Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis.

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.

NF-kappaB factor c-Rel mediates neuroprotection elicited by mGlu5 receptor agonists against amyloid beta-peptide toxicity.

Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.

[Absorption of L-lysine diatrizoate from the gastrointestinal tract: the influence of surgery, inflammation and neoplasia]. / Resorption von L-Lysin-Diatrizoat aus dem Gastrointestinaltrakt unter dem Einfluss von Operation, Entzündung und Neoplasie.

PURPOSE: To ascertain whether the absorption of L-lysine diatrizoate, a sodium-free salt of the contrast-giving diatrizoic acid, from the gastrointestinal tract is increased by surgery, inflammation or neoplasia. MATERIAL AND METHODS: Using contrast medium containing L-lysine diatrizoate for intestinal opacification, this prospective study compared 32 radiographic examinations of the upper gastrointestinal tract with 52 radiographic examination of the lower gastrointestinal tract. In blood samples taken from the patients immediately after the radiographic examinations, the concentration of diatrizoic acid was determined by high pressure liquid chromatography. The results were correlated with sex, age, surgical history and any evidence of inflammatory or neoplastic diseases. RESULTS: The serum diatrizoic acid concentration in patients after oral administration was 3.62 (95% CI, 2.86 - 10.17) microg/ml. The titer was lower in patients who had undergone abdominal surgery than in patients without surgery. Serum diatrizoic acid concentration in patients after rectal administration was 0.30 (95% CI, 0.13 - 0.60) microg/ml. The titer was significantly higher (p < 0.05) in patients suffering from inflammatory conditions or neoplasms than in the other patients. CONCLUSION: The L-lysine salt of diatrizoic acid is absorbed in larger amounts from the upper than from the lower gastrointestinal tract. Absorption is not increased after abdominal surgery. However, inflammatory conditions and neoplasms of the large bowel increase the uptake of contrast medium from the intestine.

Bcl-2 is not required in retinal ganglion cells surviving optic nerve axotomy.

Axotomy of the optic nerve in rodents induces the majority of retinal ganglion cells (RGCs) to undergo apoptosis: Only 10-15% survive 14 days past lesion. The molecular mechanism allowing this survival is not known. To test whether expression of the anti-apoptotic proto-oncogene bcl-2 gene is required in those RGCs, we examined the effect of optic nerve axotomy in bcl-2-/- mice. 7 days and 14 days post-lesion, the same number of surviving RGCs was detected in mutant and wild type retinas. Thus, the bcl-2 gene is not necessary for the survival of the subpopulation of retinal ganglion cells resisting axotomy-induced apoptosis in adult mice, nor does its normal expression delay retinal ganglion cell degeneration.

The additive effect of neurotransmitter genes in pathological gambling.

As access to gambling increases there is a corresponding increase in the frequency of addiction to gambling, known as pathological gambling. Studies have shown that a number of different neurotransmitters are affected in pathological gamblers and that genetic factors play a role. Polymorphisms at 31 different genes involved in dopamine, serotonin, norepinephrine, GABA and neurotransmitters were genotyped in 139 pathological gamblers and 139 age, race, and sex-matched controls. Multivariate regression analysis was used with the presence or absence of pathological gambling as the dependent variable, and the 31 coded genes as the independent variables. Fifteen genes were included in the regression equation. The most significant were the DRD2, DRD4, DAT1, TPH, ADRA2C, NMDA1, and PS1 genes. The r(2) or fraction of the variance was less than 0.02 for most genes. Dopamine, serotonin, and norepinephrine genes contributed approximately equally to the risk for pathological gambling. These results indicate that genes influencing a range of brain functions play an additive role as risk factors for pathological gambling. Multi-gene profiles in specific individuals may be of assistance in choosing the appropriate treatment.

Comparative evaluation of two hemagglutination tests for the detection of anti-Helicobacter pylori antibodies.

Two hemagglutination assays (IHAs) for detecting anti-H. pylori antibodies were evaluated. In 257 patients, both tests revealed sensitivity or specificity values between 71-79% or 81-82%, respectively, which were lower than those of an anti-H. pylori ELISA. Notably, the sensitivities of both IHAs varied in subgroups of patients between 48-95%. Based on the validation, the IgG-ELISA is superior to both IHAs tested, and therefore, should be used in routine diagnostics.

Association of the neutral endopeptidase (MME) gene with anxiety.

