Search PubMed⌕ Search

Biomedical subjects

K Smith

Publications and source records attributed to K Smith.

At least 19 recordsLinked to original sources
PubMed1992

Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metastases and survival in breast cancer.

Animal models suggest a role for new vessel formation (angiogenesis) in tumours with metastatic potential, and there is some evidence that this is true for human tumours. What is needed is a sensitive and specific label for endothelial cells, and one candidate would be a monoclonal antibody to platelet/endothelial cell adhesion molecule (PECAM). We have counted microvessels in 103 primary breast cancers using the JC70 antibody to PECAM (or CD31). We compared our findings with various pathological indicators (lymph node status and tumour grade, size, and type and markers (oestrogen receptor, and c-erbB-2 expression and detection of mutant p53). Tumours showed significantly higher vascularisation than normal breast tissue and the number of blood vessels/mm2 was significantly associated with node metastasis. Only 2 out of 50 tumours with 99 vessel/mm2 or less were node positive whereas 31 out of 39 tumours with counts above 140/mm2 were positive (p < 0.0001). Tumour size and grade also correlated with node metastasis and vascularisation also increased with the size of the primary and with poor differentiation. However, within each subgroup of size or differentiation tumours without node involvement had much lower vascular counts, and multivariate analysis showed that vascular count alone explains the association of size and grade with node metastasis. Other markers, conventional or novel, did not correlate with vascularisation. Even with the short follow-up in this series, vascular counts correlated with early death. These results suggest that angiogenesis is closely linked to metastasis, that it is acquired at a critical density of vessels, and that this process occurs as tumours enlarge or become more poorly differentiated. Counting of newly formed microvessels stained with endothelium-specific antibodies may prove to be a useful tool in the early detection of metastatic potential and in the selection of patients for whom anti-angiogenesis drugs might be beneficial.

Antigens, Differentiation, Myelomonocytic↗
PubMed1992

Active site of trypanothione reductase. A target for rational drug design.

The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.

Amino Acid Sequence↗
PubMed1992

Are the prejunctional alpha 2-adrenoceptors of the rat vas deferens and submandibular gland of the alpha 2A- or alpha 2D-subtype?

We have investigated the effects of antagonists at functional prejunctional alpha 2-adrenoceptors of rat was deferens and rat submandibular gland and compared potencies with affinities for the alpha 2A-, alpha 2B- and putative alpha 2D-ligand binding sites of human platelet, rat kidney, and rat submandibular gland, respectively. Prejunctional potency of antagonists was expressed as an EC30 (concentration producing a 30% increase in stimulation-evoked release of tritium in tissues pre-incubated with [3H]noradrenaline) in rat vas deferens and submandibular gland, and also as a pA2 (antagonist concentration producing a 2-fold shift in the inhibition by the alpha 2-adrenoceptor agonist xylazine of the electrically-evoked isometric twitch) in rat vas deferens. The functional prejunctional alpha 2-adrenoceptors of rat vas deferens and submandibular gland are alpha 2A-like since there was a good correlation between affinity for the alpha 2A-ligand binding site in human platelet and prejunctional potency in rat vas deferens (r = 0.90, n = 7, P less than 0.001), and submandibular gland (r = 0.84, n = 7, P less than 0.05). However, there was a better correlation between affinity for the alpha 2-ligand binding site of rat submandibular gland and prejunctional potency in rat vas deferens (r = 0.95, n = 7, P less than 0.001), and submandibular gland (r = 0.97, n = 7, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1992

Investigations of prejunctional alpha 2-adrenoceptors in rat atrium, vas deferens and submandibular gland.

