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Biomedical subjects

A M Smith

Publications and source records attributed to A M Smith.

At least 73 records · Page 4Linked to original sources
PubMed2000

Co-expression of the Bordetella pertussis leader peptidase I results in enhanced processing and expression of the pertussis toxin S1 subunit in Escherichia coli.

Bordetella pertussis is the causative agent of whooping cough. Traditional vaccines against this disease are inherently reactogenic, thus research is currently focussed on the production of less reactive, acellular vaccines. Expression of candidate antigens for these vaccines in Escherichia coli would be preferable, however, several B. pertussis antigens undergo incorrect post-translational processing in E. coli. The leader peptidase gene (lep) of B. pertussis encodes a protein of 294 amino acid residues that shares homology with other prokaryote leader peptidase I sequences. Hydrophilicity analysis based on the predicted amino acid sequence has demonstrated a similar membrane topology to that of E. coli and Salmonella typhimurium leader peptidase I. Co-expression of the B. pertussis lep gene in E. coli strain TOPP2 expressing the pertussis toxin S1 subunit was found to markedly increase the expression and post-translational processing of the S1 protein.

Amino Acid Sequence↗
PubMed2000

An event-related potential investigation of response inhibition in schizophrenia and psychopathy.

BACKGROUND: Schizophrenia and psychopathy are both characterized by impulsive, poorly planned behavior. This behavior may originate from a weak or poorly coordinated response inhibition system. We tested the hypothesis that schizophrenia and psychopathy are associated with abnormal neural processing during the suppression of inappropriate responses. METHODS: The participants were schizophrenic patients, nonpsychotic psychopaths, and nonpsychotic, nonpsychopathic control subjects (defined by the Hare Psychopathy Checklist-Revised), all incarcerated in a maximum security psychiatric facility. We recorded behavioral responses and event-related potentials (ERPs) during a Go/No Go task. RESULTS: Schizophrenic patients made more errors of commission than did the nonpsychopathic offenders. As expected, the nonpsychopathic nonpsychotic participants showed greater frontal ERP negativity (N275) to the No Go stimuli than to the Go stimuli. This effect was small in the schizophrenic patients and absent in the psychopaths. For the nonpsychopaths, the P375 ERP component was larger on Go than on No Go trials, a difference that was absent in schizophrenic patients and in the opposite direction in psychopaths. CONCLUSIONS: These findings support the hypothesis that the neural processes involved in response inhibition are abnormal in both schizophrenia and psychopathy; however, the nature of these processes appears to be different in the two disorders.

Adolescent↗
PubMed2000

Transforming growth factor-alpha-mediated growth pathways in human gastro-intestinal cell lines in relation to the gastrin autocrine pathway.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF-stimulated tyrosine kinase. Our aims were to compare EGF/TGF-alpha interactions in 2 human gastro-intestinal cell lines: MGLVA1, with a strong gastrin autocrine pathway, and C170HM2, with a weak pathway. Both cell lines expressed the TGF-alpha gene. MGLVA1 expressed TGF-alpha protein as determined by immuno-cytochemistry, which was absent in C170HM2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVA1 did not have enhanced in vitro proliferation in response to EGF or TGF-alpha, unlike C170HM2. The basal growth of MGLVA1 was inhibited by anti-sera against TGF-alpha, the EGF receptor and G17. C170HM2 was not inhibited by any of the anti-sera. Neutralisation of TGF-alpha resulted in undetectable cell-associated progastrin levels in MGLVA1 (untreated had 391.7 fmol/5 x 10(6) cells). The progastrin level of C170HM2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopeptide concentration, of unstimulated MGLVA1 was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 microg/mg protein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 compared to 0.056 microg/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphorylation of the EGF receptor, inhibited the basal growth of MGLVA1 (IC(50) 1.3 microM) and C170HM2 (9.5 microM, p < 0.05 from MGLVA1). Herbimycin, which inhibits pp60(c-src) kinase, reduced the basal growth of MGLVA1 (0.67 microM) but not C170HM2. Immunofluorescence studies confirmed the presence of tyrosine-phosphorylated proteins and pp60(c-src) within the cytoplasm of unstimulated MGLVA1 cells. There was no specific immunofluorescence for either parameter in C170HM2 cells until after treatment with EGF.

