Pharmacology of capsaicin-, anandamide-, and N-arachidonoyl-dopamine-evoked cell death in a homogeneous transient receptor potential vanilloid subtype 1 receptor population.

BACKGROUND: Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a primary pain-sensing relay at peripheral sensory nerve endings and is also widespread in the brain, where it is implicated in neurodegeneration. Previous studies of TRPV1 neurotoxicity have utilized heterogeneous receptor populations, non-selective ligands, or non-neuronal cell types. Here, we explored the pharmacology of TRPV1-induced cytotoxicity in a homogeneous, neurone-like cellular environment. METHODS: Cell death was examined in a human neurone-like cell line, stably expressing recombinant human TRPV1. Cytotoxicity was quantified in terms of nuclear morphology and mitochondrial complex II activity. Immunocytochemical markers of apoptotic cell death were also examined. RESULTS: The TRPV1-selective agonist capsaicin, and the endovanilloids anandamide and N-arachidonoyl-dopamine (NADA), induced TRPV1-dependent delayed cell death in a concentration- and time-dependent manner. Capsaicin exposure time was significantly correlated with potency (r(2)=0.91, P=0.01). Release of cytochrome c from mitochondria, activation of caspase-3, and condensed nuclear chromatin were evident 6 h after capsaicin exposure, but cytotoxicity was unaffected by a pan-caspase inhibitor (zVAD-fmk, 50 microM). CONCLUSIONS: We conclude that capsaicin, anandamide, and NADA can initiate TRPV1-dependent delayed cell death in neurone-like cells. This is an apoptosis-like process, but independent of caspase activity.

Stability, fatty acid composition and sensory properties of the M. Longissimus muscle from beef steers grazing either chicory/ryegrass or ryegrass.

Research has shown both production and health benefits for the use of chicory (Cichorium intybus) within ruminant diets. Despite this, little was known about the effects of this forage, containing differing fatty acid profiles and secondary plant compounds compared with ryegrass, on beef stability, fatty acid composition or sensory properties. An experiment was conducted to investigate whether the inclusion of chicory in the diet of grazing beef steers would alter these three properties in the M. Longissimus muscle when compared with beef steers grazing perennial ryegrass (Lolium perenne). Triplicate 2 ha plots were established with a chicory (cv. Puna II)/perennial ryegrass mix or a perennial ryegrass control. A core group of 36 Belgian Blue - cross steers were used within a 2-year beef finishing experiment (n=6/replicate plot). In the 2nd grazing year, steers were slaughtered as they reached a target fat class of 3. Muscle pH was checked 2 and 48 h post-slaughter. A section of the hindloin joint containing the M. Longissimus lumborum muscle was removed and a 20 mm-thick steak was cut and muscle samples were taken for analysis of vitamin E and fatty acid analysis. The remaining section of the loin was vacuum packed in modified atmosphere packs and subjected to simulated retail display. A section of the conditioned loin was used for sensory analysis. Data on pH, vitamin E concentration and colour stability in a simulated retail display showed there were no effects of including chicory in the diet of grazing beef steers on meat stability. There were also no differences found in the fatty acid composition or the overall eating quality of the steaks from the two treatments. In conclusion, there were no substantive effects of including chicory in the swards of grazing beef cattle on meat stability, fatty acid composition or sensory properties of the M. Longissimus muscle when compared with beef steers grazing ryegrass-only swards.

Geminivirus genes and vectors.

The geminiviruses are very small plant viruses with circular single-stranded DNA genomes. Recent advances have identified genes involved in replication, spread of virus or DNA in the plant, and insect transmission. Gene replacement experiments suggest that useful plant gene expression vectors can be constructed from these viruses.

In vivo accuracy of multispectral magnetic resonance imaging for identifying lipid-rich necrotic cores and intraplaque hemorrhage in advanced human carotid plaques.

