The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury.

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14)-prostaglandin J2 (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.

Personality and parenting style in parents of adolescents.

Since parental personality traits are assumed to play a role in parenting behaviors, the current study examined the relation between parental personality and parenting style among 688 Dutch parents of adolescents in the SMILE study. The study assessed Big Five personality traits and derived parenting styles (authoritative, authoritarian, indulgent, and uninvolved) from scores on the underlying dimensions of support and strict control. Regression analyses were used to determine which personality traits were associated with parenting dimensions and styles. As regards dimensions, the two aspects of personality reflecting interpersonal interactions (extraversion and agreeableness) were related to supportiveness. Emotional stability was associated with lower strict control. As regards parenting styles, extraverted, agreeable, and less emotionally stable individuals were most likely to be authoritative parents. Conscientiousness and openness did not relate to general parenting, but might be associated with more content-specific acts of parenting.

Bi-directional relations between anti-smoking parenting practices and adolescent smoking in a Dutch sample.

OBJECTIVE: Parenting is generally regarded a determinant of adolescent behavior, whereas the reverse is seldom considered. Reported effects of anti-smoking parenting practices on adolescent smoking are inconsistent. Cross-sectional results may have been misinterpreted and child effects have been overlooked. The main goal of this study was to explain previous inconsistent effects of anti-smoking parenting practices, by examining bi-directional relations between parenting and adolescent smoking. DESIGN AND MAIN OUTCOME MEASURES: Bi-directional relations were studied using a cross-lagged model where anti-smoking house rules, communication about smoking, and adolescent smoking were assessed at three subsequent years. RESULTS: The most prominent finding was that adolescent smoking behavior was a stronger predictor of parenting than vice versa. Anti-smoking house rules decreased as a result of adolescent smoking behavior, while communication increased. The reduction in house rules was more pronounced if parents smoked, while the increase in communication was greater for non-smoking parents. Results were independent of adolescent sex. CONCLUSION: Further research is needed to establish which aspects of parenting can be effective in deterring adolescent smoking. This study emphasizes the need for caution in interpreting cross-sectional research findings relating parenting to adolescent smoking.

Is parenting style a context for smoking-specific parenting practices?

This study examined whether global parenting style can be regarded as a context in which smoking-specific parenting practices relate to adolescent smoking cognitions and behaviors. Data were gathered through self-administered questionnaires from 482 adolescents aged 12-19 years, who participated in the Study of Medical Information and Lifestyles in Eindhoven (SMILE). We assessed parenting style dimensions (support, strict control, psychological control), smoking-specific parenting practices (parent-child communication about smoking, anti-smoking house rules, availability of tobacco products, non-smoking agreement), smoking-related cognitions according to the I-Change Model (attitude, social norm, self-efficacy, intention), and smoking behavior. Structural equation models were computed and compared for adolescents in different parenting climates. Results showed that communication and availability were related to adolescents' attitude towards smoking. Availability was additionally associated with reduced self-efficacy to refrain from smoking. Attitude and self-efficacy were subsequently related to intention to smoke, which in turn was related to smoking behavior. No direct relations were found between anti-smoking parenting practices and adolescent smoking behavior. These results were not dependent on the parenting climate. Parenting style thus did not serve as a context for smoking-specific parenting practices, indicating that these facets of parenting operate independently, and that anti-smoking parenting practices may be effective regardless of parenting climate.

Are anti-smoking parenting practices related to adolescent smoking cognitions and behavior?

The aim of this study was to explain the effects of anti-smoking parenting practices on adolescent smoking cognitions and behavior by showing the mediating effects of cognitions. Data were gathered among Dutch high school students in the control condition of the European Smoking prevention Framework Approach (ESFA). Anti-smoking parenting practices were measured by parental reactions to smoking, house rules, and frequency and content of communication about smoking. Attitudes, perceived social influences and self-efficacy made up for smoking cognitions. Additionally, intention to smoke was measured. Relations between practices and cognitions were mostly significant. While some practices were associated with less smoking (communication about health risks of smoking, health risks of breathing in smoke, addictive qualities of smoking and attention for smoking in school), others were related to increased chances of smoking (rewards for not smoking, frequency of communication about smoking, communication about being allowed to smoke, price of cigarettes and friends smoking). The effects of parenting hardly varied by parental smoking status or adolescent gender. Several practices operated through cognitions, which was more pronounced in older adolescents. Counter-productive effects of practices and the few effects in the longitudinal analyses indicate that the order in which parents and adolescents influence each other should be examined more closely.

Excitatory amino acid concentrations in ventricular cerebrospinal fluid after severe traumatic brain injury in infants and children: the role of child abuse.

