Biomedical subjects

A E Johnson

Publications and source records attributed to A E Johnson.

At least 55 records · Page 3Linked to original sources

PubMed1998

Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy.

Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.

Amino Acid Sequence↗

PubMed1998

CCK-8 inhibits ingestive behavior in rats with lateral hypothalamic 6-OHDA lesions.

Male rats were injected with 6-hydroxydopamine in the lateral hypothalamus and tested for ingestive behavior starting on the day after the injection. The rats did not eat food pellets but readily ingested an intraorally infused nutritive solution. If given three daily intraoral infusions, 6-hydroxydopamine-treated rats defended their body weight and were as sensitive to the inhibitory effect of cholecystokinin octapeptide on intake as controls. Dopamine was reduced by 94% in the dorsal striatum five days after the 6-hydroxydopamine injection. Noradrenaline and serotonin were less markedly affected. Thus, while appetitive ingestive behavior is disrupted, consummatory ingestive behavior and body weight regulatory competence are only marginally affected by massive damage to forebrain dopamine neural networks.

Animals↗

PubMed1998

Epimerization via carbon-carbon bond cleavage. L-ribulose-5-phosphate 4-epimerase as a masked class II aldolase.

Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.

Amino Acid Sequence↗

PubMed1998

BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation.

Secretory proteins are cotranslationally translocated across the mammalian ER membrane through an aqueous pore in the translocon while the permeability barrier is maintained by a tight ribosome-membrane junction. The lumenal end of the pore is also blocked early in translocation. Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore. BiP also seals translocons that are assembled but are not engaged in translocation. These ribosome-free translocons have smaller pores (9-15 A diameter versus 40-60 A in functioning translocons) and are generated when ribosomes dissociate from functioning translocons with large pores. BiP therefore maintains the permeability barrier by sealing both nontranslocating and newly targeted translocons.

Adenosine Diphosphate↗

PubMed1998

Differential modulation of dopamine D1-receptor binding and mRNA expression in the basal ganglia by the D1-receptor antagonist, SCH-23390.

Dopamine D1-receptor binding in the basal ganglia is differentially regulated by subtype nonspecific dopamine antagonists such as the antipsychotic, Fluphenazine. The purpose of the present study was to determine the relative contributions of D1 and D2 receptor systems in the regulation of basal ganglia D1-receptor binding. Rats were injected twice daily for 21 days with saline, the D1-receptor antagonist, SCH-23390, the D2-receptor antagonist, Raclopride, or both SCH-23390 and Raclopride. Dopamine D1-receptor levels (as indicated by [125I]SCH-23982 binding) and mRNA expression were measured using receptor autoradiographic and in situ hybridization histochemical techniques. [125I]NCQ-298 binding to D2-receptors was also measured as a positive control for the effects of Raclopride. SCH-23390 administration independently increased [125I]SCH-23982 binding in a region-dependent manner with the greatest increases occurring in the entopeduncular nucleus. SCH-23390 also increased D1-receptor mRNA expression in specific striatal subregions suggesting that increases in binding were related to changes in receptor synthesis. In addition, Raclopride independently enhanced D2 binding with comparable increases observed in extrastriatal regions and increases of a lesser magnitude in the striatum. These data show that the modulation of basal ganglia D1-receptor binding observed in animals treated with nonselective antagonists is due primarily to the blockade of D1-receptors. The differential enhancement in basal ganglia D1 binding observed after D1-receptor blockade may be due to anatomical or phenotypic heterogeneity within the population of striatal D1-receptor synthesizing neurons. Similarly, the differential enhancement in striatal and extrastriatal D2-receptor binding may be due to differences in the regulation of striatal and extrastriatal D2-receptor synthesizing neurons.

Animals↗

PubMed1998

Gender and living alone as determinants of fruit and vegetable consumption among the elderly living at home in urban Nottingham.

Consumption of fruits and vegetables by a sample of 369 elderly people living in Nottingham, England, was analysed in relation to whether or not they were eating five portions a day. Living status was only of significance to men who, if single, consumed 2.66 portions of fruits and vegetables per day compared with an overall mean of 4.1. The salient question is therefore not "Are you living alone?" but " s there a woman in the household?". Those respondents who were older and less educated ate less vegetables and those respondents who had a lower income or social grade ate less fruit. Men were less likely to be able to cook a range of meals, to have had a job that involved cooking or to watch cookery programmes on television. Single men were more likely than single women to say that eating food that was easy to cook and prepare was an important influence on their food choice. Single women on the other hand were more influenced by body image. Finding foods that were the right portion size and easy to open, prepare and cook was more important to single men than married men, as was the amount of money left after paying the bills.

Aged↗

PubMed1998

Measurement of the performance of air cleaners against the particulate element of rosin-based solder flux fume.

