carcinoma cell lines expressing P-glycoprotein (P-gp) with an average EC50 of 0.032 microM. Inhibition of MDR by OC144-093 was reversible, but the effect persisted for at least 12 h after removal of compound from the culture medium. OC144-093 had no effect on the response to cytotoxic agents by cells in vitro lacking P-gp expression or expressing a multidrug resistance-associated protein (MRP-1). OC144-093 was not cytotoxic by itself against 15 normal, nontransformed, or tumor cell lines, regardless of P-gp status, with an average cytostatic IC50 of >60 microM. OC144-093 blocked the binding of [3H]azidopine to P-gp and inhibited P-gp ATPase activity. The compound was >50% p.o. bioavailable in rodents and dogs and did not alter the plasma pharmacokinetics of i.v.-administered paclitaxel. OC144-093 increased the life span of doxorubicin-treated mice engrafted with MDR P388 leukemia cells by >100% and significantly enhanced the in vivo antitumor activity of paclitaxel in MDR human breast and colon carcinoma xenograft models, without a significant increase in doxorubicin or paclitaxel toxicity. The results demonstrate that OC144-093 is an orally active, potent, and nontoxic inhibitor of P-gp-mediated multidrug resistance that exhibits all of the desired properties for treatment of P-gp-mediated MDR, as well as for prevention of MDR prior to selection and/or induction of refractory disease.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

The neurotoxicity of the venom phospholipases A(2), notexin and taipoxin.

The presynaptically active, toxic phospholipases known as notexin and taipoxin are principal components of the venom of the Australian tiger snake and the Australian taipan respectively. The inoculation of the toxins into one hind limb of rats caused, within 1 h, the depletion of transmitter from the motor nerve terminals of the soleus muscle. This was followed by the degeneration of the motor nerve terminals and of the axonal cytoskeleton. By 24 h 70% of muscle fibers were completely denervated. Regeneration and functional reinnervation were almost fully restored by 5 days, but collateral innervation was common in the regenerated muscles, and this abnormality persisted for at least 9 months. The data provide an explanation for both the severity of neuromuscular paralysis that can accompany envenoming bites by tiger snakes and taipans and the difficulty experienced by physicians in managing the envenomed subjects.

Acetylcholinesterase↗
PubMed2000

Cultural identity and psychological adjustment of adolescent Chinese immigrants in New Zealand.

Based on an integration of acculturation and developmental models, this study examined the relationship between psychological adjustment and cultural identity determined by the relative strength of identification with own ethnic group, and the mainstream society. Over 400 immigrant Chinese students completed questionnaires and 24 participated in interviews. Results indicated that adolescents who were integrated had higher self-esteem than their peers who were separated or marginalized p<0.001). This finding is consistent with research on ethnic minority adolescents in other countries, and suggests that integration has important implications on the psychological well-being of these adolescent immigrants.

Acculturation↗
PubMed2000

Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed.

Animals↗
PubMed2000

Is there a learning curve in diagnosing urolithiasis with noncontrast helical computed tomography?

OBJECTIVE: To report one department's experience with helical computed tomographic (HCT) evaluation of patients with suspected renal colic to diagnose ureteral calculi; to determine whether there is a learning curve in performing HCT in this context; and to determine whether HCT for the evaluation of renal colic exposes patients to more radiation than the standard intravenous pyelography (IVP) combined with nephrotomography. METHODS: All patients presenting to the emergency department with flank or abdominal pain were evaluated with nonreformatted noncontrast HCT. To determine changes in diagnostic accuracy, patients were divided into 2 groups: those evaluated between September 1996 and January 1997 (group 1, 67 patients), and those seen from February to June 1997 (group 2, 53 patients). A radiation exposure study was performed using phantoms, and radiation exposure for HCT, IVP and nephrotomography was measured. RESULTS: Review of HCT scans to diagnose ureteral calculi had a sensitivity of 91.7%, specificity of 82.6%, and accuracy of 87.2% in group 1, and a sensitivity of 95.5%, specificity of 86.7%, and accuracy of 91.9% in group 2. Patients undergoing IVP with nephrotomography were exposed to an effective dose equivalent of 343 mrem (dSv) (for men) and 664 mrem (for women). The effective dose equivalent for an HCT scan was 180 mrem. CONCLUSION: HCT offers excellent, rapid diagnostic accuracy without the need for intravenous contrast medium and with a lower radiation exposure level than IVP in evaluating patients with acute flank pain. There is a small but real learning curve in evaluating patients with acute flank pain with HCT.

Adolescent↗
PubMed1999

The upstream region of the nodD3 gene of Sinorhizobium meliloti carries enhancer sequences for the transcriptional activator NtrC.

In Sinorhizobium meliloti the expression of the nodulation genes nodABC is regulated in response to the level of fixed nitrogen (ammonia). Previous results suggested that the response to the nitrogen status is mediated by the two-component NtrB/NtrC system which controls transcription of the nodD3 gene, encoding a positive regulatory protein for the activation of nodABC transcription. Here we confirm by DNase I footprinting and gel shift assays that NtrC, when phosphorylated by NtrB, is able to interact with the enhancer sequences present upstream of nodD3. A model is proposed whereby NtrC functions to control the transcription from the two promoters in the upstream region of nodD3 in response to nitrogen status.

Bacterial Proteins↗
PubMed1999

Comparison of the kinetics of the specific cellular immune response to duck hepatitis B virus in infected and immune ducks.

The kinetics of the cell mediated immune response by ducks acutely and chronically infected with, or immune to infection by duck hepatitis B virus (DHBV) was determined. This was measured by an antigen specific blastogenesis assay to duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B core antigen (DHBcAg) using peripheral blood mononuclear cells (PBMC). The three outcomes of acute infection by DHBV were either clearance from both serum and liver, clearance from serum but not liver, or the development of persistent viraemia. Acutely infected ducks that failed to clear the infection also failed to develop a significant cellular immune response to both antigens. Ducks with chronic infection acquired as neonates or as the result of the failure to clear acute infection had an increasing cellular immune response over time. Two groups of immune ducks were examined. These were either ducks that had become immune following infection or that had been vaccinated. Both groups of ducks demonstrated significant cellular responses following challenge with DHBV irrespective of the level of their responses before challenge. However, there was a reduction in the response of their PBMC over a 4-week-period postchallenge. The range of cellular immune responses to DHBV antigens observed in this study has a number of counterparts in hepatitis B infection of humans. Coupled with the defined clinical outcomes that can be established in the duck/DHBV model, further study of the cellular immune response to DHBV is warranted.

Animals↗