Enkephalins have been implicated in the regulation of mood, anxiety, reward, euphoria and pain. One of the major enzymes for enkephalin degradation is neutral endopeptidase [enkephalinase, membrane metalloendopeptidase (MME)]. We identified a dinucleotide polymorphism in the 5' region of the MME gene. Subjects were placed into three genotypes, 3/3, 3/x, and x/x since the 3 allele was the most common of the six alleles. Using one-way analysis of variance, we examined the association of these genotypes with the mean SCL-90 scores for anxiety, depression, obsessive-compulsive and phobic anxiety symptoms in 120 Caucasian males from an addiction treatment unit. There was a significant association between the MME genotypes and the SCL-90 scores for phobic anxiety, obsessive-compulsive and anxiety at a Bonferroni corrected alpha value of 0.0125. These results support a role of genetic variants of enkephalin metabolism in anxiety.

Multivariate analysis of associations of 42 genes in ADHD, ODD and conduct disorder.

In a previous study (Comings DE et al. Comparison of the role of dopamine, serotonin, and noradrenergic genes in ADHD, ODD and conduct disorder. Multivariate regression analysis of 20 genes. Clin Genet 2000: 57: 178-196) we examined the role of 20 dopamine, serotonin and norepinephrine genes in attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and conduct disorder (CD), using a multivariate analysis of associations (MAA) technique. We have now brought the total number of genes examined to 42 by adding an additional 22 candidate genes. These results indicate that even with the inclusion of these additional genes the noradrenergic genes still played a greater role in ADHD than any other group. Six other neurotransmitter genes were included in the regression equation - cholinergic, nicotinic, alpha 4 receptor (CHNRA4), adenosine A2A receptor (ADOA2A), nitric oxide synthase (NOS3), NMDAR1, GRIN2B, and GABRB3. In contrast to ADHD and ODD, CD preferentially utilized hormone and neuropeptide genes These included CCK, CYP19 (aromatase cytochrome P-450), ESR1, and INS (p = 0.005). This is consistent with our prior studies indicating a role of the androgen receptor (AR) gene in a range of externalizing behavors. We propose that the MAA technique, by focusing on the additive effect of multiple genes and on the cummulative effect of functionally related groups of genes, provides a powerful approach to the dissection of the genetic basis of polygenic disorders.

Comparison of the role of dopamine, serotonin, and noradrenaline genes in ADHD, ODD and conduct disorder: multivariate regression analysis of 20 genes.

The present study is based on the proposal that complex disorders resulting from the effects of multiple genes are best investigated by simultaneously examining multiple candidate genes in the same group of subjects. We have examined the effect of 20 genes for dopamine, serotonin, and noradrenergic metabolism on a quantitative score for attention deficit hyperactivity disorder (ADHD) in 336 unrelated Caucasian subjects. The genotypes of each gene were assigned a score from 0 to 2, based on results from the literature or studies in an independent set of subjects (literature-based scoring), or results based on analysis of variance for the sample (optimized gene scoring). Multivariate linear regression analysis with backward elimination was used to determine which genes contributed most to the phenotype for both coding methods. For optimized gene scoring, three dopamine genes contributed to 2.3% of the variance, p = 0.052; three serotonin genes contributed to 3%, p = 0.015; and six adrenergic genes contributed to 6.9%, p = 0.0006. For all genes combined, 12 genes contributed to 11.6% of the variance, p = 0.0001. These results indicate that the adrenergic genes play a greater role in ADHD than either the dopaminergic or serotonergic genes combined. The results using literature-based gene scoring were similar. An examination of two additional comorbid phenotypes, conduct disorder and oppositional defiant disorder (ODD), indicated they shared genes with ADHD. For ODD different genotypes of the same genes were often used. These results support the value of the simultaneous examination of multiple candidate genes.

A multivariate analysis of 59 candidate genes in personality traits: the temperament and character inventory.

Cloninger (Cloninger CR. Neurogenetic adaptive mechanisms in alcoholism. Science 1987: 236: 410-416) proposed three basic personality dimensions for temperament: novelty seeking, harm avoidance, and reward dependence. He suggested that novelty seeking primarily utilized dopamine pathways, harm avoidance utilized serotonin pathways, and reward dependence utilized norepinephrine pathways. Subsequently, one additional temperament dimension (persistence) and three character dimensions (cooperativeness, self-directedness, and self-transcendence) were added to form the temperament and character inventory (TCI). We have utilized a previously described multivariate analysis technique (Comings DE, Gade-Andavolu R, Gonzalez N et al. Comparison of the role of dopamine, serotonin, and noradrenergic genes in ADHD, ODD and conduct disorder. Multivariate regression analysis of 20 genes. Clin Genet 2000: 57: 178-196; Comings DD, Gade-Andavolu R, Gonzalez N et al. Multivariate analysis of associations of 42 genes in ADHD, ODD and conduct disorder. Clin Genet 2000: in press) to examine the relative role of 59 candidate genes in the seven TCI traits and test the hypothesis that specific personality traits were associated with specific genes. While there was some tendency for this to be true, a more important trend was the involvement of different ratios of functionally related groups of genes, and of different genotypes of the same genes, for different traits.