BACKGROUND: High-resolution MRI has been shown to be capable of identifying plaque constituents, such as the necrotic core and intraplaque hemorrhage, in human carotid atherosclerosis. The purpose of this study was to evaluate differential contrast-weighted images, specifically a multispectral MR technique, to improve the accuracy of identifying the lipid-rich necrotic core and acute intraplaque hemorrhage in vivo. METHODS AND RESULTS: Eighteen patients scheduled for carotid endarterectomy underwent a preoperative carotid MRI examination in a 1.5-T GE Signa scanner using a protocol that generated 4 contrast weightings (T1, T2, proton density, and 3D time of flight). MR images of the vessel wall were examined for the presence of a lipid-rich necrotic core and/or intraplaque hemorrhage. Ninety cross sections were compared with matched histological sections of the excised specimen in a double-blinded fashion. Overall accuracy (95% CI) of multispectral MRI was 87% (80% to 94%), sensitivity was 85% (78% to 92%), and specificity was 92% (86% to 98%). There was good agreement between MRI and histological findings, with a value of kappa=0.69 (0.53 to 0.85). CONCLUSIONS: Multispectral MRI can identify the lipid-rich necrotic core in human carotid atherosclerosis in vivo with high sensitivity and specificity. This MRI technique provides a noninvasive tool to study the pathogenesis and natural history of carotid atherosclerosis. Furthermore, it will permit a direct assessment of the effect of pharmacological therapy, such as aggressive lipid lowering, on plaque lipid composition.

Structure of tobraviral particles: a model suggested from sequence conservation in tobraviral and tobamoviral coat proteins.

Comparisons of the coat protein sequences of four tobraviruses with those of seven tobamoviruses indicate that these proteins share a common evolutionary origin. Numerous amino acids for which specific functions have been identified in the molecular structure of the tobacco mosaic virus vulgare protein have identical or closely similar counterparts among the tobraviral proteins. These include those with roles in the hydrophobic core of the protein, those that contribute to the RNA binding site and those involved in the control of virus assembly. We suggest a model for the structure of the tobraviral particle that not only offers an explanation for the greater diameter of the tobraviral particle but also confirms an early suggestion for RNA placement within this particle.

Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing.

Virus resistance in Nicotiana benthamiana plants containing a translatable Pea early browning virus (PEBV) 54K sequence from the 201K replicase gene has been reported previously. Resistant plants contain multiple transgene copies divided between two loci. Analysis of a genetic series containing the two loci in separate homozygous or heterozygous condition suggest that only one of the loci is necessary to induce the resistance. The resistance observed in R2 and R3 generations of lines containing both transgene loci in homozygous condition became less consistent in R4 and R5 generations. This inversely correlated with steady-state transgene transcript levels of the segregating populations. The use of recombinant Potato virus X vectors carrying PEBV 54K sequences showed that the resistance is based upon posttranscriptional gene silencing, is non-strand specific, and recognizes 3' located sequences within the PEBV 54K sequence.

Discrete regions of the sensor protein virA determine the strain-specific ability of Agrobacterium to agroinfect maize.

The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection. In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection. The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection. The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348,to agroinfection competence. The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6. In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background. The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined.

Cell-cycle, phase-specific activation of Maize streak virus promoters.

It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize streak virus (MSV) in transgenic maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing "long and short" C-sense promoter sequences upstream of the GUS (beta-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas the C-sense promoter sequences produced two peaks of activity in the S and G2 cell-cycle phases.

Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA.

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome.

Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.

The discovery of capsazepine, the first competitive antagonist of the sensory neuron excitants capsaicin and resiniferatoxin.

Capsaicin and resiniferatoxin are natural products which act specifically on a subset of primary afferent sensory neurons to open a novel cation-selective ion channel in the plasma membrane. These sensory neurons are involved in nociception, and so, these agents are targets for the design of a novel class of analgesics. Although synthetic agonists at the capsaicin receptor have been described previously, competitive antagonists at this receptor would be interesting and novel pharmacological agents. Structure-activity relationships for capsaicin agonists have previously been rationalized, by ourselves and others, by dividing the capsaicin molecule into three regions--the A (aromatic ring)-, B (amide bond)-, and C (hydrophobic side chain)-regions. In this study, the effects on biological activity of conformational constraint of the A-region with respect to the B-region are discussed. Conformational constraint was achieved by the introduction of saturated ring systems of different sizes. The resulting compounds provided agonists of comparable potency to unconstrained analogues as well as a moderately potent antagonist, capsazepine. This compound is the first competitive antagonist of capsaicin and resiniferatoxin to be described and is active in various systems, in vitro and in vivo. It has recently attracted considerable interest as a tool for dissecting the mechanisms by which capsaicin analogues evoke their effects. NMR spectroscopy and X-ray crystallography experiments, as well as molecular modeling techniques, were used to study the conformational behavior of a representative constrained agonist and antagonist. The conformation of the saturated ring contraint in the two cases was found to differ markedly, dramatically affecting the relative disposition of the A-ring and B-region pharmacophores. In agonist structures, the A- and B-regions were virtually coplanar in contrast to those in the antagonist, in which they were approximately orthogonal. A rationale for agonist and antagonist activity at the capsaicin receptor is proposed, based on the consideration of these conformational differences.

