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G Kemp

Publications and source records attributed to G Kemp.

At least 19 recordsLinked to original sources
PubMed2004

Polygalacturonase-inhibiting proteins can function as activators of polygalacturonase.

The interaction between fungal endopolygalacturonases (EPGs) and polygalacturonase-inhibiting proteins (PGIPs) found in plant cell walls has been well established. The typical EPG/PGIP interaction is characterized by high affinity, reversibility, and a 1:1 stoichiometry that results in lowering the catalytic rate of a particular endopolygalacturonase by up to 99.7%. Various EPG and PGIP isoforms and glycoforms have been isolated and characterized, and combinations of EPGs and PGIPs demonstrate a range of enzyme inhibition. EPG/PGIP interactions have prompted many researchers to suspect the involvement of these proteins in the production of specific signals (oligosaccharins) during plant pathogenesis. We have recently reported on initial studies in our laboratory indicating that, for certain EPG/PGIP combinations, the specific activity of EPG is increased beyond that characteristic of the enzyme alone. In this paper, we present a detailed analysis of the product of the interaction of native Phaseolus vulgaris PGIP-2 with five EPGs from Aspergillus niger, namely PGI, PGII, PGA, PGB, and PGC in the presence of homogalacturonan. We demonstrate that for PGA and PGC, the interaction with PGIP-2 may result in either inhibition or activation in a manner that is pH dependent. This data suggests the need for a reevaluation of the conventional description applied to PGIPs; suggestions include polygalacturonase-binding protein and polygalacturonase-modulating protein.

Aspergillus niger↗
PubMed1999

Biodistribution and absorbed radiation dose estimates of 99mTc-labeled dimercaptopropionyl human serum albumin.

UNLABELLED: The use of 99mTc-labeled red blood cells (RBC) for the evaluation of left ventricular function using equilibrium-gated blood-pool imaging suffers from several problems and potential risks. In this study, we estimated the absorbed radiation dose of 99mTc-labeled dimercaptopropionyl human serum albumin (DMP-HSA) as a potential alternative to 99mTc-RBC. METHODS: After the administration of 99mTc-DMP-HSA, whole-body imaging was performed up to 48 h after injection in five volunteers. The heart contents, liver and remainder of the body were used as source organs. Multicompartment modeling of the biodistribution was performed and absorbed radiation dose estimates for 99mTc-DMP-HSA were obtained using the Medical Internal Radiation Dose (MIRD) calculation. RESULTS: Residence times of 0.62 and 0.43 h were obtained for the heart contents and liver, respectively. Radiation dose estimates yielded an effective dose of 0.0055 mSv/MBq. CONCLUSION: 99MTC-DMP-HSA yielded absorbed radiation doses comparable with those of 99mTc-RBC. Therefore, the radiation properties of 99mTc-DMP-HSA are such that it can be used for clinical diagnostic studies.

Absorption↗
PubMed1998

Capillary electrophoresis: a versatile family of analytical techniques.

Capillary electrophoresis is a sensitive and versatile technique which has emerged over the past 10 years into the forefront of analytical methodology. Its applicability is enhanced by short separation times, facile and rapid methods of development and a requirement for very small amounts of analyte. This article reviews basic principles behind the technique and the range of options which allow the same instrument to be used for the separation and analysis of a wide range of analytes from large biomolecules, such as proteins and nucleic acids, to metal and organic ions while having the capability to resolve enantiomers and non-charged molecules.

Biotechnology↗
PubMed1998

Rainbow trout cytokine and cytokine receptor genes.

Transforming growth factor-beta (TGF-beta) and interleukin-1 beta (IL-1 beta) have been cloned from rainbow trout (Oncorhynchus mykiss) in our laboratory. The trout TGF-beta is typical of members of the cysteine knot cytokine family, and has highest homology to TGF-beta 1, TGF-beta 4 and TGF-beta 5. The gene organisation is different to other TGF-beta genes despite the presence of seven exons. Trout IL-1 beta has less homology to known IL-1 beta s (49-56% amino acid similarity) but shows clear conservation of the secondary structure of the "mature peptide". An interleukin-converting enzyme cut site is not apparent however, and it remains to be determined whether fish IL-1 beta must be cleaved for biological activity. The trout IL-1 beta gene consists of six exons, one less than in mammals, with the missing exon at the 5' end of the gene. Two trout chemokine receptors have also been isolated in our laboratory, having high homology to CXC-R4 and CC-R7 (67% and 56% nucleotide identity respectively). With a view to studying the biological effects of fish cytokines we have also cloned the inducible nitric oxide (iNOS) and cyclooxygenase (COX-2) genes in trout. Both have high homology to known mammalian and chicken genes, and have been shown to be inducible in trout.

