Influence of high inorganic selenium and manganese diets for fattening pigs on oxidative stability and pork quality parameters.

Data are scarce regarding combined high Se and Mn supplementation in livestock diets, however, as Se and Mn are functionally related as cofactors of glutathione peroxidase (GPx) and Mn-superoxide dismutase (SOD), respectively, beneficial synergistic effects on oxidative stability of tissues may result. This experiment evaluated the effect of an oversupply of Se and Mn within European legal limits compared with recommendations on performance, oxidative stability of the organism and meat quality in a randomised complete block design. A total of 60 crossbred gilts were fed maize-barley-soya bean meal diets formulated in a 2×2 factorial approach with inorganic Se (0.2 v. 0.5 mg/kg Se dry matter (DM)) or inorganic Mn (20 v. 150 mg/kg Mn DM) from 31 to 116 kg BW. Se supplementation reduced feed intake, whereas high Mn diets impaired average daily gain (P<0.05). Qualitative carcass characteristics were impaired by Se and Mn predominantly in the semimembranosus muscle. Activity of GPx in liver was increased by high Se diets (P<0.05). Mn supplementation increased catalase (CAT) activity in liver, GPx in plasma and total antioxidative capacity (TAC) in muscle, whereas it decreased CAT activity in plasma (P<0.05). Cu/Zn-SOD in muscle showed higher activity in high-Se-low-Mn diets but lower activity when both high Se and Mn were combined (Se×Mn P<0.05). Mn supplementation increased Mn concentration in longissimus thoracis et lumborum, but simultaneously reduced Se concentration (P<0.05). Upon retail display, Mn increased lipid oxidation more pronouncedly (higher thiobarbituric acid reactive substances; P<0.05) than Se (P<0.10). Despite some positive effects (Mn increased TAC, Se increased GPx, Se and Mn increased tenderness), no synergistic effects of high Se and Mn diets or an overall beneficial impact on meat quality, especially during storage, could be observed. Including the negative effects on performance, feeding Se and Mn up to the maximum legal level cannot be recommended.

Inhibitory function of the dorsomedial hypothalamic nucleus on the hypothalamic-pituitary-adrenal axis response to an emotional stressor but not immune challenge.

Accumulating evidence implicates the dorsomedial hypothalamic nucleus (DMH) in the regulation of autonomic and neuroendocrine stress responses. However, although projections from the DMH to the paraventricular hypothalamic nucleus (PVN), which is the critical site of the neuroendocrine stress axis, have been described, the impact of DMH neurones in the modulation of hypothalamic-pituitary-adrenal (HPA) axis activation during stress is not fully understood. The present study aimed to investigate the role of the DMH in HPA axis responses to different types of stimuli. Male Sprague-Dawley rats fitted with a chronic jugular venous catheter were exposed to either an emotional stressor (elevated platform-exposure) or immune challenge (systemic interleukin-1ß administration). Bilateral electrolytic lesions of the DMH disinhibited HPA axis responses to the emotional stressor, as indicated by higher plasma adrenocorticotrophic hormone levels during and after elevated platform exposure in lesioned animals compared to sham-lesioned controls. Moreover, DMH-lesioned animals showed increased neuronal activation in the PVN, as indicated by a higher c-Fos expression after elevated-platform exposure compared to controls. By contrast, DMH-lesions had no effects on HPA axis responses to immune challenge. Taken together, our data suggest an inhibitory role of DMH neurones on stress-induced HPA axis activation that is dependent upon the nature of the stimulus being important in response to an emotional stressor but not to immune challenge.

The role of substance P in stress and anxiety responses.

Substance P (SP) is one of the most abundant peptides in the central nervous system and has been implicated in a variety of physiological and pathophysiological processes including stress regulation, as well as affective and anxiety-related behaviour. Consistent with these functions, SP and its preferred neurokinin 1 (NK1) receptor has been found within brain areas known to be involved in the regulation of stress and anxiety responses. Aversive and stressful stimuli have been shown repeatedly to change SP brain tissue content, as well as NK1 receptor binding. More recently it has been demonstrated that emotional stressors increase SP efflux in specific limbic structures such as amygdala and septum and that the magnitude of this effect depends on the severity of the stressor. Depending on the brain area, an increase in intracerebral SP concentration (mimicked by SP microinjection) produces mainly anxiogenic-like responses in various behavioural tasks. Based on findings that SP transmission is stimulated under stressful or anxiety-provoking situations it was hypothesised that blockade of NK1 receptors may attenuate stress responses and exert anxiolytic-like effects. Preclinical and clinical studies have found evidence in favour of such an assumption. The status of this research is reviewed here.

