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Biomedical subjects

C M Hill

Publications and source records attributed to C M Hill.

At least 19 recordsLinked to original sources
PubMed1999

Purified recombinant Escherichia coli ketol-acid reductoisomerase is unsuitable for use in a coupled assay of acetohydroxyacid synthase activity due to an unexpected side reaction.

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme purified by metal-ligand affinity chromatography. The pure enzyme was tested for its ability to provide a sensitive and continuous coupled assay for acetohydroxyacid synthase (EC 4.1.3.18), the preceding enzyme in the pathway of branched-chain amino acid biosynthesis. An unexpected side reaction of ketol-acid reductoisomerase was observed in which it catalyzes the reduction of pyruvate. Although relatively slow, this side reaction is high enough to prohibit the use of this enzyme in a coupled assay for acetohydroxyacid synthase.

Acetolactate Synthase↗
PubMed1999

Insurance ovulation, embryo mortality and twinning.

The 'insurance ova' hypothesis (Anderson, 1990) views dizygotic twinning as a by-product of selection for multiple ovulation which sometimes--in error--results in the birth of twins. From this viewpoint, polyovulation is a mechanism which reduces the risks of fertilization failure or embryo defect/mortality. If DZ twin births are a 'side-effect' of a mechanism which compensates for defective embryos one might predict that embryo defect rates and twinning rates will covary. This prediction is tested using national-level data on twinning rates and rates of trisomy-21 (Down's syndrome), and a strong positive correlation is found, even when controlling for maternal age. One suggestion that follows from this finding is that intra- and interpopulation variation in both twinning rates and Down's syndrome rates may result, in part, from individual variation in pre-implantation rejection of embryos in the very early stages of pregnancy. In this paper it is proposed that the 'insurance ova' explanation for twinning in humans could be expanded to incorporate a model of rejection of anomalous embryos, be they anomalies of number or type. Variation in the efficiency of an embryo rejection mechanism, combined with variation in frequency of polyovulation, would have consequences for individual reproductive success.

Adult↗
PubMed1999

The efficacy of a novel adenosine agonist (WAG 994) in postoperative dental pain.

AIMS: To determine the comparative efficacy of a new novel adenosine agonist (WAG 994) in postoperative pain after third molar surgery. METHODS: One hundred and twenty-two patients with postoperative pain after third molar surgery were randomised in a placebo double-blind trial with an active control group. In the early postoperative period patients received either a single dose of WAG 994 1 mg, ibuprofen 400 mg or matched placebos. Pain intensity score was recorded on serial visual analogue scales over a 6 h investigation period. Similarly, pain relief was completed on a 4 point categorical scale at each evaluation point. Patients had access to escape analgesic and if these were taken, the time and dosage were recorded. A sparse sampling technique was used to investigate the relationship between analgesic effects and plasma concentrations of WAG 994. RESULTS: All three treatment groups were matched for various demographic variables. For all efficacy measures, WAG 994 was not significantly different from placebo (P >0.05), whereas ibuprofen 400 mg was significantly superior to placebo (P<0.001). No significant relationships (P<0.05) were found between WAG 994 pharmacokinetic variables and efficacy measures. Conclusion WAG 994, an adenosine agonist, did not show efficacy in the management of postoperative pain after third molar surgery. Although this pain responds well to nonsteroidal anti-inflammatory drugs, it appears to be resistant to compounds that interact with purinergic receptors.

Adenosine↗
PubMed1999

The Banff 97 working classification of renal allograft pathology.