Association of the enkephalinase gene with low amplitude P300 waves.

Low amplitude of the P300 evoked potential waves has been linked to substance abuse. Defects in opioidergic genes regulating reward pathways have been implicated as risk factors in substance abuse. Since the rate of degradation of enkephalins regulates their CNS level, we focused on the MME gene for metallo-membrane endopeptidase (neutral endopeptidase, enkephalinase). We identified a GT repeat polymorphism 5' to the gene and examined its potential association with P300 wave amplitude in 25 male subjects with substance abuse. There was significant association of low mol. wt alleles with low amplitude of the P300 wave at the parietal (p = 0.0087) and coronal (p = 0.009) leads. These results support a role of endogenous opioids in the regulation of P300 wave amplitude.

The proenkephalin gene (PENK) and opioid dependence.

We tested the hypothesis that the alleles at the (CA)n repeat of the proenkephalin gene (PENK) might be associated with opioid addiction in 31 non-Hispanic Caucasian subjects with opioid dependence (heroin), 89 ethnically matched subjects with substance dependence other than opioid dependence and 132 controls. Among the subjects with opioid dependence, 66% carried the > or = 81 bp allele compared with 40% of subjects with other types of substance abuse (chi2 = 11.31, p < 0.004) and 49% of controls (chi2 = 6.0, p < 0.015). These results are consistent with a role of the PENK gene in opioid dependence.

Supernatant from stored red cells activates neutrophils.

Bioreactive substances including cytokines and lipids accumulate during storage of red blood cells (RBCs) but their clinical importance is uncertain. The goal of this study was to evaluate the effect of stored RBC supernatant on neutrophil activity in vitro. Packed RBCs (PRBCs) were collected and divided into two aliguots, one leukodepleted and the other nonleukodepleted. Plasma supernatant from PRBCs were collected on days 1, 8, 15, 29 and 35 and its effect on neutrophil expression of CD11b, CD16 and oxidative burst was measured by flow cytometry. Levels of tumour necrosis factor alpha (TNF alpha) and interleukin-8 (IL8) were also measured. The supernatant from PRBC units stored for greater than 15 days activated and primed neutrophils as evidenced by an increase CD11b and CD16 expression and oxidative burst. The greatest effect was seen in the oldest concentrates (35-day-old) (P < 0.008). Leukodepletion abrogated the effects of stored supernatant on CD11b and CD16 expression (P < 0.02) but did not reduce priming of the neutrophil oxidative burst (P > 0.1). Very low levels of IL8 and TNF alpha were detected in stored supernatants. Stored PRBC supernatant contains substances which directly enhance neutrophil expression of adhesion protein CD11b, CD16 and prime neutrophil oxidative burst. The exceedingly low level of IL8 and TNF alpha found in this study suggests that other factors may play a more important role in neutrophil priming and activation.

Transfusing red blood cells stored in citrate phosphate dextrose adenine-1 for 28 days fails to improve tissue oxygenation in rats.

OBJECTIVE: To determine whether the time that red blood cells are stored in citrate phosphate dextrose adenine-1 solution before transfusion alters the ability to improve tissue oxygenation. DESIGN: Prospective, randomized, controlled study. SETTING: University research institute laboratory. SUBJECTS: Male Sprague-Dawley rats (350 to 450 g). INTERVENTIONS: Twenty-four hours after randomization to sham laparotomy (n = 21) or cecal ligation and perforation (n = 16)1 supply-dependency of systemic oxygen uptake (VO2) was induced in rats by isovolemic hemodilution. Rats were then re-randomized to receive either rat red blood cells stored in citrate phosphate dextrose adenine-1 for 3 days ("fresh" n = 17) or rat red blood cells stored in citrate phosphate dextrose adenine-1 for 28 days ("old" n = 20). MEASUREMENTS AND MAIN RESULTS: Changes in systemic VO2 were measured for 90 mins to determine the efficiacy of the treatment. Statistical analysis included a fully factorial repeated-measures, generalized linear model. No significant interaction was found between cecal ligation and perforation or sham animals and transfusion with fresh or old red blood cells. However, comparing the combined groups of animals receiving either fresh or old red blood cells, we found that after the transfusion of old red blood cells, systemic VO2 was not significantly improved (after hemodilution 1.68 +/- 0.27 mL/100 g/min, after transfusion 1.86 +/- 0.17 mL/100 g/min; p > .05). In contrast, transfusion with fresh red blood cells acutely increased systemic VO2 (after hemodilution 1.62 +/- 0.06 mL/100 g/min, after transfusion 2.10 +/- 0.09 mL/100 g/min; p = .049). CONCLUSION: Storage of rat red blood cells for 28 days in citrate phosphate dextrose adenine-1 impaired their ability to improve tissue oxygenation when transfused into either control or septic rats placed into supply dependency of systemic VO2.