BACKGROUND: Excitotoxicity is an important mechanism in secondary neuronal injury after traumatic brain injury (TBI). Excitatory amino acids (EAAs) are increased in cerebrospinal fluid (CSF) in adults after TBI; however, studies in pediatric head trauma are lacking. We hypothesized that CSF glutamate, aspartate, and glycine would be increased after TBI in children and that these increases would be associated with age, child abuse, poor outcome, and cerebral ischemia. METHODS: EAAs were measured in 66 CSF samples from 18 children after severe TBI. Control samples were obtained from 19 children who received lumbar punctures to rule out meningitis. RESULTS: Peak and mean CSF glycine and peak CSF glutamate levels were increased versus control values. Subgroups of patients with TBI were compared by using univariate regression analysis. Massive increases in CSF glutamate were found in children <4 years old and in child abuse victims. Increased CSF glutamate and glycine were associated with poor outcome. A trend toward an association between high glutamate concentration and ischemic blood flow was observed. CONCLUSIONS: CSF EAAs are increased in infants and children with severe TBI. Young age and child abuse were associated with extremely high CSF glutamate concentrations after TBI. A possible role for excitotoxicity after pediatric TBI is supported.

Vaccination against coccidiosis: host strain-dependent evocation of protective and suppressive subsets of murine lymphocytes.

BALB/c mice are normally more resistant than C57BL/6 (B6) mice to infection with Eimeria vermiformis, but these phenotypes can be reversed by oral or parenteral vaccination with a crude antigen prepared from the parasite. Treatment of mice with antibodies specific for CD4+ or CD8+ T cells showed that the increased susceptibility of vaccinated BALB/c mice was associated with the presence of CD4+ T cells. This finding was confirmed when the recipients of CD4+ T cells selected from the mesenteric lymph nodes (MLN) of vaccinated BALB/c mice produced more oocysts after challenge than the recipients of a similar population of cells from sham-vaccinated mice. The residual population of cells (presumably enriched for CD8+ T cells, 'CD8+'), on the other hand, conferred some protection and, in B6 mice, the findings were reversed. Thus, vaccination induced suppressive or protective CD4+ cells and protective or suppressive 'CD8+' cells, depending upon the normal resistance/susceptibility phenotype of the host. Examinations of the isotypes (IgG1, IgG2a) of specific serum antibodies, and of the levels of IFN-gamma and IL-5 cytokines released by MLN cells stimulated ex vivo, did not allow any further characterization of the mechanisms involved.

Opioid neurotoxicity: fentanyl-induced exacerbation of cerebral ischemia in rats.

We tested the hypothesis that fentanyl would worsen ischemia-induced brain damage. In two sequential protocols forty rats were physiologically monitored and controlled. In protocol 1, rats were randomized (n=10/group) to 30 min of control (N2O plus 0.4% halothane), low dose fentanyl (loading dose [LD] 50 micrograms kg-1, maintenance dose [MD] 2 micrograms kg-1 min-1), or high-dose fentanyl (LD 800 micrograms kg-1, MD 32 micrograms kg-1 min-1). After 15 min of fentanyl or sham infusion trimethaphan 0.5 mg was given i.v. and 3 min later bilateral carotid artery occlusion and blood withdrawal-induced hypotension were maintained for 12 min. At 18 h postischemia rats underwent cerebral perfusion fixation. Brain areas were graded from 0 (normal) to 5. In addition to analysis of specific regions, neuropathologic scores were also summated over all brain regions and analyzed to compute a summed neuropathologic score. In protocol 2, five control and five high-dose fentanyl rats were treated identically except that post-ischemic oxygenation was maintained for 6 h and cerebral perfusion-fixation was performed 6 h post-ischemia. Only the caudate/putamen was examined in protocol 2. Fentanyl worsened lesions in both fentanyl groups' summed neuropathologic scores (P=0.002) in protocol 1 and specifically, in the caudate/putamen (P<0.01) in both protocols. Fentanyl in both high and low doses can exacerbate incomplete forebrain ischemia in rats.

Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia.

The inducible isoform of the enzyme cyclooxygenase-2 (COX2) is an immediate early gene induced by synaptic activity in the brain. COX2 activity is an important mediator of inflammation, but it is not known whether COX2 activity is pathogenic in brain. To study the role of COX2 activity in ischemic injury in brain, expression of COX2 mRNA and protein and the effect of treatment with a COX2 inhibitor on neuronal survival in a rat model of global ischemia were determined. Expression of both COX2 mRNA and protein was increased after ischemia in CA1 hippocampal neurons before their death. There was increased survival of CA1 neurons in rats treated with the COX2-selective inhibitor SC58125 [1-[(4-methylsulfonyl) phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole] before or after global ischemia compared with vehicle controls. Furthermore, hippocampal prostaglandin E2 concentrations 24 h after global ischemia were decreased in drug-treated animals compared with vehicle-treated controls. These results suggest that COX2 activity contributes to CA1 neuronal death after global ischemia.