BACKGROUND: Standardization of renal allograft biopsy interpretation is necessary to guide therapy and to establish an objective end point for clinical trials. This manuscript describes a classification, Banff 97, developed by investigators using the Banff Schema and the Collaborative Clinical Trials in Transplantation (CCTT) modification for diagnosis of renal allograft pathology. METHODS: Banff 97 grew from an international consensus discussion begun at Banff and continued via the Internet. This schema developed from (a) analysis of data using the Banff classification, (b) publication of and experience with the CCTT modification, (c) international conferences, and (d) data from recent studies on impact of vasculitis on transplant outcome. RESULTS: Semiquantitative lesion scoring continues to focus on tubulitis and arteritis but includes a minimum threshold for interstitial inflammation. Banff 97 defines "types" of acute/active rejection. Type I is tubulointerstitial rejection without arteritis. Type II is vascular rejection with intimal arteritis, and type III is severe rejection with transmural arterial changes. Biopsies with only mild inflammation are graded as "borderline/suspicious for rejection." Chronic/sclerosing allograft changes are graded based on severity of tubular atrophy and interstitial fibrosis. Antibody-mediated rejection, hyperacute or accelerated acute in presentation, is also categorized, as are other significant allograft findings. CONCLUSIONS: The Banff 97 working classification refines earlier schemas and represents input from two classifications most widely used in clinical rejection trials and in clinical practice worldwide. Major changes include the following: rejection with vasculitis is separated from tubulointerstitial rejection; severe rejection requires transmural changes in arteries; "borderline" rejection can only be interpreted in a clinical context; antibody-mediated rejection is further defined, and lesion scoring focuses on most severely involved structures. Criteria for specimen adequacy have also been modified. Banff 97 represents a significant refinement of allograft assessment, developed via international consensus discussions.

Acute Disease↗
PubMed1998

Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzyme II and characterization of three herbicide-insensitive forms.

Sulphonylurea and imidazolinone herbicides act by inhibiting acetohydroxyacid synthase (AHAS; EC 4.1.3.18), the enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids. AHAS requires as cofactors thiamin diphosphate, a bivalent metal ion and, usually, FAD. Escherichia coli contains three isoenzymes and this study concerns isoenzyme II, the most herbicide-sensitive of the E. coli forms. A plasmid containing the large and small subunit genes of AHAS II was mutagenized using hydroxylamine and clones resistant to the sulphonylurea chlorimuron ethyl were selected. Three mutants were isolated; A26V, V99M and A108V. A26V has been described previously whereas the equivalent mutation of A108V has been reported in a herbicide-insensitive variant of yeast AHAS. The V99M mutation has not been discovered previously in AHAS from any source. The mutants were each over-expressed in E. coli, and the enzymes were purified to homogeneity. Some differences from wild type in the kinetic properties (kcat, Km and cofactor affinities) were observed, most notably a 28-fold decrease in the affinity for thiamin diphosphate of V99M. None of the mutants shows marked changes from the wild type in sensitivity to three imidazolinones, with the largest increase in the apparent inhibition constant being a factor of approximately 5. The A26V mutant is weakly resistant (6- to 20-fold) to six sulphonylureas, whereas stronger resistance is seen in V99M (20- to 250-fold) and A108V (35- to 420-fold). Resistance as a result of these mutations is consistent with a molecular model of the herbicide-binding site, which predicts that mutation of G249 might also confer herbicide insensitivity. Three G249 mutants were constructed, expressed and purified but all are inactive, apparently because they cannot bind FAD.

Acetolactate Synthase↗
PubMed1998

The amino terminus of human CCR5 is required for its function as a receptor for diverse human and simian immunodeficiency virus envelope glycoproteins.

The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.

Amino Acid Sequence↗
PubMed1998

Reliability of the hip distraction index in two-month-old German shepherd dogs.