Animal models for blood transfusion: a model for sterile blood sampling in the rat.

Rat blood is frequently used for experimental transfusion. However, no data are available concerning the quality of the blood used, although bacterial contamination could severely alter results. To obtain large quantities of sterile rat blood for a transfusion study, we tested carotid artery cannulation, known as a standard procedure. Blood cultures from the collected blood showed polymicrobial overgrowth even after sterility measures were improved. In contrast, the puncture of the abdominal aorta proved to be a simple and reliable method for the collection of sterile blood. We conclude that studies using blood collected from donor rats should be controlled and the quality of such blood be tested before transfusion.

Studies of the potential role of the dopamine D1 receptor gene in addictive behaviors.

Abnormalities in the dopaminergic reward pathways have frequently been implicated in substance abuse and addictive behaviors. Recent studies by Self and coworkers have suggested an important interaction between the dopamine D1 and D2 receptors in cocaine abuse. To test the hypothesis that the DRD1 gene might play a role in addictive behaviors we examined the alleles of the Dde I polymorphism in three independent groups of subjects with varying types of compulsive, addictive behaviors-Tourette syndrome probands, smokers and pathological gamblers. In all three groups there was a significant in the frequency of homozygosity for the DRD1 Dde I 1 or 2 alleles in subjects with addictive behaviors. The DRD1 11 or 22 genotype was present in 41.3% of 63 controls and 57.3% of 227 TS probands (P = 0.024). When 23 quantitative traits were examined by ANOVA those carrying the 11 genotype consistently had the highest scores. Based on these results, we examined the prevalence of the 11 genotype in controls, TS probands without a specific behavior, and TS probands with a specific behavior. There was a progressive, linear increase, significant at alpha < or = 0.005 for scores for gambling, alcohol use and compulsive shopping. Problems with three additional behaviors, drug use, compulsive eating and smoking were significant at alpha < or = 0.05. All six variables were related to addictive behaviors. In a totally separate group of controls and individuals attending a smoking cessation clinic, and smoking at least one pack per day, 39.3% of the controls versus 66.1% of the smokers carried the 11 or 22 genotype (P = 0.0002). In a third independent group of pathological gamblers, 55.8% carried the 11 or 22 genotype (P = 0.009 vs the combined controls). In the TS group and smokers there was a significant additive effect of the DRD1 and DRD2 genes. The results for both the DRD1 and DRD2 genes, which have opposing effects on cyclic AMP, were consistent with negative and positive heterosis, respectively. These results support a role for genetic variants of the DRD1 gene in some addictive behaviors, and an interaction of genetic variants at the DRD1 and DRD2 genes.

Degradabilidade ruminal da fibra da cana-de-açúcar, pela técnica dos sacos de náilon in situ, quando suplementada por várias fontes proteicas / Ruminal degradability of sugar cane fiber, when supplemented with different protein sources through in situ bag technique

Quatro bovinos dotados de cânulas de rúmen foram utilizados em um delineamento change over 4 x 4, para testar os efeitos dos seguintes tratamentos: A) farelo de algodäo; B) farelo de soja; C) soja crua e D) soja torrada, sobre a degradabilidade ruminal da fibra em detergente neutro (FDN) da cana-de-açúcar, empregada como único alimento volumoso, com utilizaçäo da técnica dos sacos de náilon in situ. A degradabilidade da fibra foi inferior quando suplementada por soja gräos e superior, quando por farelo de algodäo (p < 0,05). Näo houve diferença estatística nos efeitos dos gräos de soja crus e torrados sobre a degradabilidade da fibra da cana-de-açúcar

Exon and intron variants in the human tryptophan 2,3-dioxygenase gene: potential association with Tourette syndrome, substance abuse and other disorders.

Defects in serotonin metabolism, and abnormalities in both blood serotonin and tryptophan levels, have been reported in many psychiatric disorders. Tryptophan 2,3-dioxygenase (TDO2) is the rate limiting enzyme for the breakdown of tryptophan to N-formyl kenurenine. Functional variants of this gene could account for the observed simultaneous increases or decreases of both serotonin and tryptophan in various disorders. We have identified four different polymorphisms of the human TDO2 gene. Association studies show a significant association of one or more of these polymorphisms and Tourette syndrome (TS), attention deficit hyperactivity disorder (ADHD) and drug dependence. The intron 6G-->T variant was significantly associated with platelet serotonin levels. Only the association with TS was significant with a Bonferroni correction (p = 0.005). Our purpose here is not to claim these associations are proven, but rather to report preliminary results and show that easily testable polymorphisms are available. We hope to encourage additional research into the potential role the TDO2 gene in these and other psychiatric disorders.