Major risk factors for cardiovascular disease mortality in adults: results from the Nutrition Canada Survey cohort.

The cardiovascular mortality experience of over 7000 Canadians ages 35-79 years free of self-reported heart disease or stroke who participated in the Nutrition Canada survey is presented. The effects of various risk factors on cardiovascular disease mortality were assessed using multivariate Poisson regression analyses. Factors associated with a significantly increased risk of dying included cigarette smoking, hypertension, diabetes and, for women, serum cholesterol. Relative risks were similar for those ages 35-64 years compared to those 65-79 years for diabetes but were higher among those 35-64 years for cigarette smoking, diastolic hypertension, obesity and serum cholesterol (females only). Individuals drinking three or more drinks daily had a relative risk of 3.18 for stroke. Population attributable risks for smoking, hypertension, elevated serum cholesterol and diabetes, respectively, were 47%, 21%, 7% and 8% for men and 10%, 21% 18% and 16% for women.

The utilisation of peripherally-administered intravenous nutrient solutions.

The utilisation of three peripherally-administered intravenous nutritional regimens has been evaluated in 42 patients on the first four days following surgery for colorectal cancer. A standard dextrose-saline (DS) regimen (n = 16) has been compared with an amino-acid (AA) regimen (n = 12) and a regimen consisting of glucose, amino-acid and fat (GAF) (n = 14). Fat and carbohydrate oxidation was calculated pre- and post-operatively using indirect calorimetry. Patients receiving AA showed a fall in carbohydrate oxidation (p < 0.01) and a rise in fat oxidation (p < 0.05) post-operatively, whereas no significant changes in fat and carbohydrate oxidation occurred in the DS and GAF groups. Cumulative nitrogen balance (NB) for the first four post-operative days was significantly better (p < 0.01) in the AA group (-10.3 +/- 3.8 g; mean +/- s.e.m.) than in the DS group (-25.3 +/- 3.1 g), due to an improved NB in the AA group on the first and second days only. Cumulative NB in the GAF group (+7.7 +/- 2.3 g) was significantly better (p < 0.01) than in the other two groups. Where the provision of peripheral intravenous nutritional support is desired, the use of a combination of glucose, amino-acid and fat is recommended.

Comparison of various predictive formulae for the estimation of resting energy expenditure.

The accuracy of the Harris-Benedict (HB), Kleiber and Robertson-Reid predictive formulae and the Fleisch tables in estimating resting energy expenditure (REE) has been assessed in 114 cancer patients and 54 patients with nonmalignant illness. The effects of weight status and disease on the predictive ability of the formulae were assessed by comparison of the estimated REE value with that measured by indirect calorimetry. Underestimation of measured REE by greater than 10% occurred in 6-62% of patients, depending on the formula used. Weight loss did not affect predictive ability, whereas the presence of cancer resulted in inaccurate prediction of REE in female patients. Using a multiple comparison procedure, the Fleisch tables were the most accurate in all groups, while the HB formula was least accurate in male patients, irrespective of weight status or disease status. The use of predictive formulae and tables are inappropriate for the accurate estimation of REE in individual patients.

Narcissus mosaic virus: a potexvirus with an encapsidated subgenomic messenger RNA for coat protein.

Narcissus-mosaic-virus RNA is translated into a coat-protein-sized product in wheat-germ cell-free extracts. This protein was shown to be very similar to authentic coat protein by partial proteolysis in SDS/polyacrylamide-gel electrophoresis, and by serology. Fractionation of the RNA revealed a small RNA molecule of approx. 840 nucleotides, which alone coded for the coat protein. This subgenomic RNA was found to be encapsidated in a short virus particle.

An encapsidated, subgenomic messenger RNA encodes the coat protein of carnation mottle virus.

The translation strategy of carnation mottle virus (CarMV) in vitro has been generally assumed to involve internal initiation events on full-length, genomic RNA (4.3 kb). We suggest that this is, at least in part, incorrect. Encapsidated RNA, fractionated on denaturing sucrose gradients, or total RNA from CarMV-infected leaves, fractionated under non-denaturing conditions, was translated in an mRNA-dependent rabbit reticulocyte cell-free system. Evidence for subgenomic RNAs which encode a polypeptide of Mr 38 000 was found. This product was shown to be related to authentic CarMV coat protein by partial proteolysis with alpha-chymotrypsin and SDS/polyacrylamide-gel electrophoresis.