OBJECTIVE: To determine whether distraction index (DI), a measure of passive hip joint laxity, at 2 months of age was predictive of DI at 4 or 12 months of age in German Shepherd Dogs. DESIGN: Prospective cohort study. ANIMALS: 45 German Shepherd Dogs. PROCEDURE: DI was measured at 2, 4, and 12 months of age. At the same times, a standard ventrodorsal radiographic projection of the pelvis with the hip joints extended was obtained and examined for evidence of degenerative joint disease (DJD). To facilitate radiographic positioning, dogs were sedated or anesthetized. RESULTS: DI at 2 months of age was not significantly correlated with DI at 4 or 12 months of age. However, DI at 4 months of age was correlated with DI at 12 months of age. The proportion of dogs with DI > or = 0.3 at 12 months of age that had radiographic evidence of DJD by 12 months of age (13/22; 59%) was significantly greater than the proportion of dogs with DI < 0.3 at 12 months of age that had radiographic evidence of DJD by 12 months of age (1/9; 11%). CLINICAL IMPLICATIONS: For German Shepherd Dogs, DI at 2 months of age was not sufficiently reliable to predict DI at 4 and 12 months of age; however, DI at 4 and 12 months of age were comparable. We recommend that, for German Shepherd Dogs, DI not be measured before 4 months of age and that particularly for breeding dogs, DI be remeasured after maturity to confirm DI obtained at earlier ages. Studies including other breeds of dogs should be done to determine the youngest reliable age to initiate hip joint screening.

Age Factors↗
PubMed1998

Spectrum of surgical risk for left main coronary stenoses: benchmark for potentially competing percutaneous therapies.

Coronary artery bypass grafting is the preferred therapy for left main trunk coronary stenoses because of a demonstrated survival advantage compared with medical therapy. Recently, however, advocates of percutaneous intervention have suggested that stenting or atherectomy may provide acceptable results for these patients. To determine the spectrum of surgical outcome among patients with left main trunk coronary stenoses, we analyzed 14 potential covariates of in-hospital and 3-year mortality for a consecutive series of patients without prior coronary artery bypass grafting treated at our institution from January 1990 to December 1995. In-hospital mortality (2.3%) for 1585 consecutively treated patients correlated independently with renal dysfunction, age, and class III to IV heart failure. A model was constructed that allowed prediction of outcome (c-statistic = 0.77, p < 0.0001), and only the 9% of patients at highest risk were found to be at risk for death >5%. Mortality at 3 years (15.6%+/-2.2%) in a previously specified subgroup (n = 285) correlated independently with baseline age, renal dysfunction, and chronic pulmonary disease. A model was constructed that allowed prediction of outcome for the validation sample (four risk groups with 3-year mortality rates of 4.5%+/-2.5%, 6.5%+/-2.8%, 20.0%+/-4.2%, and 39.8%+/-8.5%; p < 0.0001. These data provide a contemporary benchmark of clinical outcome against which possible competing therapies may be compared.

Aged↗
PubMed1998

A one-year review of maxillofacial sports injuries treated at an accident and emergency department.

To assess the aetiology and demand for oral and maxillofacial surgery services associated with sports injuries, a prospective study was undertaken. Data were collected from consecutive patients (with maxillofacial injuries associated with sports) attending the accident and emergency department at the Cardiff Royal Infirmary in the UK during a 12-month period and analysed. Data relating to demography, aetiology, site and extent of injury, treatment and outcome were collected. There were 790 attendances (695M:85F) arising principally from injuries related to rugby (n = 206), cycling (n = 189) and football (n = 109) but few from recognized contact sports (n = 26). The principal causes of these injuries were direct bodily contact (n = 260) and falls (n = 219). The commonest injury was soft-tissue laceration (n = 604); 80 patients had dentoalveolar fractures and 64 patients had fractures of the facial skeleton. Injuries were located in the upper- (n = 257), middle- (n = 201) and lower third of the face (n = 124) with 188 lip/intraoral injuries. Repair of lacerations (n = 600) was the commonest treatment; only 46 fractures required interventive treatment. Follow-up was performed for most of these patients at the Department of Oral and Maxillofacial Surgery at the Dental Hospital (n = 404) and general medical/dental practitioners (n = 258). These data highlight the importance of oral and maxillofacial surgery staff in the management of sports injuries in accident and emergency departments. Moreover, they suggest the need for prioritization of rule and legislation changes and the continuing need to improve safety standards to prevent maxillofacial injuries.

Accidental Falls↗
PubMed1998

Treatment of renal failure in idiopathic membranous nephropathy with azathioprine and prednisolone.