We have examined the effects of a series of alpha 2-adrenoceptor antagonists on the stimulation-evoked release of tritium from rat atrium, vas deferens and submandibular gland pre-incubated with [3H]noradrenaline, and correlated these potencies with affinities for the alpha 2A-ligand binding site of human platelet and the alpha 2B-ligand binding site of rat kidney. The alpha 2B-selective adrenoceptor antagonists prazosin and ARC 239 showed significantly higher, and the alpha 2A-selective antagonist BRL 44408 showed significantly lower, potency in atrium than in vas deferens and submandibular gland. Yohimbine and BDF 8933 failed to distinguish between prejunctional alpha 2-adrenoceptors or between ligand binding sites. It is concluded that the prejunctional alpha 2-adrenoceptor of rat atrium resembles the alpha 2B-ligand binding site, and differs from the prejunctional alpha 2-adrenoceptors of rat vas deferens and submandibular gland, which resemble the alpha 2A-ligand binding site.

Adrenergic alpha-Antagonists↗
PubMed1992

Preparation of a chemiluminescent imidoester for the non-radioactive labelling of proteins.

A chemiluminescent aryl acridinium ester was synthesized which possesses an imidate ester group capable of reacting with proteins under mild conditions. The compound can be detected at levels as low as 5.2 x 10(-19) mol using commercially available luminometers and can therefore be used to produce high specific activity labelled antibodies for use in immunochemiluminometric assays. The imidate ester compares favourably with a previously reported N-succinimidyl ester in terms of its labelling properties but is easier to synthesize, requiring one less step. The compound was used to label affinity purified to synthesize, requiring one less step. The compound was used to label affinity purified sheep antibodies to human parathyroid hormone to demonstrate its utility in a two-site immunochemiluminometric assay for the measurement of intact parathyroid hormone.

Acridines↗
PubMed1992

Psychological screening for non-specific abdominal pain.

Non-specific abdominal pain (NSAP) may have a detectable psychological component that could be used to predict outcome. To test this hypothesis, 131 patients aged 14-40 years admitted with acute abdominal pain were assessed using the General Health Questionnaire (GHQ) and Hospital Anxiety and Depression (HAD) scale, and a structured interview. Of 61 patients with NSAP, more had a psychosocial problem identified by the admitting registrar (P < 0.01) and marginally more had high questionnaire scores. The risk of having NSAP was high if an abnormality on interview accompanied high questionnaire scores (relative risk 1.93 (95 per cent confidence interval (c.i.) 1.35-2.77)) or if prodromal pain had lasted > 7 days (relative risk 2.13 (95 per cent c.i. 1.55-2.92)). After 2 years, patients with continuing pain had higher HAD and Spielberger Anxiety Trait scores (both P < 0.02); NSAP was associated with persisting pain (relative risk 2.22 (95 per cent c.i. 1.10-4.48)). Psychosocial factors are implicated in NSAP and in chronic pain, but the sensitivity and specificity of questionnaire assessment are too low to be useful in diagnosis.

Abdominal Pain↗
PubMed1992

Measurement of muscle protein fractional synthetic rate by capillary gas chromatography/combustion isotope ratio mass spectrometry.

The measurement of skeletal muscle protein fractional synthetic rate using an infusion of (1-13C)leucine and measuring the isotopic abundance of the tracer in skeletal muscle protein by preparative gas chromatography (GC)/ninhydrin isotope ratio mass spectrometry (IRMS) is laborious and subject to errors owing to contamination by 12C. The purpose of this study was to compare muscle (13C)leucine enrichment measured with the conventional preparative GC/ninhydrin IRMS approach to a new, continuous-flow technique using capillary GC/combustion IRMS. Quadriceps muscles were removed from four Sprague-Dawley rats after each was infused at a different rate with (1-13C)leucine for 6-8 h. Muscle leucine enrichment (at. % excess) measured by both methods differed by less than 4%, except at low (13C)leucine enrichments (less than 0.03 at. % excess). In addition, capillary GC/combustion IRMS was used to assess muscle (13C)leucine enrichment and fractional muscle protein synthesis rate in ten normal young men and women infused with (1,2-13C2)leucine for 12-14 h. This approach reduced the variability of the isotope abundance measure and gave estimates of muscle protein synthesis rate (0.050 +/- 0.011% h-1 (mean +/- SEM); range = 0.023-0.147% h-1) that agree with published values determined using the standard analytical approach. The measurement of (13C)leucine enrichment from skeletal muscle protein by capillary GC/combustion IRMS provides a simple, acceptable and practical alternative to preparative GC/ninhydrin IRMS.

