Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription.

Herpes simplex virus (HSV) immediate early (IE) transcription is known to be stimulated by a structural component of the virion which interacts, either directly or indirectly, with specific regulatory sequences located far upstream from IE messenger RNA 5'-termini. The aim of the work described in this paper is the mapping and identification of the virion component. Cloned HSV DNA fragments derived from various parts of the genome were cotransfected into BHK cells together with chimaeric plasmids which contained the thymidine kinase gene under IE control. Stimulation of thymidine kinase synthesis was elicited by cloned EcoRIi (0.63 to 0.72 map units), BamHIf (0.64 to 0.69) or EcoRIb (0.72 to 0.87). Cloned BamHIf had the same specificity as the virion component, since it stimulated thymidine kinase expression only from chimaeric plasmids which contained functional IE-specific regulatory sequences. The effect of EcoRIb was not confined to plasmids with IE-specific regulatory regions, suggesting a more general stimulatory role for one or more of the polypeptides encoded by this fragment. A subclone containing a 2.7 X 10(3) base-pair fragment of BamHIf (pMC1) was active in the cotransfection assay, and the effect was abolished by an eight base-pair insertion into the middle of this fragment. The only polypeptide known to map entirely within the HSV genome region defined by pMC1 was identified as the major tegument species Vmw65. The results therefore suggest that Vmw65 is the virion component which trans-activates HSV IE transcription.

Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons.

The role of p38 and c-jun-N-terminal kinases 1/2, members of the mitogen-activated protein kinase family, in mediating the toxic effects of human immunodeficiency virus-1 transactivator of transcription (Tat) and gp120 were explored in primary mouse striatal neurons in vitro. Both Tat and gp120 caused significant increases in p38 and c-jun-N-terminal kinase mitogen-activated protein kinase phosphorylation, caspase-3 activity, neurite losses and cell death in striatal neurons. Tat-induced increases in caspase-3 activity were significantly attenuated by an inhibitor of c-jun-N-terminal kinase (anthra[1,9-cd]pyrazol-6(2H)-one), but not by an inhibitor of p38 ([4-(4-fluorophenyl)-2-(4-methylsul-finylphenyl)-5-(4-pyridyl)1 H-imidazole]), mitogen-activated protein kinase. However, despite preventing increases in caspase-3 activity, c-jun-N-terminal kinase inhibition failed to avert Tat-induced neuronal losses suggesting that the reductions in caspase-3 activity were insufficient to prevent cell death caused by Tat. Alternatively, gp120-induced increases in caspase-3 activity, neurite losses and neuronal death were prevented by p38, but not c-jun-N-terminal kinase, mitogen-activated protein kinase inhibition. Our findings suggest that gp120 induces neuronal dysfunction and death through actions at p38 mitogen-activated protein kinase, while Tat kills neurons through actions that are independent of p38 or c-jun-N-terminal kinase mitogen-activated protein kinase, or through the concurrent activation of multiple proapoptotic pathways.

Maternal pregnancy weight gain and cord blood iron status are associated with eosinophilia in infancy.

OBJECTIVE: Allergic disease is multifactorial in origin. Because iron nutrition affects immune responses and maternal pregnancy weight gain impairs fetal iron delivery while increasing fetal demands for growth, the study examined maternal pregnancy weight gain, newborn iron status and an index of atopic disease, infant eosinophilia. STUDY DESIGN: Within a larger prospective study of healthy newborns at risk for developing iron deficiency anemia, umbilical cord iron indicators were compared to infant eosinophil counts. RESULT: Infants who developed eosinophilia exhibited higher cord reticulocyte-enriched zinc protoporphyrin/heme ratio, P<0.05 and fewer cord ferritin values in the highest (best) quartile, P<0.05. If cord ferritin was in the upper three quartiles, the negative predictive value for infant eosinophilia was 90%. High maternal pregnancy weight gain predicted infant eosinophil counts, P<0.04, and contributed to cord ferritin predicting eosinophilia, P<0.003. CONCLUSION: Poor fetal iron status may be an additional risk factor for infant eosinophilia.