BACKGROUND: Progressive deterioration in renal function occurs in 20-50% of patients with idiopathic membranous nephropathy (IMN). Several treatment regimens have been used to reverse this with varying effect and toxicity. METHODS: Thirteen patients (10 males, 3 females, median age 56 years) with IMN and progressive renal failure were treated with oral prednisolone 20-60 mg/day and azathioprine 1.3-2.7 mg/kgBW/day. All patients were followed up for a minimum of 2 years with a median follow-up of 73 months (range 24-103 months). RESULTS: Ten patients responded to treatment with a fall in serum creatinine and renal function stabilized in the remainder. Two patients relapsed, one of whom responded to an increase in immunosuppression, the other is now on dialysis. Proteinuria has significantly reduced in 10 patients, and only four patients still have nephrotic-range proteinuria. Mean (+/- SE) peak pretreatment serum creatinine of 229 (+/- 161) mumol/l and urinary protein of 11.8 (+/- 1.8) g/24 have fallen to 163 (+/- 65) mumol/l and 3.25 (+/- 1.0) g/24 h after 12 months treatment (P < 0.005, Wilcoxon matched pairs test). Immunosuppressive treatment has been successfully withdrawn in four patients after intervals ranging from 12 to 60 months. Adverse effects, which occurred in 10 patients, have been mild and have not led to treatment withdrawal though dose reductions have been necessary in some patients. CONCLUSIONS: Oral prednisolone and low-dose azathioprine is an effective therapy for progressing renal failure due to IMN, and induces remission of nephrotic syndrome. Side-effects are less than other immunosuppressive regimens.

Adolescent↗
PubMed1997

GnRH antagonist inhibition of luteinizing hormone secretion and ovulation in the pig.

A GnRH antagonist, Ac-D-p-Cl-Phe1,2, D-Trp3, D-Arg6, D-Ala10 GnRHb (Organon), was utilized to determine the effective dosage and duration to inhibit LH secretion in the pig. In a preliminary trial, barrows received either 10, 50, or 250 micrograms/kg BW of the GnRH antagonist. Secretion of LH was inhibited within 30 min for a duration of 12 h with the 100 micrograms/kg dose but persisted for greater than 48 h with the 250 micrograms/kg treatment. A second study determined effectiveness of the antagonist for inhibiting ovulation in cyclic gilts. At first detection of standing estrus, cyclic gilts were treated with either saline (control), 100, or 200 micrograms/kg BW of the GnRH antagonist (GnRH1). A second group of GnRH antagonist gilts received 200 micrograms/kg BW of the GnRH antagonist approximately 8 h prior to standing estrus (GnRH2). The GnRH1-treatment failed to inhibit or delay ovulation. Ovulation was inhibited and estrous cycles lengthened in GnRH2-treated gilts. These preliminary results suggest that ovulation in the gilt can be inhibited if the GnRH antagonist is administered prior to the LH surge.

Animals↗
PubMed1997

Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

CD4 Antigens↗
PubMed1997

Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits.

The first step in the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (EC 4.1.3.18). The reaction involves the decarboxylation of pyruvate followed by condensation with either a second molecule of pyruvate or with 2-oxobutyrate. The enzyme requires as cofactors thiamine diphosphate, a divalent metal ion and, usually, FAD. In most bacteria the enzyme is a heterotetramer of two large and two small subunits. Escherichia coli contains three active isoenzymes and the present study concerns isoenzyme II, whose large and small subunits are encoded by the ilvG and ilvM genes respectively. Cloning these genes into a plasmid vector and overexpression in E. coli allowed a two-step purification procedure for the native enzyme to be developed. The level of expression is considerably higher from a vector that introduces a 50 residue N-terminal fusion containing an oligohistidine sequence on the large subunit. Purification to homogeneity was achieved in a single step by immobilized-metal-affinity chromatography. The kinetic properties of the native and fusion enzyme are indistinguishable with respect to the substrate pyruvate and the inhibitor chlorsulfuron. The individual subunits were expressed as oligohistidine-tagged fusion proteins and each was purified in a single step. Neither subunit alone has significant enzymic activity but, on mixing, the enzyme is reconstituted. The kinetic properties of the reconstituted enzyme are very similar to those of the fusion enzyme. It is proposed that the reconstitution pathway involves successive, and highly co-operative, binding of two small subunit monomers to a large subunit dimer. None of the cofactors is needed for subunit association although they are necessary for the restoration of enzymic activity.