A number of commercially available air cleaners designed for use against solder fume have been examined. The fume capture efficiencies have been measured with a tracer gas comprising a neutrally buoyant mixture of sulphur hexafluoride and helium, and the filtration efficiencies have been measured using solder fume produced at a constant concentration, laboratory-generated monodisperse aerosols, and standard aerosols. The extractor volume flow rates of the simpler types of fume collection units, measured by us, differed from those reported by manufacturers. The capture efficiency of these units was good when the soldering iron was less than about 20 cm from the air intake, but the filtration efficiency against particulates and, therefore, the protection offered, was poor. More robust LEV units containing a HEPA filter had a similar capture efficiency, but a filtration efficiency that was well above the standard strictly necessary; such units will give good protection if properly used. The tip-extraction type of system was found to be very effective but the extraction nozzle rapidly became blocked with the rosin in the solder. Subsidiary measurements of vapour filtration efficiency were made. Activated carbon filters in the form of foams, used with the simpler type of cleaner, had negligible filtration efficiency against either particulates or vapours and would, therefore, offer no protection against any hazardous component of the fume. The granular carbon filter in the tip-extraction system was an effective vapour filter.

Aerosols↗

PubMed1998

Riposte to Guest Commentaries on 'Problems associated with randomized controlled clinical trials in breast cancer.

This paper addresses the objections of Professor M. Baum and Mr W. J. Cunliffe to my thesis that the randomized controlled clinical trial is a poor tool for the investigation of the treatment of breast cancer, argued in a discussion paper entitled 'Problems associated with randomized controlled clinical trials in breast cancer' (A.E. Johnson, 1998, Journal of Evaluation in Clinical Practice 4, 119-126). The objections range from those that have a philosophical basis, through those founded on differing concepts of the classification of primary tumours and the nature of the metastatic tumour, to those that question the reliability and usefulness of the clinical evaluation of response to treatment in terms of histological grade and rate of tumour shrinkage. An alternative approach to research through primary systemic therapy with selection of treatment according to predicted tumour behaviour was severely criticized, both on the preceding grounds and because it was assumed that the alternative to randomization is management by anecdote. These objections are examined and evidence in support of reliable and useful clinical measurement of response is presented in some detail. The problems associated with randomization as a technique for the evaluation of treatments, when the intrinsic variability of tumours is very large without the intervention of treatment, remain unsolved.

Breast Neoplasms↗

PubMed1998

Fruit and vegetable consumption in later life.

OBJECTIVE: to assess levels of fruit and vegetable consumption in elderly people, and to examine the socio-economic, physical and psychological factors which influence this consumption. METHODS: a three-phase survey: face to face interviews; self-completed dietary diaries with a food frequency questionnaire; and follow-up face-to-face interviews. PARTICIPANTS: 445 elderly people (aged 65+) randomly selected from general practitioner lists in urban Nottingham and rural Nottinghamshire, Lincolnshire and Leicestershire. RESULTS: the recommended target of five portions of fruit and vegetables a day was achieved by less than half the respondents: 37% of those living in the urban area and 51% of those living in the rural area. Low fruit and vegetable consumption was particularly associated with being male, smoking and having low levels of social engagement. CONCLUSIONS: most elderly people consume less than the recommended levels of fruit and vegetables. Health programmes promoting fruit and vegetable consumption may not be successfully reaching elderly people and need to target those particularly at risk of low consumption.

Aged↗

PubMed1998

Food safety knowledge and practice among elderly people living at home.

OBJECTIVE: To assess the food storage knowledge and practice of elderly people living at home. METHODS: Three phase survey data collection: face to face interviews; dietary diaries with a food frequency questionnaire; and follow up interviews. SETTING: Urban Nottingham. PARTICIPANTS: 809 elderly people (aged 65+) randomly selected from general practitioner lists. MAIN OUTCOME MEASURES: Respondent's refrigerator temperature; knowledge of freezer star rating; understanding of "use by" and "sell by" dates; reported ability to read food product safety labels. RESULTS: From a weighted total of 645 refrigerators measured, 451 (70%) were too warm for the safe storage of food (> or = 6 degrees Celsius). Only 41% of respondents (n = 279) knew the star rating of their freezer. Within a smaller sub-sample knowledge of the "use by" and "sell by" dates was good, but 45% of these respondents reported difficulty reading food labels. The storage of foods at inappropriate temperatures was not independent of socioeconomic or demographic status, and tended to be more likely among the poorer and those not living alone. CONCLUSIONS: Food storage practices among the majority of elderly people interviewed in this study do not meet recommended safety standards to minimise the risk of food poisoning.

Aged↗

PubMed1998

One medicine.

Explore the source record for details and available documents.