Blotting, Southern↗
PubMed2000

Gadolinium-enhanced arterial-phase MR imaging of hypervascular liver tumors: comparison between tailored and fixed scanning delays in the same patients.

The purpose of this study was to compare in the same patients tailored and fixed scanning delays during gadolinium-enhanced arterial-phase magnetic resonance imaging of hypervascular liver tumors. Tailored scanning delays were obtained with automated region of interest threshold triggering. A delay of 23 seconds between the start of contrast material injection and imaging was used for fixed delay examinations. Quantitative and qualitative evaluation was performed in 21 patients with normal cardiac function referred for MR assessment of hypervascular liver tumors. In the tailored examinations, the median time delay between the start of contrast material injection and the start of magnetic resonance imaging was 21 seconds (range, 18-34 seconds). The median tumor-to-liver contrast during tailored examinations was 19.1 versus 14.7 during fixed delay examinations. This difference, however, was not significant. Similarly, the enhancement in the aorta, the portal vein, the liver, and the tumor did not differ significantly between examinations performed with tailored and fixed delays. It is concluded that in our group of patients with hypervascular liver tumors and normal cardiac function, no significant improvement in tumor-to-liver contrast and enhancement during the arterial phase was found when gadolinium-enhanced magnetic resonance imaging was performed with a tailored scanning delay rather than with a fixed delay.

Adult↗
PubMed2000

Whole brain quantitative CBF and CBV measurements using MRI bolus tracking: comparison of methodologies.

Three different deconvolution techniques for quantifying cerebral blood flow (CBF) from whole brain T*(2)-weighted bolus tracking images were implemented (parametric Fourier transform P-FT, parametric single value decomposition P-SVD and nonparametric single value decomposition NP-SVD). The techniques were tested on 206 regions from 38 hyperacute stroke patients. In the P-FT and P-SVD techniques, the tissue and arterial concentration time curves were fit to a gamma variate function and the resulting CBF values correlated very well (CBF(P-FT) = 1.02 x CBF(P-SVD), r(2) = 0.96). The NP-SVD CBF values (i.e., original unfitted curves were used) correlated well with the P-FT CBF values only when a sufficient number of time series volumes were acquired to minimize tracer time curve truncation (CBF(P-FT) x 0.92 x CBF(NP-SVD), r(2) = 0.88). The correlation between the fitted CBV and the unfitted CBV values was also maximized in regions with minimal tracer time curve truncation (CBV(fit) = 1.00 x CBV(unfit), r(2) = 0.89). When a sufficient number of time series volumes could not be acquired (due to scanner limitations) to avoid tracer time curve truncation, the P-FT and P-SVD techniques gave more reliable estimates of CBF than the NP-SVD technique.

Acute Disease↗
PubMed2000

Whole brain quantitative CBF, CBV, and MTT measurements using MRI bolus tracking: implementation and application to data acquired from hyperacute stroke patients.

A robust whole brain magnetic resonance (MR) bolus tracking technique based on indicator dilution theory, which could quantitatively calculate cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) on a regional basis, was developed and tested. T2*-weighted gradient-echo echoplanar imaging (EPI) volumes were acquired on 40 hyperacute stroke patients after gadolinium diethylene triamine pentaacetic acid (Gd-DTPA) bolus injection. The thalamus, white matter (WM), infarcted area, penumbra, and mirror infarcted and penumbra regions were analyzed. The calculation of the arterial input function (AIF) needed for absolute quantification of CBF, CBV, and MTT was shown to be user independent. The CBF values (ml/min/100 g units) and CBV values (% units, in parentheses) for the thalamus, WM, infarct, mirror infarct, penumbra, and mirror penumbra (averaged over all patients) were 69.8 +/- 22.2 (9.0 +/- 3.0 SD); 28.1 +/- 6.9 (3.9 +/- 1.2); 34.4 +/- 22.4 (7.1 +/- 2.7); 60.3 +/- 20.7 (8.2 +/- 2.3); 50.2 +/- 17.5 (10.4 +/- 2.4); and 64.2 +/- 17.0 (9.5 +/- 2.3), respectively, and the corresponding MTT values (in seconds) were 8.0 +/- 2.1; 8.6 +/- 3.0; 16.1 +/- 8.9; 8.6 +/- 2.9; 13.3 +/- 3.5; and 9.4 +/- 3.2. The infarct and penumbra CBV values were not significantly different from their corresponding mirror values, whereas the CBF and MTT values were (P < 0.01). Quantitative measurements of CBF, CBV, and MTT were calculated on a regional basis on data acquired from hyperacute stroke patients, and the CBF and MTT values showed greater sensitivity to areas with perfusion defects than the CBV values. J. Magn. Reson. Imaging 2000;12:400-410.