Amino Acid Sequence↗
PubMed1997

Activation of the adenovirus protease requires sequence elements from both ends of the activating peptide.

The adenovirus protease requires activation by an 11-residue peptide, GVQSLKRRRCF, to achieve maximum proteolytic activity. Derived from the C terminus of the viral protein pVI, the activating peptide (pVI-CT) forms a disulfide bond with cysteine 104 of the protease and causes a conformational change that accompanies the development of proteolytic activity. Results presented here show that the interaction of pVI-CT with the protease is dependent not only on the cysteine 10 but also on glycine 1 and valine 2. Removal of these residues, acetylation of the N-terminal glycine, or mutation of the valine to alanine or threonine significantly reduces or abolishes activation. Peptides lacking Gly-1 and Val-2 still form a disulfide bond with the protease but do not cause a conformational change in the protease also they are not effective inhibitors of activation as the interaction is readily reversed by full-length pVI-CT. These results suggest that pVI-CT causes activation by binding to two distinct regions of the protease and in doing so stabilizes the catalytic site. The reversible nature of the activation, suggested by the results presented here, may well reflect an in vivo regulatory mechanism.

Cysteine Endopeptidases↗
PubMed1997

Optimisation of a peptide-based indirect ELISA for the detection of antibody in the serum of HIV-1 seropositive patients.

A modified peptide-based indirect ELISA technique for the detection of HIV-1 specific antibodies in the sera of HIV-1 seropositive individuals is described. We found that the reduction of non-specific binding of HIV-1 seropositive sera to the ELISA plate was essential for the reliable detection of serum antibodies in the peptide based indirect ELISA. Optimal results were obtained using Immulon microtitre plates, different concentrations of denatured. purified grade of casein in the blocking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropositive sera 1 in 1600. These conditions reduced non-specific binding and improved assay sensitivity. We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and false negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA without compromising its sensitivity and have particular relevance for those wishing to apply the peptide-based indirect ELISA technique to serum samples which exhibit high levels of non-specific binding. To illustrate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a conserved region of HIV-1 gp120 sharing homology with the FAS apoptosis antigen were analysed using this technique.

Amino Acid Sequence↗
PubMed1996

Activation of the protease from human adenovirus type 2 is accompanied by a conformational change that is dependent on cysteine-104.

Adenovirus codes for a protease the activity of which can be regulated in vitro by an 11 residue peptide (GVQSLKRRRCF) derived from another viral protein, pVI. Three cysteine residues, one in the activating peptide and two in the protease (C104 and C122), play a central role in both activation and catalysis. Expression of protease mutants in insect cells has shown that C104 is not essential for proteolytic activity. GVQSLKRRRCF also caused a concentration-dependent increase in tryptophan fluorescence of protease expressed in Escherichia coli that paralleled the increase in proteolytic activity, indicating that activation was accompanied by a conformational change. Tryptophan fluorescence of C104S was not increased by the addition of GVQSLKRRRCF, nor was the fluorescence of wild-type protease increased by the addition of the peptide analogues where cysteine is replaced by aspartic acid or serine, suggesting that C104 is involved in activation and C122 in catalysis.

Adenoviruses, Human↗
PubMed1996

Amifostine pretreatment for protection against cyclophosphamide-induced and cisplatin-induced toxicities: results of a randomized control trial in patients with advanced ovarian cancer.