Purification and characterization of a human bradykinin binding protein from inflammatory cells.

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

A urinary metabolite ratio that reflects systemic caffeine clearance.

Systemic caffeine clearance and urinary metabolite profiles were determined in 15 subjects with diverse exposure histories to cytochrome P-450 inducers (cigarette smoke) and inhibitors (oral contraceptive steroids). A correlation was observed between caffeine clearance and a urinary ratio based on the molar recovery of paraxanthine 7-demethylation products relative to a paraxanthine 8-hydroxylation product (r = 0.91; P less than 0.001). Analysis of urinary metabolites was undertaken in a larger population to assess the effects of gender, age, oral contraceptives, and smoking on the ratio. No gender differences were observed in either adults or children; children (n = 21) showed a higher (P less than 0.001) mean metabolite ratio than adults (n = 61), oral contraceptive users (n = 9) had lower (P less than 0.05) ratios than women not taking oral contraceptives (n = 30), and smokers (n = 26) had higher (P less than 0.001) ratios than nonsmokers (n = 61). The data indicate that a urinary metabolite ratio based on paraxanthine 7-demethylation/8-hydroxylation products reflects systemic caffeine clearance and likely monitors cytochrome P-450 activity inducible by polycyclic aromatic hydrocarbons.

Copper absorption by remnant small bowel after extensive intestinal resection in the rat.

Rats were subjected to 50% proximal or 50% distal small bowel resection or sham operation. Four weeks later intestinal absorption of copper was measured in vivo using a recirculation technique. In each rat 10 cm in situ segments of duodenum, midgut, and ileum were perfused through the lumen for 2 h with 15 ml of an isotonic solution containing 2 micrograms/ml copper together with 14C-polyethylene glycol as volume marker. Absorption was expressed per g dry mucosa and per cm segment length. After proximal enterectomy absorption of copper per g mucosa decreased in the duodenum and midgut but remained unchanged in the ileum compared to sham-operated controls. However, there were adaptive increases in mucosal mass in all segments. Copper absorption per cm segment length in duodenum and midgut remained unchanged from controls and ileal absorption per cm increased in proportion to mucosal growth. After distal enterectomy absorption of copper per g mucosa remained unchanged in the duodenum but decreased in the midgut and ileum compared to sham-operated controls. Adaptive mucosal growth now occurred only in the midgut and ileum. In the ileum mucosal mass more than doubled. Copper absorption per cm segment length in the duodenum and midgut did not change from controls. However, in the ileum absorption per cm increased above that of controls because of the marked mucosal growth. Therefore, in response to either proximal or distal small bowel resection, the ileum was the site for adaptive copper absorption but adaptive mechanisms differed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotransformation of caffeine by microsomes from human liver. Kinetics and inhibition studies.

The nature of the cytochrome P-450-dependent enzyme reactions giving rise to four primary metabolites of caffeine was investigated using microsomes isolated from livers of human kidney donors. Metabolite formation proceeded at a lower rate than that predicted from in vivo caffeine elimination half-lives, as has been observed in other species using this compound as a substrate in microsomal incubations. Kinetic experiments indicated that the formation of each of the N-demethylated metabolites paraxanthine, theobromine and theophyline was mediated by both a high- and a low-affinity catalytic site over a substrate concentration range from 0.05 mM to 80.0 mM, although only the high-affinity component is likely to be of any importance at normally encountered in vivo caffeine concentrations. 7-Ethoxyresorufin and acetanilide, selective substrates for two polycyclic aromatic hydrocarbon (PAH)-inducible isozymes of cytochrome P-450 in the mouse (P1-450 and P3-450, respectively) were each able to inhibit competitively the formation of caffeine metabolites by human liver microsomes, while caffeine could in turn similarly inhibit the biotransformations of these two compounds. The isozyme-selective P-450 inhibitor alpha-naphthoflavone (ANF) potently inhibited the high-affinity component of caffeine N-demethylations, while 1-phenylimidazole (PI) was a more potent inhibitor of the low-affinity component. The inhibition studies also indicated that the formation of 1,3,7-trimethyluric acid was mediated by both ANF-sensitive and PI-sensitive sites. Taken together, the data support suggestions from in vivo studies that a PAH-inducible isozyme of cytochrome P-450 plays a significant role in the biotransformation of caffeine in man.