Blood Flow Velocity↗
PubMed2000

H(2)(15)O PET validation of steady-state arterial spin tagging cerebral blood flow measurements in humans.

Steady-state arterial spin tagging approaches can provide quantitative images of CBF, but have not been validated in humans. The work presented here compared CBF values measured using steady-state arterial spin tagging with CBF values measured in the same group of human subjects using the H(2)(15)O IV bolus PET method. Blood flow values determined by H(2)(15)O PET were corrected for the known effects of incomplete extraction of water across the blood brain barrier. For a cortical strip ROI, blood flow values determined using arterial spin tagging (64+/-12 cc/100 g/min) were not statistically different from corrected blood flow values determined using H(2)(15)O PET (67+/-13 cc/100 g/min). However, for a central white matter ROI, blood flow values determined using arterial spin tagging were significantly underestimated compared to corrected blood flow values determined using H(2)(15)O PET. This underestimation could be caused by an underestimation of the arterial transit time for white matter regions.

Adult↗
PubMed2000

Nature of immobilized antibody layers linked to thioctic acid treated gold surfaces.

Utilization of 125I-labeled IgG enables an investigation of protein immobilized to gold electrodes sputter deposited on microporous nylon membranes, including the precise nature of the surface-protein bond (i.e. covalent or non-specific adsorption), physical location of the immobilized protein (i.e. on the surface of the gold electrode or within the pores of the membrane), and the amount of protein immobilized. This is accomplished by comparing the mass of protein immobilized to gold surfaces that have been treated in several different fashions, as well as, deposition of the gold on nylon membranes that have been treated differently. It is shown that these microporous gold electrodes, proposed previously for conducting novel non-separation electrochemical enzyme immunoassays, consist of multiple protein layers non-specifically adsorbed. Approximately, half of the total adsorbed protein is immobilized to the gold surface with the remaining protein bound within the pores on the nylon membrane.

Adsorption↗
PubMed2000

Selenate-supplemented nutritional formula increases plasma selenium in hemodialysis patients.

OBJECTIVE: The purpose of this study was to determine the short-term effect of feeding selenium-supplemented formulas on the selenium status of end-stage renal disease patients on hemodialysis. DESIGN AND SETTING: The prospective, randomized, single-blind study of parallel design was conducted at three hemodialysis clinics. PATIENTS: A total of 79 hemodialysis patients were randomly assigned into one of three groups. INTERVENTION: Liquid nutritional formula supplemented with either selenite (28 microg Se/8 oz, n = 26), selenate (28 microg Se/8 oz, n = 26), or nonfortified (7 microg Se/8 oz, n = 27) was fed to hemodialysis patients as their sole source of nutrition for 14 days. MAIN OUTCOME MEASURE: Plasma and red blood cell (RBC) selenium and glutathione peroxidase (GPX) activities were measured in predialysis blood both before (day 1) and after (day 8) a 7-day baseline period, and after subjects received the formula as the sole source of nutrition (approximately 35 kcal/kg/d) for 14 days (day 22). RESULTS: Selenium intake (Mean +/- SEM, microg/d) was 134 +/- 9, 140 +/- 9, and 35 +/- 2 for patients receiving selenite-, selenate-, or non-supplemented formula, respectively. On day 22, plasma selenium (micromol/L) was greater (P <.032) in the selenate-supplemented group (1.5 +/- 0.1) compared with the nonsupplemented group (1.2 +/- 0.1), but not compared with the selenite-supplemented group (1.4 +/- 0.1). Plasma GPX activity was 44% to 60% that of healthy controls and not different among groups. RBC selenium and GPX activities were within the normal range and were not different among groups. CONCLUSION: The results of this study indicate that a liquid formula supplemented with selenium as selenate is successful at maintaining selenium concentrations within normal range, as well as significantly increasing plasma selenium levels compared with nonsupplementation.