Breast↗

PubMed1998

The interaction of physical, psychological, socioeconomic and sociodemographic variables on the Body Mass Index (MINDEX) of the community-dwelling elderly.

Two cohorts of older people 65-74, and 75 or more years of age living at home in Nottingham or in a nearby rural area in the UK were interviewed and anthropometric measurements were taken (n=1037). Bivariate analyses showed significant relationships between mindex and variables previously associated with food choice. Those with a mindex above the 95th percentile were more likely to live in a rural area, and were less likely to smoke than those between the 5th and 95th percentiles. Those with a low mindex, below the 5th percentile, were more likely to smoke than those between the 5th and 95th percentiles (45% compared to 17%), and were also more likely to live in an urban area, to have difficulty carrying shopping bags, not to use a car for shopping, to have a poorer appetite, and to live alone. Those overweight and underweight had lower levels of social engagement and were more likely to be of a lower social class. In multivariate analysis, socioeconomic variables and psychosocial variables were not significantly associated with mindex, when controlling for physical and sociodemographic variables. Advancing age, female gender, smoking and decreased appetite were significantly associated with decreased mindex (Adjusted R2 = .168).

Aged↗

PubMed1998

Early syphilis in an active duty military population and the surrounding civilian community, 1985-1993.

Syphilis among active duty soldiers at Fort Bragg, North Carolina, and the nonmilitary population of Cumberland County was examined during a 9-year period encompassing the most recent nationwide syphilis epidemic. A total of 762 cases of primary and secondary syphilis were recorded between 1985 and 1993, 27% of which occurred in soldiers. The epidemic struck both military and civilian populations simultaneously; epidemic curves in the two populations were parallel, peaking in 1990-1991, with highest annual incidences of 122.6/ 100,000 (military) and 48.0/100,000 (civilian). Individual risk factor data were not available for analysis, but a relationship was observed between primary and secondary syphilis diagnoses in both populations and cocaine arrests in Cumberland County. Our findings provide epidemiological support for a high degree of interplay between the military and the surrounding civilian communities that has significant implications for control of sexually transmitted diseases. Enhanced collaboration between military and civilian public health authorities is essential to the control of syphilis and other sexually transmitted diseases.

Adult↗

PubMed1998

Problems associated with randomized controlled clinical trials in breast cancer.

The domination of the randomized controlled clinical trial in clinical research in breast cancer is questioned in the context of adjuvant therapy. The criteria for selection of cases by TNM classification are shown to be poorly related to tumour behaviour. The factors that determine outcome--the presence or absence of metastases, their size at the time of presentation of the primary cancer and their growth rate--are not measured. Metastases cannot be detected at sizes below about 10 mm in diameter. This represents about 30 doublings of volume or three-quarters of the life span of the tumour. There is no identifiable starting point for a trial. It is possible to estimate growth rates from the rates of shrinkage in response to primary medical treatment. Modelling of the times at which metastases may appear in the clinic from different possible starting sizes reveals that the usual end points of time from primary diagnosis to metastasis or death have an enormous intrinsic variability. Treatment is merely another confounding variable. The randomized controlled clinical trial is shown to be a poor tool for the investigation of adjuvant treatments. An alternative approach, based upon observation of individual tumour response to primary medical treatment, is commended.

Breast Neoplasms↗

PubMed1997

Tissue factor positions and maintains the factor VIIa active site far above the membrane surface even in the absence of the factor VIIa Gla domain. A fluorescence resonance energy transfer study.

Coagulation factor VIIa (fVIIa), a soluble serine protease, exhibits full proteolytic activity only when bound to its cofactor, tissue factor (TF). Both proteins interact with membranes; TF is an integral membrane protein, while fVIIa binds reversibly to phospholipid surfaces via its Gla domain. In this study, we examine the extent to which the location of the fVIIa active site in the fVIIa.TF complex is determined by the fVIIa Gla domain. A fluorescein dye was covalently attached to the active site of fVIIa lacking the Gla domain (Gla domainless fVIIa, GD-fVIIa) via a tripeptide tether to yield fluorescein-D-Phe-Pro-Arg-GD-fVIIa (Fl-FPR-GD-fVIIa). The location of the active site of GD-fVIIa relative to the membrane surface was determined using fluorescence resonance energy transfer between the fluorescein dye in the active site of GD-fVIIa and octadecylrhodamine (OR) at the surface of phospholipid vesicles. As expected, no energy transfer was observed between Fl-FPR-GD-fVIIa and OR in vesicles composed of phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) because the Gla domain is required for the binding of fVIIa to phospholipid. However, when Fl-FPR-GD-fVIIa was titrated with PC or PC/PS vesicles into which purified TF had been reconstituted, energy transfer was observed. Based on the dependence of fluorescence resonance energy transfer on OR density, the average distance of closest approach between fluorescein in the active site of Fl-FPR-GD-fVIIa.TF and OR at the vesicle surface was determined to be 78 A (kappa2 = (2)/(3)). Since this value is nearly the same as that obtained with intact Fl-FPR-fVIIa bound to TF, the presence or absence of the fVIIa Gla domain has only a small effect on the location of the active site in the fVIIa.TF complex. The extracellular domain of tissue factor therefore must be fairly rigid and fixed relative to the surface to position and maintain the fVIIa active site far above the membrane even in the absence of the fVIIa Gla domain.