Oral vaccination against coccidiosis: responses in strains of mice that differ in susceptibility to infection with Eimeria vermiformis.

Four strains of mice with different susceptibilities to Eimeria vermiformis were orally dosed with a crude antigen prepared from sporulated oocysts of the parasite, with or without cholera toxin as adjuvant. The effect on subsequent challenge infections depended on the resistance and susceptibility phenotypes of the host: oocyst production was reduced in susceptible C57BL/6 and NIH mice but increased in resistant BALB/c and C3H mice. Despite this contrast, no fundamental differences were detected between the immune responses of BALB/c and C57BL/6 mice, either to vaccination or after superimposed infection, but the suppressing and enhancing effects of vaccination were transmissible to naive recipients via suspensions of mesenteric lymph node cells. The results obtained are compared with those previously reported for parenterally immunized BALB/c and C57BL/6 mice.

Hederagenin glycosides from the flowers of Cephalaria transsylvanica.

Three new triterpenoidal saponins, 3-O-[beta-D-glucopyranosyl(1-->4)-beta-D-Glucopyranosyl(1-->3)-alpha-L- rhamnopyranosyl (1-->4)-beta-D-xylopyranosyl]-hederagenin 28-O-[beta-D-glucopyranosyl (1-->4)-beta-D-glucopyranosyl] ester (1), 3-O-[beta-D-glucopyranosyl (1-->3)-alpha-L-rhamnopyranosyl(1-->2)-beta-D-xylopyranosyl]-hederage nin 28-O-[beta-D-glucopyranosyl(1-->4)-beta-D-glucopyranosyl] ester (2), and 3-O-[alpha-L-rhamnopyranosyl(1-->4)-beta-D-xylopyranosyl]-hederagenin (3) were isolated from the flowers of Cephalaria transsylvanica. Two new prosapogenins, 3-O-[beta-D-glucopyranosyl (1-->4)-beta-D-glucopyranosyl(1-->3)-alpha-L-rhamnopyranosyl(1-->4)- beta-D-xylopyranosyl]-hederagenin (4) and 3-O-[beta-D-glucopyranosyl (1-->3)-alpha-L-rhamnopyranosyl(1-->2)-beta-D-xylopyranosyl]-hederage nin (5) were also isolated after cleavage of the ester-glycosidic linkage of the bisdesmosidic compounds. Structure elucidation was carried out chemically and spectroscopically.

Opioid neurotoxicity: fentanyl dose-response effects in rats.

Opioids, when administered in large doses, produce brain damage, primarily in the limbic system and association areas in rats. This investigation examined the relationship between opioid dose and severity and frequency of brain damage in rats. Forty male Sprague-Dawley rats were anesthetized with halothane/N2O and underwent tracheal intubation, mechanical ventilation, arterial/venous cannulation, and insertion of a rectal temperature probe and biparietal electroencephalogram electrodes. After surgery, halothane was discontinued and O2/N2O 30%/70% was administered for 1 h. Rats were then randomly assigned to one of eight groups. The control group received a loading dose (LD) of 4 mL/kg of 0.9% normal saline solution (NSS) and a maintenance dose (MD) of 4 mL.kg-1.h-1 NSS. The other groups were given fentanyl lypophilized and reconstituted in NSS with the LD ranging from 50 to 3200 micrograms/kg and the MD from 2 to 128 micrograms.kg-1.min-1. After 2 h of fentanyl or NSS infusion; all rats received 100% O2 and, when alert, their tracheas were extubated; after 7 days the rats underwent cerebral perfusion fixation, followed by light microscopic evaluation. Histopathologic lesions (primarily eosinophilic neuron degeneration) were subjectively graded by a pathologist unaware of the experimental treatment; the grades were based on the percentage of dead neurons. There were no lesions observed in the brain areas in any of the control or 200-8 (LD, microgram/kg; MD, microgram.kg-1.min-1) groups. Eleven of 20 rats in the 400-16, 800-32, 1600-64, and 3200-18 groups showed evidence of brain damage primarily in limbic system structures and association areas (P < 0.05). Our data confirm that fentanyl produces limbic system brain damage in rats, and that the damage occurs over a broad range of doses.

T-cell receptor gamma--delta lymphocytes and Eimeria vermiformis infection.