Adult↗
PubMed2000

Review article: gastrin and colorectal cancer.

The polypeptide hormone gastrin was identified nearly a hundred years ago and its role in the regulation of acid secretion is well established. Gastrin also acts as a growth factor and is trophic for the normal gastric oxyntic mucosa. This growth promoting action has led to the extensive investigation of its role in carcinogenesis, in particular colorectal neoplasia. The relationship between gastrin and colorectal adenocarcinoma has been subject to controversy, however the findings from several recent studies have resulted in a clearer understanding of the mechanism of action of gastrin in this is common cancer. The majority of colorectal cancers produce their own gastrin, which may act in an autocrine manner. The tumour cells also express gastrin/CCKB receptors (and/or a combination of isoforms) which mediate the proliferative action. This locally produced gastrin gives rise to a small increase in systemic gastrin levels. Autocrine gastrin may also have a role in tumour development, as expression occurs early in the adenoma-carcinoma sequence. In addition, several studies using animal models have shown that systemic hypergastrinaemia promotes the proliferation of both normal and neoplastic colonic epithelium. Hyperproliferative colonic epithelium in the presence of hypergastrinaemia has been recorded in humans and a well-designed epidemiological study has demonstrated an increased incidence of colorectal cancer. Gastrin is a potential therapeutic target in the treatment of colorectal cancer and several approaches have been assessed. Receptor antagonists and antisecretory agents have been demonstrated to be ineffectual. Novel methods of inhibition, including the use of anti-gastrin antibodies, are currently being evaluated.

Amino Acid Sequence↗
PubMed2000

Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met). A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.

Acetylcholinesterase↗
PubMed2000

Structures of prevention: a safe-sex/safe-injecting audit of Mount Alexander Shire, a methodological pilot.

This paper reports the findings of a methodological pilot project that examined the availability of safe-sex and safe-injecting products in a rural shire in central Victoria. The study mapped the distribution and audited the availability of condoms, water-based lubricants, needles/syringes and needle disposal units to provide baseline data on access to and the availability of products that are directly related to the prevention of the human immunodeficiency virus, sexually transmissible infections and blood-borne viruses. Clear disparities were observed in the availability and pricing of safe sex/safe use materials between regional centres and outlying areas in the Shire. The findings make it clear that an emphasis on overcoming structural impediments to the adoption of safe sex/safe injecting practices is required.

Condoms↗
PubMed2000

Recombinant allergens for diagnosis and therapy of allergic disease.

Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome with use of genetically engineered recombinant allergens. Over the past 10 years, the most important allergens from mites, pollens, animal dander, insects, and foods have been cloned, sequenced, and expressed. In many cases the three-dimensional allergen structure has been determined and B-cell and T-cell epitopes have been mapped. These studies show that allergens have diverse biologic functions (they may be enzymes, enzyme inhibitors, lipocalins, or structural proteins) and that as a rule the allergen function is unrelated to its ability to cause IgE antibody responses. High-level expression systems have been developed to produce recombinant allergens in bacteria, yeast, or insect cells. Recombinant allergens show comparable IgE antibody binding to their natural counterparts (where available) and show excellent reactivity on skin testing and in in vitro diagnostic tests. Cocktails of recombinant allergens can be formulated with predetermined and uniform allergen levels, which could replace natural allergens and result in the development of innovative, patient-based tests for allergy diagnosis. Recombinant allergens also offer the exciting possibility of developing new forms of allergen immunotherapy, including the use of hypoallergens, allergens coupled to IgE suppressive adjuvants, and peptide-based therapies. The production of recombinant allergens as defined molecular entities makes it feasible to consider the possibility of developing prophylactic allergen vaccines. The introduction of recombinant allergens in research and in clinical trials should lead to significant improvements in allergy diagnosis and treatment.

Allergens↗
PubMed2000

When always isn't enough: implications of the late application of condoms for the validity and reliability of self-reported condom use.