Continuous increase in blood lactate concentration during different ramp exercise protocols.

The applicability of a continuous model description of the blood lactate concentration [( La-]) vs. O2 uptake (VO2) relationship was studied in nine healthy male volunteers during three different ramp exercise protocols. The work rate was increased at either 8, 15, or 50 W/min. The continuous model for [La-] = â + b exp(cVO2) was compared statistically with a previously proposed log-log transformation model for the [La-] and VO2 variables. It was found that the mean square error was significantly less for the continuous as opposed to the log-log model (P less than 0.01) by analysis of variance pooled across all three ramp slopes. The mean square errors from the individual ramp slopes were also significantly less for the continuous model by paired t test (P less than 0.05). It was observed that the major contributor to the increased error of the log-log model was at VO2's at or above the intersection point (lactate threshold) of the two linear log-transformed segments. The log-log transformation does not appear to relate to any physiological process. The lactate slope index, taken as the point where the slope of the relationship between [La-] and VO2 (i.e., d[La-]/dVO2) equaled 1, occurred at a mean VO2 of 2.25 and 2.37 l/min for the 15- and 8-W/min ramp slopes, respectively, but at 2.76 l/min for the 50-W/min ramp (P less than 0.05). It is concluded that [La-] increases as a continuous function with respect to VO2 across a wide range of ramp work rate slopes.

Hospital epidemiology of Pseudomonas aeruginosa from patients with cystic fibrosis.

Pseudomonas aeruginosa colonizes the respiratory tract of most older patients with cystic fibrosis. The means by which these bacteria are acquired and the risk for patient-to-patient spread among subjects with cystic fibrosis are poorly understood. We studied the spread of Ps. aeruginosa within a hospital environment. Pseudomonas was rarely recovered from the inanimate environment surrounding patients with cystic fibrosis or from hand or rectal cultures of patients who were colonized in the oropharynx. There was transient cross-colonization with Ps. aeruginosa between patients with cystic fibrosis sharing a hospital room in three of seven pairs studied. In all cases the "new" isolate was recoverable only once and was not found during a 2-year follow-up. Three of four sibling pairs with cystic fibrosis shared the same Ps. aeruginosa serotype(s). The risk of sustained cross-colonization by Ps. aeruginosa between patients with cystic fibrosis appears to be minimal, except under conditions of prolonged close contact.

Detection of canine herpesvirus 1 in a wide range of tissues using the polymerase chain reaction.

Canine herpesvirus 1 (CHV-1), a member of the alphaherpesvirus sub-family, is known to cause fatal infections in litters of puppies and may also be involved in infertility, abortion, and stillbirths in adult dogs. The purpose of this study was to determine the presence of CHV-1 DNA using the polymerase chain reaction (PCR) in twelve key sites that have been associated with latency for the other herpesviruses. A 605 base pair portion of the viral glycoprotein B (gB) gene was amplified using degenerate primers, cloned, and sequenced. Conventional 20 mer primers were designed using this sequence information to amplify a 120 bp fragment of gB situated between the original degenerate primers. The specificity of amplification was confirmed by Southern Blot hybridisation using an internal oligonucleotide probe. DNA was extracted from tissue samples taken from twelve dogs at post mortem and from twenty-four blood samples. Nine out of twelve dogs showed evidence of infection with CHV-1; the tissues most commonly affected were lumbo-sacral ganglia (5/12 dogs), tonsil (5/12), parotid salivary gland (4/9), and liver (4/9). No positive results were detected within the twenty-four blood samples. These results indicate that exposure to CHV-1 may be much more common than previously suggested.