Binding Sites↗

PubMed1997

Modulation of basal ganglia neurotransmission by the classical antipsychotic fluphenazine is due in part to the blockade of dopamine D1-receptors.

Classical antipsychotics, such as fluphenazine, influence neurotransmission by blocking both dopamine D1- and D2-receptors which in turn results in widespread adaptive changes in the neurochemistry of the basal ganglia. The purpose of the present study was to determine the role of D1-receptors in mediating some of these neurochemical events, including changes in D1- and D2-receptor binding, and the expression of preproenkephalin and glutamic acid decarboxylase mRNAs. For these experiments, rats were given a depot injection of fluphenazine decanoate or injected twice daily for 21 days with the D1-receptor antagonist SCH-23390. An additional group received both fluphenazine and SCH-23390 and controls were given saline. Fluphenazine administration decreased D2-receptor binding throughout the basal ganglia while SCH-23390 was without effect. In contrast to the uniform reduction in D2-receptor binding, fluphenazine altered D1-receptor binding in a region-dependent manner. Region-dependent changes were also observed in animals given SCH-23390 which increased binding in the entopeduncular nucleus and posterior caudate-putamen without affecting other brain regions. Both fluphenazine and SCH-23390 significantly enhanced preproenkephalin and glutamic acid decarboxylase (GAD) mRNA expression in the anterior striatum. Fluphenazine also increased GAD mRNA levels in the entopeduncular nucleus. Together, these results indicate that the attenuation of D1-receptor-mediated neurotransmission modulates a number of clinically relevant neurochemical processes in the basal ganglia.

Analysis of Variance↗

PubMed1997

Protein S alters the active site location of activated protein C above the membrane surface. A fluorescence resonance energy transfer study of topography.

The location of the active site of membrane-bound activated protein C (APC) relative to the phospholipid surface was determined both in the presence and absence of its cofactor, protein S, using fluorescence resonance energy transfer (FRET). APC was chemically modified to create the FRET donor species, Fl-FPR-APC, with a fluorescein dye (Fl) covalently attached to the active site via a D-Phe-Pro-Arg (FPR) tether and located in the active site near S4. FRET was observed when Fl-FPR-APC was titrated in the presence of Ca2+ ions with phosphatidylcholine/phosphatidylserine (4:1) vesicles containing the FRET acceptor, octadecylrhodamine (OR). Assuming a random orientation of transition dipoles (kappa2 = 2/3), the average distance of closest approach between the fluorescein in the active site of the membrane-bound APC and the OR at the membrane surface is 94 A. The same calcium-dependent distance was obtained for both small and large unilamellar vesicles and for vesicles that contained phosphatidylethanolamine. The active site of membrane-bound APC is therefore located far above the phospholipid surface. Upon addition of protein S, the efficiency of Fl-FPR-APC to OR energy transfer increased due to a protein S-dependent rotational and/or translational movement of the APC protease domain relative to the surface. If this movement were solely translational, then the average height of the fluorescein in the membrane-bound APC.protein S complex would be 84 A above the surface. The extent of Fl-FPR-APC to OR energy transfer was unaltered by the addition of thrombin-inactivated protein S. The protein S effect was also specific for APC, since the addition of protein S to similarly-labeled derivatives of factor Xa, factor IXa, or factor VIIa did not alter the locations of their active sites. This direct measurement demonstrates that the binding of the protein S cofactor to its cognate enzyme elicits a relocation of the active site of APC relative to the membrane surface and thereby provides a structural explanation for the recently observed protein S-dependent change in the site of factor Va cleavage by APC.

Animals↗

PubMed1997

Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration.

Portions of the nascent chain are exposed to the lumen, the cytosol, or neither at different stages during the cotranslational integration of a protein into the ER membrane, as shown by compartment-specific collisional quenching of fluorophores incorporated into the polypeptide. The opening or closing of each end of the aqueous translocon pore is tightly controlled and occurs in a sequence that does not compromise the membrane's permeability barrier. Surprisingly, these structural changes at the membrane are effected by the transmembrane segment in the nascent protein from inside the ribosome. Thus, the ribosome, not the translocon, first recognizes the transmembrane segment and triggers long-range structural changes at the translocon that may be involved in shifting its function from translocation to integration.

Animals↗