Condoms are most effective when they are applied before intercourse. This study assessed the prevalence of 'condom cheats'--instances of condom use in which the condom is put on after initial penetration. As part of a prospective study of safer sexual behaviour, 103 heterosexual tertiary students completed a condom use diary over a period of up to 6 months. Of the 464 condoms used by study participants, 13% were put on after initial penetration. Thirty-eight percent of condom users reported at least one instance of late application of a condom. Late application of condoms is common and places individuals at risk of infection with HIV and many other STDs. The widespread practice of this behaviour casts doubt on the validity of self-reports of condom use. Assessments of risk based on self-reported condom use may lead to underestimates of the risk entailed in heterosexual young adults' condom use behaviour, and may also lead to underestimates of condom efficacy.

Adolescent↗
PubMed2000

Non-Penicillin-Binding protein mediated high-level penicillin and cephalosporin resistance in a Hungarian clone of Streptococcus pneumoniae.

A clone of Hungarian pneumococcal strains has recently been isolated with notably high levels of beta-lactam resistance (penicillin MIC, 16 microg/mL; cefotaxime MIC, 4 microg/mL). The role that each penicillin-binding protein (PBP) plays in the development of resistance in these strains was investigated via transformation of susceptible strain R6 with pbp DNA from resistant strain 3191. Transformation of strain R6 with pbp2X DNA resulted in transformants with penicillin and cefotaxime MICs of 0.06 and 0.25 microg/mL, respectively. Further introduction of pbp2B and 1A DNA increased penicillin MICs to 0.25 and 4 microg/mL, respectively. Transformation of strain R6 with a combination of pbp2X and pbp1A DNA produced R63191/2X/1A strains with an unexpected low cefotaxime MIC of 0.5 microg/mL. This low-level of cefotaxime resistance was surprisingly increased from 0.5 to 2 microg/mL in R63191/2X/2B/1A strains. This suggests the involvement of altered PBP 2B in cefotaxime resistance. Therefore, within this particular setting of resistance, the environmental presence of selectors for altered PBP 2B (penicillin or piperacillin) are required for the development of high-level cefotaxime resistance. The MICs of R63191/X/2B/1A strains never reached the MICs of the donor strain. Full MICs of the donor were eventually reached by transforming R63191/2X/2B with chromosomal3191 DNA. Resultant transformants revealed the introduction of altered PBP 1A, while unaltered PBPs 1B, 2A, and 3 proved that these PBPs were not involved in resistance. A non-PBP resistance determinant has therefore made up the difference in resistance between R63191/2X/2B/1A and donor strain 3191. Beta-lactamase activity and efflux systems have so far been eliminated as causes of resistance. This resistance determinant represents a novel mechanism for beta-lactam resistance in clinical isolates of pneumococci, operates at the highest level of resistance, and remains to be identified.

Aminoacyltransferases↗
PubMed2000

Analysis of penicillin-binding protein lb and 2a genes from Streptococcus pneumoniae.

Fifty clinical isolates (penicillin MICs, 0.03-8 microg/mL) of Streptococcus pneumoniae were randomly selected from hospitals throughout South Africa, together with seven strains isolated in Hungary (penicillin MICs, 16-32 microg/mL). Penicillin-binding protein (pbp) 1b and 2a genes were amplified by PCR, and the purified DNA was digested with HinfI, StyI, and MseI + DdeI restriction enzymes. The fragments were radioactively end-labeled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. A collection of isolates was further selected for sequence analysis of pbp1b and 2a. DNA fingerprint analysis revealed a uniform profile amongst all isolates for both genes. All isolates revealed a maximum of only seven nucleotide substitutions in their pbp1b genes, resulting in a maximum of three amino acid substitutions in PBP 1B. In the case of the pbp2a gene, up to 13 nucleotide substitutions were observed randomly distributed amongst penicillin-susceptible and resistant isolates, revealing a maximum of five amino acid substitutions in PBP 2A. No amino acid substitutions were found to be common amongst all penicillin-resistant isolates. Transformation experiments with pbp1b and 2a genes isolated from two resistant strains (MICs, 4 and 16 microg/mL) failed to transform pneumococcal strains to increased levels of penicillin resistance. These results show that the pbp1b and 2a genes examined here do not display the typical mosaic gene patterns observed in the pbp2x, 2b, and 1a genes of penicillin-resistant pneumococci. In addition, the transformation studies suggest that PBPs 1B and 2A may not play a role in the development of penicillin resistance in some pneumococci.

Amino Acid Sequence↗