PURPOSE: Serious cumulative toxicity is a well-recognized consequence of chemotherapy. Amifostine, an organic thiophosphate, has demonstrated the ability to protect selectively a broad range of normal, but not neoplastic, tissues from the cytotoxic effects of chemotherapy and radiotherapy. This study was designed to determine if amifostine could reduce the serious toxicities associated with cyclophosphamide and cisplatin (CP), without reducing antitumor efficacy in patients with ovarian cancer. PATIENTS AND METHODS: Two hundred forty-two patients with advanced ovarian cancer were randomized to receive six cycles of cyclophosphamide (1,000 mg/m2) and cisplatin (100 mg/m2) with or without amifostine (910 mg/m2) every 3 weeks for six cycles. The occurrence of hematologic, renal, neurologic, and ototoxicity was evaluated. Antitumor efficacy was assessed by pathologic tumor response and survival. RESULTS: Pretreatment with amifostine before each cycle of chemotherapy resulted in a reduction of cumulative toxicities. Hematologic toxicity consisted of grade 4 neutropenia associated with fever and/or infection that required antibiotic therapy (P = .005), days in hospital (P = .019), and days on antibiotics (P = .031). Platinum-specific toxicities consisted of protracted serum creatinine elevations (P = 0.004), > or = 40% reduction from baseline in creatinine clearance (P = .001), and severity of neurologic toxicity (P = .029). Twenty-four percent of CP patients compared with 9% of amifostine plus CP patients discontinued therapy because of protocol-specified toxicity (P = .002). Pathologic tumor response rates were 37% with amifostine and 28% in controls, with comparable median survival times of 31 months. Amifostine was generally well tolerated; the principal side effects were emesis and a transient decrease in blood pressure. CONCLUSION: Pretreatment with amifostine reduces the cumulative hematologic, renal, and neurologic toxicities associated with the CP regimen, with no reduction in antitumor efficacy.

Adult↗
PubMed1995

Assessment of mitochondrial function and control in normal and diseased states.

Mitochondrial function in muscle in vivo can be quantitatively evaluated using 31-phosphorus nuclear magnetic resonance. In resting muscle, the concentrations of ions (e.g. H+, Na+) and two of the major bioenergetic components (inorganic phosphate and creatine) are determined by regulated transcellular transport processes. During recovery after exercise the kinetics and control of mitochondrial ATP synthesis can be established. During exercise the relative contributions to ATP synthesis of phosphocreatine (using creatine kinase), anaerobic glycogenolysis and oxidative phosphorylation are dissected and have been shown to change with time. The consequences of mitochondrial lesions and dysfunctions on these processes have been summarised.

Adenosine Triphosphate↗
PubMed1994

The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis.

Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95%. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.

Adenoviruses, Human↗
PubMed1993

The adenovirus protease is activated by a virus-coded disulphide-linked peptide.

In common with many other viruses, adenoviruses code for a protease essential for the development of infectivity. Recombinant adenovirus protease was active in crude in vitro complementation assays but was inactive with peptide or purified protein substrates. Activity was reconstituted by a component of adenovirus virions, which was identified as GVQSLKRRRCF, a peptide derived from the virus protein pVI. Synthetic peptides were used to demonstrate that the cysteine is essential and that the disulphide-linked dimer is required for activity. It is proposed that the adenovirus protease is a cysteine protease and that its activation by the peptide involves thiol-disulphide interchange, which serves to expose the active site cysteine. This represents a novel strategy for controlling the activity of a protease that is required for virus maturation.

Adenoviruses, Human↗
PubMed1993

The active adenovirus protease is the intact L3 23K protein.

The L3 23K protein was isolated from adenovirus type 2 and shown to cleave purified substrates, confirming that this protein is the adenovirus protease. Separate antisera, prepared against the amino- and carboxy-terminal regions of the 23K protein react with active protease, demonstrating that, contrary to previous reports, zymogen activation is not involved in the regulation of this enzyme. Molecular exclusion chromatography indicated that the protease is active as a monomer. Purified protease was shown to be inhibited by Zn2+ and Cu2+ and by some, but not all, recognized cysteine protease inhibitors, indicating participation of a thiol group and providing additional support to the suggestion that regulation of the enzyme involves a form of thiol-disulphide interchange.

Adenoviruses, Human↗
PubMed1993

A prospective evaluation of transvaginal sonography for detection of ovarian disease.