Tonometry to estimate intestinal perfusion in newborn piglets.

AIM: To determine the correlation between gastric intramucosal pH and superior mesenteric artery (SMA) flow in newborn piglets. METHODS: Fourteen newborn piglets were randomly assigned to either a control or to an epinephrine group which received 0,1,2,4,0 microg/kg/min of epinephrine for 60 minutes, each dose. Gastric tonometry was performed, SMA flow was measured, and intramucosal pH and the ratio of tonometer pCO(2) over arterial pCO(2) (rCO(2)) were calculated. RESULTS: Intramucosal pH decreased over time in both groups, but tended to be lower in the epinephrine group. With increasing dose of epinephrine, SMA flow decreased; this in turn increased rCO(2) (p = 0.04) with a tendency to decrease intramucosal pH (p = 0.06). CONCLUSIONS: Gastric tonometry may be useful in human neonates to evaluate gut ischaemia.

An overview of municipal state of the environment reporting in Canada.

State of the Environment (SOE) reporting is an emerging municipal management tool designed to monitor and increase awareness of the current status, changes and trends in the condition of the local environment. A multifaceted investigation was undertaken to examine municipal SOE reporting in Canada and to identify barriers to its widespread implementation. Highlights of the case study and survey components are summarized and a conceptual model for municipal SOE reporting is proposed. Overall, the study revealed considerable interest in environmental reporting, however, the lack of common municipal indicators, organizing frameworks and environmental data accessible at the local level impedes its widespread implementation. Future needs to enhance SOE reporting include: development of common municipal indicators, including environmental sustainability indicators; enhancement of the compatibility of SOE reporting frameworks across municipal, provincial and national levels; and re-examination of the data collected by diverse levels of government to optimize their utilization at the local level.

A program to reduce pesticide spraying in the indoor environment: evaluation of the 'Roach Coach' project.

PURPOSE: To assess the effectiveness of a pilot integrated pest management (IPM) program in controlling cockroaches in an apartment complex, without pesticide sprays. METHODS: A brief educational session and booklet were provided to tenants. Non-spray pest control methods were promoted. A telephone questionnaire was administered at pre- and post-test to assess knowledge, attitudes and practices of tenants. Cockroach counts were determined at pre- and post-test. The type and frequency of pesticide treatments were monitored prior to and during the 8-month IPM demonstration period. RESULTS: Overall, the knowledge, attitudes and practices of tenants improved after the IPM intervention. There was a significant shift in treatment type away from spraying towards more paste/gel treatments. Cockroach levels tended to be lower after IPM compared with before. CONCLUSION: A brief educational session and booklet can influence building residents to accept and comply with an IPM program. This program can be effective in controlling cockroaches without pesticide sprays.

Effectiveness of public health interventions in food safety: a systematic review.

PURPOSE: To summarize evidence on the effectiveness of public health interventions regarding food safety at restaurants, institutions, homes and other community-based settings. METHOD: This systematic review of published and unpublished studies involved a comprehensive literature search, screening for relevance, quality assessment of relevant studies, data extraction and synthesis. RESULTS: The interventions identified in 15 studies included in this review were grouped into three categories: inspections, food handler training, and community-based education. The evidence suggests that: routine inspection (at least once per year) of food service premises is effective in reducing the risk of foodborne illness; food handler training can improve the knowledge and practices of food handlers; and selected community-based education programs can increase public knowledge of food safety. DISCUSSION: There is some evidence for the effectiveness of multiple public health interventions on food safety. Future research needs include evaluation of HACCP and community-based education programs.

Preliminary development of the Body Image Affect Scale.

A new body-image questionnaire was developed to measure the affect associated with a negative body image. Responses showed a Cronbach coefficient alpha of .84 and a negative correlation of -.21 with using humor in times of stress.