Acetolactate Synthase↗
PubMed1997

Correlation between subjective and objective measures used to determine severity of postoperative pain in dogs.

OBJECTIVE: To determine the association between subjective and objective variables commonly used to evaluate severity of postoperative pain in dogs. DESIGN: Prospective double-blind study. ANIMALS: 36 dogs with unilateral rupture of the cranial cruciate ligament that underwent surgery to stabilize the stifle. PROCEDURE: Each dog was assessed to determine severity of pain before and after surgery, using various subjective and objective criteria. RESULTS: Subjective measures of pain (scores for visual analogue and numerical rating scales) correlated poorly or were not correlated with heart rate, respiratory rate, blood pressure, and results of a pain threshold test. Scores for visual analogue and numerical rating scales correlated with each other and with the amount of vocalization at most time periods. CLINICAL IMPLICATIONS: We detected a weak association between commonly employed subjective and objective measures of pain. This indicated that some of these measurement techniques do not predictably reflect severity of postoperative pain in dogs. Therefore, clinicians should not rely too heavily on these variables when assessing severity of postoperative pain in dogs.

Analgesics↗
PubMed1997

An audit of antibiotic prescribing in third molar surgery.

The use of antibiotics as a prophylactic measure against infection is widespread following the removal of impacted third molars. The advantages of using such prophylaxis appear to be marginal and there is little substantial evidence to support the use of second and third generation antibiotics for routine prophylaxis. An audit of antibiotic prescribing practices in the University Dental Hospital National Health Service Trust (Cardiff) was undertaken and demonstrated the potential for saving large sums of money whilst apparently incurring no clinical disadvantage. The value of such audit process in oral and maxillofacial surgery is in identifying best practice followed by measuring and, where possible, improving standards.

Adult↗
PubMed1997

Autosomal dominant Alport syndrome linked to the type IV collage alpha 3 and alpha 4 genes (COL4A3 and COL4A4).

BACKGROUND: Alport syndrome is a hereditary nephritis that may lead to end-stage renal disease (ESRD) in young adult life and is often associated with sensorineural deafness and/or ocular abnormalities. The majority of families are X-linked due to mutations in the COL4A5 gene at Xq22. Autosomal forms of the disease are also recognized with recessive disease, having been shown to be due to mutations in the COL4A3 and COL4A4 genes on chromosome 2. Familial benign haematuria has also been mapped to this region in some families. SUBJECTS AND METHODS: We describe a large family with autosomal dominant Alport syndrome in which males and females are equally severely affected and one member with a mild sensorineural deafness reached ESRD aged 35 years. Renal biopsy in four affected patients demonstrated characteristic thickened and split glomerular basement membranes on electron-microscopy. RESULTS: Genetic linkage analysis using markers on chromosome 2q demonstrated co-segregation of the disease with the markers D2S351 and D2S401 with a maximum lod score of 3.4 at zero recombination. Linkage to the COL4A4 gene was confirmed using an intragenic COL4A4 polymorphism. Mutation analysis has revealed a missense Leu36Pro mutation in exon 5 of the adjacent COL4A3 gene in the unaffected mother, which may lead to a more severe phenotype in affected family members carrying this mutation. CONCLUSION: Mutations in the COL4A3 and COL4A4 genes can cause a spectrum of glomerular basement membrane disease ranging from autosomal recessive Alport syndrome to autosomal dominant Alport syndrome and familial benign haematuria.

Adult↗
PubMed1997

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.

3T3 Cells↗