The role of T-cell receptor gamma--delta T lymphocytes in coccidiosis was examined by determining the course of infection with Eimeria vermiformis in BALB/c mice depleted of gamma--delta lymphocytes by treatment with GL3 monoclonal antibody. The replication of the parasite in primary infections was not greatly, or consistently, affected by this treatment, and there was no correlation between the extent of depletion of small intestinal intraepithelial lymphocytes and the number of oocysts produced. The resistance of immunized mice to challenge was not compromised by depletion of intraintestinal epithelial lymphocytes when their depletion was effected at the time of primary infection and/or administration of the challenge inoculum. Thus, T-cell receptor gamma--delta T lymphocytes do not appear to be crucial to the establishment, or the control, of primary infection with E. vermiformis and are not principal mediators of the solid immunity to challenge that this infection induces.

Opioid neurotoxicity: neuropathologic effects in rats of different fentanyl congeners and the effects of hexamethonium-induced normotension.

We tested the hypotheses that convulsant doses of opioids would produce limbic system damage exacerbated by hexamethonium. Ventilated paralyzed rats received intravenous (IV) isovolumic infusion of fentanyl loading dose (LD) 1000 micrograms/kg, maintenance dose (MD) 40 micrograms.kg-1.min-1 (n = 10), sufentanil LD 400 micrograms/kg, MD 13.3 micrograms.kg-1.min-1 (n = 10), alfentanil LD 1500 micrograms/kg, MD 150 micrograms.kg-1.min-1 (n = 10), or 0.9% saline control LD 4 mliter/kg, MD 4 mliter.kg-1.h-1 (n = 10), with O2/N2 30%/70% during opioid infusion and O2/N2O in controls during saline infusion. Hexamethonium (LD 20 mg/kg, MD 40-120 mg.kg-1.h-1) was given IV during opioid infusion to half of the rats. Cerebral perfusion-fixation with formalin was performed 24 h later, followed by histopathologic assessment. None of the control rats showed any histologic abnormalities. Overall summed neuropathologic severity was worse in opioid treated groups (P = 0.01). Lesions occurred primarily in cortical regions and limbic system structures. When arterial blood pressure was controlled to a lower level with hexamethonium (147 vs 100 mm Hg), rats had less severe lesions (P = 0.02). These data indicate that fentanyl, sufentanil, and alfentanil all can produce histopathologic evidence of brain injury in rats mitigated by hexamethonium.

Triterpenoid saponins from Cephalaria transsylvanica.

Two new triterpenoid saponins, transsylvanosides G and H, were isolated from Cephalaria transsylvanica and their structures established as 3-O-[beta-D-glucopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->3)-alpha-L- rhamnopyranosyl(1-->2)-beta-D-xylopyranosyl] hederagenin 28-O-[beta-D-glucopyranosyl(1-->4)-beta-D-xylopyranosyl] ester (1) and 3-O-[beta-D-xylopyranosyl(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-beta-D- xylopyranosyl] hederagenin (2), respectively. A novel prosapogenin (3) was obtained on the alkaline hydrolysis of 1 and its structure defined as 3-O-[beta-D-glucopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->3)-alpha-L- rhamnopyranosyl(1-->2)-beta-D-xylopyranosyl] hederagenin. The structures of compounds 1-3 were established by spectral and chemical methods.

Responses to vaccination in strains of mice that differ in susceptibility to coccidiosis.

BALB/c mice are normally resistant to infection with Eimeria vermiformis than C57BL/6 (B6) mice, but these phenotypes were reversed by prior vaccination with crude antigens prepared from developmental stages of the parasite: B6 mice were protected, and BALB/c mice were made more susceptible. Infections with a heterologous species, E. pragensis, were unaffected when this was given either alone or together with E. vermiformis. In both strains of mice, vaccination induced serum antibody responses to E. vermiformis and the levels were boosted by superimposed infection, the highest values being found in BALB/c mice. Cellular responses in the mesenteric lymph nodes (MLN), as indicated by cellularity and proliferation, either unstimulated or restimulated in vitro with E. vermiformis antigen, were decreased in both strains, but markedly more in BALB/c than B6. The capacity of MLN cells to transfer immunity to naive recipients was lowered by vaccination of BALB/c donors but unimpaired in vaccinated B6 mice. Responses to the mitogen, concanavalin A, and to unrelated antigens (human erythrocytes and fowl gamma globulin) were unaffected. Thus, parenteral vaccination, which increased the susceptibility of BALB/c mice to infection with E. vermiformis, had a depressing effect on some specific immune responses in the MLN. It was surprising to find some reduction in the cellular responses of the MLN of B6 mice also, although they were protected by vaccination.