OBJECTIVE: Despite the expanding role of transvaginal sonography in routine gynecologic examinations, as well as in screening for ovarian cancer, recent reports have noted problems when using the procedure for detecting ovarian masses and visualizing the ovaries in postmenopausal women. Our study was designed to assess prospectively the capability of transvaginal sonography in evaluating the ovary and detecting adnexal masses. SUBJECTS AND METHODS: Transvaginal sonography was used to examine 113 ovaries in 59 women within 72 hr before gynecologic surgery. Ovarian size and echo texture were assessed, and a search was made for adnexal masses. Sonograms were interpreted without knowledge of the clinical history or results of physical examination, and the sonographic findings were compared with surgical and pathologic data. RESULTS: In the 22 premenopausal patients, 16 (76%) of 21 histologically normal ovaries were identified on sonograms, but only 13 (59%) of 22 adnexal masses. Lesions as large as 177 cm3 were not detected. In the 37 postmenopausal patients, 12 (20%) of 59 normal ovaries and six (54%) of 11 adnexal masses were identified. Five malignant masses (largest, 113 cm3) were not detected. CONCLUSION: In this selected population, our ability to detect normal postmenopausal ovaries and ovarian masses was suboptimal in a number of cases. Practitioners should be aware of potential limitations in the use of this technique alone to evaluate the ovary.

Adnexa Uteri↗
PubMed1992

A novel acetylcholine receptor-related peptide blocks canine cardiac ganglia and inhibits the nicotinic receptor of PC-12 cells.

A 13 amino acid peptide from the calf muscle acetylcholine receptor has been previously shown to bind both snake neurotoxins and acetylcholine. In the experiments reported here a modified complementary peptide (cAChR) derived from that acetylcholine receptor peptide was tested for biological activity in a canine heart preparation. It was expected that the modified complementary peptide would exhibit either acetylcholine-like effects or acetylcholine inhibiting effects, since, as a complementary peptide to the receptor, it should resemble acetylcholine. In these studies cAChR was administered via the sinus node artery of dog hearts in intact animals which were anesthetized with pentobarbital, intubated, and prepared with local cardiac electrograms and force gauges. cAChR was also injected directly into thoracic sympathetic ganglia. Both approaches demonstrated cAChR inhibition of neural transmission, cAChR was added to the medium of carbachol stimulated PC-12 cells. In these cells, derived from a rat pheochromocytoma, sodium flux is controlled by neural nicotinic receptors. With or without preincubation cAChR inhibited carbachol stimulation of sodium flux, exhibiting a Ki of approximately 9 x 10(-5) (similar to that of hexamethonium). Thus cAChR appears to be a novel synthetic peptide which interrupts nicotinic cholinergic neural transmission by acting as an antagonist of the neural nicotinic receptor.

Amino Acid Sequence↗
PubMed1992

Proteolysis is a key process in virus replication.

Proteases were amongst the first enzymes to be isolated and crystallized, and the discovery of their existence in viruses and the realization of the vital role they play has given a new lease of life to one of the oldest topics in biochemistry. Already we have seen the discovery of new variations on well-studied reaction mechanisms and there is the promise of others to come that may be totally novel. When this is allied to the prospect of developing the knowledge which is beginning to accrue into a much needed antiviral therapy, it is clear that viral proteases and their key role in viral replication will be an increasing focus of attention for some time to come.

Animals↗
PubMed1991

A total ban on workplace smoking is acceptable and effective.

The acceptability and effectiveness of a total workplace smoking ban in Telecom Australia was evaluated in a series of studies. Staff in a sample of representative areas were surveyed prior to the introduction of the bans, then 6 and 18 months afterward. By 18 months 81% of all staff, including 53% of smokers, approved of the bans. Sixty-six percent of staff reported a total ban was operative, and 31% reported a total ban except for a smoking room, leaving only 3% reporting lesser restrictions. Smokers were smoking between three and four less cigarettes per work day, and the numbers of smokers had decreased by about double the community rate. The policy had little perceived effect on productivity but resulted in some tension between staff that progressively decreased and now is limited to the few areas where there were problems with compliance. A subsample of managers and staff were interviewed and factors relevant to successful implementation of the policy were identified. These included a clear statement of policy, strong managerial support via equipping managers with leadership and negotiating skills, and the use of occupational health nurses. It is important to provide assistance to affected staff to help them adjust to the ban both before as well as in the months after implementation.

Adult↗