Adult↗
PubMed1992

Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo.

Amide Synthases↗
PubMed1992

Evaluation of the Multi-Test device for immediate hypersensitivity skin testing.

Test-to-test reproducibility, user variability, and the effect of positive reactions on adjacent negative sites were evaluated for the Multi-Test skin testing device. Twenty-five subjects had skin tests with 24 histamine (1 mg/ml), four glycerosaline controls, and four "dry" controls on two testings 7 days apart. To assess reproducibility from large and smaller histamine reactions and/or their effect on adjacent negative controls, 10 of the 25 subjects were retested once with the same testing format, but histamine at 10 mg/ml was substituted. To determine whether large allergen reactions affect adjacent negative controls differently than histamine reactions, 24 additional patients were retested on arms and back with negative controls adjacent to allergens to which they had prior 3+ to 4+ skin test reactions. Conclusions from 2688 skin tests on 49 patients are as follows: large (mean > 10 mm) histamine reactions reproduced better than smaller (mean < 7 mm) responses--coefficients of variation were 12.3 and 21.4 respectively. A 14% user variability occurred when comparing mean wheal sizes from histamine produced by each nurse and 1.2% when comparing their coefficients of variation. Neither small histamine reactions nor large reactions from histamine and allergens affected adjacent negative controls. We conclude that Multi-Test is a highly reliable skin testing technique that provides good reproducibility of results and low user variability.

Adolescent↗
PubMed1992

Characterisation of melarsen-resistant Trypanosoma brucei brucei with respect to cross-resistance to other drugs and trypanothione metabolism.

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.

Animals↗
PubMed1992

The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei.

D,L-dihydrolipoamide and D,L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10(9) and 4.51 x 10(9) M-1, respectively. These complexes possess 6-membered cyclic dithioarsenite rings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500-fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione. L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate. The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 +/- 4.3 and 9.7 +/- 2.9 pmol (10(8) cells)-1, respectively). The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 +/- 1.4 and 47.8 +/- 3.7 mU mg-1, respectively). However, the Km for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains. Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane. These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs.

Animals↗
PubMed1992

Reactogenicity and immunogenicity of a double-strength acellular pertussis vaccine.

The reactogenicity and immunogenicity of a double-strength acellular pertussis vaccine were evaluated after administration to 16 4-6-year-old children. The vaccine contained toxoided lymphocytosis-promoting factor (6.0 micrograms/dose), filamentous haemagglutinin (70 micrograms/dose), agglutinogens (1.4 micrograms/dose) and the 69 kDa protein (approximately 8.0 micrograms/dose). The vaccine was extremely well tolerated with few minor side effects following immunization. Significant increases in antibodies to all pertussis vaccine components were noted. In summary, this double-strength acellular pertussis vaccine, containing a very high dose of filamentous haemagglutinin, had minimal reactogenicity and was immunogenic. These findings, as well as other studies with this vaccine, indicate that filamentous haemagglutinin is not a major determinant of vaccine reactogenicity.

Antibodies, Bacterial↗
PubMed1992

Replication of human herpesvirus 6 in epithelial cells in vitro.

Mink lung epithelial (NBL-7) cells were shown to be permissive for human herpesvirus 6 (HHV-6) by four independent methods of analysis: detection of infectious virus, viral antigens, viral DNA sequences, and herpesvirus particles. Infection was serially passaged, with minimal cytopathology, for several months demonstrating for the first time that a cell of epithelial origin can support HHV-6 replication.