Avian coccidiosis: changes in intestinal lymphocyte populations associated with the development of immunity to Eimeria maxima.

The effect of infection and subsequent challenge with Eimeria maxima on the populations of lymphocytes in the small intestine of Light Sussex chickens was assessed by immunohistochemistry. T cells were characterized for CD3, CD4, CD8, TCR1 (gamma delta heterodimer) or TCR2 (alpha beta 1 heterodimer) markers, and B cells for the expression of IgM, IgA and IgG. After a primary inoculum there were, in both the epithelium and the lamina propria, two distinct increases in the numbers of T lymphocytes. The first peaked on days 3-5 and the second, greater influx, on day 11 after infection. CD4+ and CD8+ cells were represented in both peaks but, whereas CD4+ cells were found almost exclusively in the lamina propria, CD8+ cells were present in both sites. The area staining positive for CD8+ cells was somewhat greater than the value obtained for CD4+ cells. In the epithelium there was an early, small increase in TCR1(+)-staining, followed by a larger rise to the second peak, at which time there was also an increase in the lamina propria. Staining for TCR2+ cells followed the same pattern with a reversed distribution between epithelium and lamina propria. Changes after challenge were minimal and confined to the epithelium. The most notable changes in the expression of immunoglobulins were, in the lamina propria, a biphasic increase in the amount of IgM(+)-staining in the course of primary infection (corresponding approximately to that of the T cells), and in IgA+ cells shortly after challenge.

Comparison of extracellular dopamine concentration in awake unstressed and postsurgical nitrous oxide sedated rats.

Nitrous oxide (N2O), 70%, in O2 is often used as a control condition after surgical preparation in rodents undergoing neuroscience investigations. Concern has been expressed that this constitutes a stressful condition. Microdialysis was used in 15 rats to assess extracellular striatal dopamine concentrations during overnight soundproof isolation and on the following day after vascular cannulation and halothane excretion under N2O sedation with concomitant neuromuscular blockade. The overnight dialysate dopamine concentration was 22.8 +/- 8.7 pg/40 microliters. Thirty minutes after stopping halothane, the dialysate concentration was 362.6 +/- 91.6 pg/40 microliters during postsurgical N2O sedation. These data indicate that (a) compared to an unstressed baseline, significant brain dopamine effects occur with N2O sedation after surgery with halothane N2O anesthesia, and (b) baseline conditions can have a major effect on microdialysis data expressed as percentage of baseline.

Cytotoxic effects of natural killer cells have no significant role in controlling infection with the intracellular protozoon Eimeria vermiformis.

The course of infection with Eimeria vermiformis in C57BL/6J; NK cell-defective C57BL/6J bg/bg; BALB/c; T-cell-defective BALB/c nu/nu; and T-cell-, B-cell-, and NK cell-defective BALB/c x C57BL/6 scid/scid bg/bg mice was monitored. For young C57BL/6J mice, the bg/bg mutants consistently produced fewer oocysts than the controls; there were no differences between older mice of these strains. Wild-type BALB/c mice were more resistant to infection than the nu/nu and scid/scid bg/bg mutants, but there was no difference between the mutants. Treatment of BALB/c mice with poly(I.C) had no effect on the course of infection. These findings confirm the ineffectiveness of NK cells in this system.

Triterpenoid glycosides from Cephalaria transsylvanica.

On the basis of spectroscopic and chemical methods, the structures of two new triterpenoid glycosides, transsylvanoside E and F, isolated from Cephalaria transsylvanica have been established as 3-O-[beta-D-xylopyranosyl (1-->3)-alpha-L-rhamnopyranosyl (1-->4)-beta-D-glucopyranosyl (1-->4)-beta-D-glucopyranosyl (1-->2)-beta-D-xylopyranosyl]-3 beta,23- dihydroxy delta 12-oleanen-28-carboxylic acid and 3-O-[beta-D-glucopyranosyl (1-->3)-alpha-L-rhamnopyranosyl (1-->4)-beta-D-xylopyranosyl]-28-O-[beta- D-glucopyranosyl (1-->4)-beta-D-glucopyranosyl]-3 beta,23-dihydroxy delta 12-oleanen- 28-carboxylic acid, respectively. A new proglycoside was isolated from the cleavage of the ester-glycoside linkage and it's structure characterized as 3-O-[beta-D-glucopyranosyl (1-->3)-alpha-L- rhamnopyranosyl (1-->4)-beta-D-xylopyranosyl]-3 beta,23-dihydroxy delta 12-oleanen-28-carboxylic acid.