The effects of an anabolic steroid and peripherally administered intravenous nutrition in the early postoperative period.

Sixty patients undergoing colorectal surgery for malignancy were randomized to receive the anabolic steroid stanozolol (n = 30) or to a control group (n = 30). Patients were further randomized to receive on the first 4 postoperative days a) a standard dextrose-saline regimen (DS), b) an amino acid regimen (AA), or c) a glucose-amino acid-fat regimen (GAF) via a peripheral vein. Fat and carbohydrate oxidation rates were calculated pre- and postoperatively using indirect calorimetry. Postoperative nitrogen balance (NB) in patients receiving amino acids was significantly improved (p less than 0.02) by the administration of stanozolol. Fat and carbohydrate oxidation rates were not significantly affected by stanozolol. Patients in the stanozolol and control AA groups showed a fall in carbohydrate oxidation (p less than 0.01) and a rise in fat oxidation (p less than 0.05) postoperatively, whereas no significant changes in fat and carbohydrate oxidation occurred in the two DS and two GAF groups. Cumulative NB for the first 4 postoperative days was significantly better (p less than 0.01) in the two AA groups than in the two DS groups, due to an improved NB in the two AA groups on the 1st and 2nd days only. Cumulative NB in the two GAF groups was significantly better (p less than 0.01) than in all the other groups. This study shows that stanozolol improves postoperative NB in patients receiving amino acids alone, whereas the provision of a more complete nutritional regimen containing glucose, amino acids, and fat results in a positive NB unaffected by stanozolol.

Cardiovascular disease mortality trends and related risk factors in Canada.

Large declines have occurred in cardiovascular disease mortality in both sexes in Canada during the past two decades. However, there are many countries with substantially lower rates at the present time. Ischemic heart disease mortality accounts for about 60% of cardiovascular disease mortality. Cardiovascular disease rates have declined progressively since the mid-1960s in males 35 to 64 and 65 or more years of age, as well as in females in the latter age group. In younger females, 35 to 64 years of age, a slowly progressive reduction in rates started as early as 1930. Trends for stroke mortality also reveal similar long term reductions in rates since 1930 in both sexes. Relative risks for smoking, hypertension, elevated serum cholesterol and diabetes were analyzed. Almost one-third of the cardiovascular deaths in males were attributable to smoking. Population attributable risks for the four risk factors together were 53% for both sexes. Attention is drawn to the increased risks for persons in lower socioeconomic groups and those with low standards of education and the importance of recognition of these factors in intervention programs.

Histological and bacteriological studies of burn wounds treated with boiled potato peel dressings.

Histological and clinical studies have been made on comparable burn wounds covered with either boiled potato peels affixed to gauze bandages or gauze dressings alone; both dressings were applied over a thin layer of 5 per cent silver sulphadiazine. Compared with treatment with plain gauze dressings, the application of the potato peel dressing reduced or eliminated dessication, permitted the survival of superficial skin cells and hastened epithelial regeneration. Bacteriological studies showed that the potato peels had no intrinsic antibacterial activity, the wounds beneath both dressings showing either no growth or, on most occasions, the same bacterial species. The easy availability of potato peels and gauze bandages on to which they can be affixed, the simplicity of the preparation of this dressing, the ease of sterilization and its low cost of production make this the dressing of choice for burn wounds in our developing country.

Suicide in Canada: an epidemiological assessment.

Suicide rates in Canada rapidly increased during the 1960s and 1970s. More recent analysis of these trends indicates that in males suicide rates have stabilized and in females a notable decrease has been identified. The greatest changes in suicide rates have occurred among the youngest age groups (15 to 19), while little change has occurred in suicide mortality rates for males aged 50 years and over. The age-specific death rates in 1986 are uniformly distributed in males above age 20, while in females an inverted "U" curve is demonstrated with the peak at age 45-50. Males continue to have higher rates and the difference between males and females is expanding. A birth cohort analysis indicates that the contribution of the birth cohort to explaining suicide rates has diminished and been replaced by a more recent period effect. Suicide remains the second most important cause of death of persons between 15 and 34 years of age. Provincial variation is discussed through geographic variation, cause-specific rankings and potential years of life lost. In contrast to national trends, suicide mortality in Alberta, Quebec and New Brunswick continues to increase. An atlas is provided to display Canadian census divisions that exhibit elevated rates of suicide.