Animals↗
PubMed1992

Effect of impaired renal function on the pharmacokinetics of coadministered cefoperazone and sulbactam.

The pharmacokinetics of cefoperazone 2 g combined with sulbactam 1 g after a single dose administered intravenously were evaluated in 24 subjects with normal and impaired renal function. Subjects were categorized into four groups based on endogenous creatinine clearance Clcr. Patients in groups 1, 2 and 3 had ClcrS of greater than 60, 31 to 60, and 10 to 30 mL/min/1.73 m2, respectively. Patients in group 4 required maintenance haemodialysis and were assumed to have Clcr less than 10 mL/min/1.73 m2. Pharmacokinetic parameters were determined by noncompartmental methods. No significant differences (P greater than 0.05) in mean peak serum cefoperazone-sulbactam concentrations for group 1 (208.4/29.0 mg/L), group 2 (199.0/34.1 mg/L), group 3 (163.2/35.0 mg/L), and group 4 (234.0/66.0 mg/L) were noted. Correlations between both total serum (r = 0.58) and renal (r = 0.35) clearance and creatinine clearances were negative for cefoperazone, although both were shown to decline with diminished renal function. Correlations between serum (r = 0.85) and renal (r = 0.72) clearances and creatinine clearance for sulbactam were, on the other hand, both positive and declined in a linear fashion. No significant differences in steady state volumes of distribution were noted for either cefoperazone (P = 0.53) or sulbactam (P = 0.85) amongst the four groups. After 24 h, urinary recovery was also comparable for both cefoperazone (P = 0.64) and sulbactam (P = 0.85) amongst the four groups. The concentrations of cefoperazone and sulbactam remained at or above the MICs (16/8 mg/L) for common bacterial pathogens for 2.5, 3, 7 and 14 h in groups 1, 2, 3 and 4, respectively.

Adolescent↗
PubMed1992

Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic mechanism.

Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.

Amino Acid Sequence↗
PubMed1992

Investigations of the subtype of alpha 2-adrenoceptor mediating contractions of the human saphenous vein.

1. We have examined the effects of a series of alpha 2-adrenoceptor antagonists against isometric contractions to noradrenaline in human saphenous vein, and correlated these potencies with affinities for the alpha 2A-ligand binding site of human platelet and the alpha 2B-ligand binding site of rat kidney. 2. The alpha 2B-selective adrenoceptor antagonists, prazosin, ARC 239 and HV 723 showed high, and the alpha 2A-selective antagonist BRL 44408 showed low, potency in human saphenous vein. 3. Potency in human saphenous vein correlated better with affinity for the alpha 2B-ligand binding site (r = 0.71, n = 12, P less than 0.01) than with affinity for the alpha 2A-ligand binding site (r = 0.56, n = 12, non significant). 4. It is concluded that the postjunctional alpha 2-adrenoceptor of human saphenous vein resembles an alpha 2B-ligand binding site more than an alpha 2A-ligand binding site.

Adrenergic alpha-Antagonists↗
PubMed1992

Lives in a balance: perceived family functioning and the psychosocial adjustment of adolescent cancer survivors.

Childhood cancer patients have a greater likelihood of long-term survival than ever before. This study examined both the perceived family functioning of adolescents who had successfully completed treatment for pediatric cancer and the relationship between family functioning and post-treatment adjustment. Eighty-eight adolescent survivors of hematologic malignancies were assessed regarding their family functioning, mental health, self-esteem, global competence, and problem behaviors. Contrary to expectations about the influence of cancer on these families, adolescent cancer survivors reported lower levels of family cohesion than the normative sample of healthy adolescents and their families. While current age, gender, age at diagnosis, and time since treatment completion were generally not associated with adolescents' adjustment, perceived family cohesion and adaptability were strongly related to post-treatment psychological adjustment.

Adaptation, Psychological↗