Detection and monitoring of virus infections by real-time PCR.

The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.

Comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications.

The adenovirus (AdV) hexon constitutes the major virus capsid protein. The epitopes located on the hexon protein are targets of neutralizing antibodies in vivo, serve in the recognition by cytotoxic T cells, and provide the basis for the classification of adenoviruses into the 51 serotypes known to date. We have sequenced the entire hexon gene from human serotypes with incomplete or no sequence information available (n = 34) and performed a comparative analysis of all sequences. The overall sequence divergence between the 51 human serotypes ranged from 0.7 to 25.4% at the protein level. The sequence information has been exploited to assess the phylogeny of the adenovirus family, and protein distances between the six AdV species (A to F) and among individual serotypes within each species were calculated. The analysis revealed that the differences among serotypes within individual species range from 0.3 to 5.4% in the conserved regions (765 amino acids [aa]) and from 1.5 to 59.6% in the variable regions (154 to 221 aa). Serotypes of different species showed an expectedly greater divergence both in the conserved (5.9 to 12.3%) and variable (49.0 to 74.7%) regions. Construction of a phylogenetic tree revealed three major clades comprising the species B+D+E, A+F, and C, respectively. For serotypes 50 and 51, the original assignment to species B and D, respectively, is not in accordance with the hexon DNA and protein sequence data, which placed serotype 50 within species D and serotype 51 within species B. Moreover, the hexon gene of serotype 16, a member of species B, was identified as the product of recombination between sequences of species B and E. In addition to providing a basis for improved molecular diagnostics and classification, the elucidation of the complete hexon gene sequence in all AdV serotypes yields information on putative epitopes for virus recognition, which may have important implications for future treatment strategies permitting efficient targeting of any AdV serotype.

Molecular detection and quantitative analysis of the entire spectrum of human adenoviruses by a two-reaction real-time PCR assay.

Adenoviruses (AdV) can cause life-threatening infections in immunosuppressed patients. Reliable diagnostic tests are therefore of paramount importance. Apparently, any of the six AdV species (A to F), currently comprising 51 different serotypes, can play a clinically important role in patients with impaired immune response. It is imperative therefore that diagnostic assays cover the entire spectrum of these viruses. We have sequenced presumably conserved regions of the adenoviral genome in all AdV serotypes. Based on the complete sequence information of the hexon gene, we were able to develop a two-reaction real-time PCR assay covering all human adenoviruses with equally high specificity and sensitivity. The detection systems were tested using reference strains for all 51 serotypes and >1,000 clinical samples derived from peripheral blood and stool specimens from pediatric patients after allogeneic stem cell transplantation. The two-reaction assay presented permits highly specific detection and quantification of adenoviral DNA of any serotype. From the perspective of routine clinical diagnosis, the assay represents an important improvement over existing approaches by providing a sensitive and economic technique for early detection and monitoring of adenoviral infections.

Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.

A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.

Differential involvement of rat medial prefrontal cortex dopamine receptors in modulation of hypothalamic-pituitary-adrenal axis responses to different stressors.

Recent investigations have implicated the medial prefrontal cortex (mPFC) in modulation of subcortical pathways that contribute to the generation of behavioural, autonomic and endocrine responses to stress. However, little is known of the mechanisms involved. One of the key neurotransmitters involved in mPFC function is dopamine, and we therefore aimed, in this investigation, to examine the role of mPFC dopamine in response to stress in Wistar rats. In this regard, we infused dopamine antagonists SCH23390 or sulpiride into the mPFC via retrodialysis. We then examined changes in numbers of cells expressing the c-fos immediate-early gene protein product, Fos, in subcortical neuronal populations associated with regulation of hypothalamic-pituitary-adrenal (HPA) axis stress responses in response to either of two stressors; systemic injection of interleukin-1 beta, or air puff. The D1 antagonist, SCH23390, and the D2 antagonist, sulpiride, both attenuated expression of Fos in the medial parvocellular hypothalamic paraventricular nucleus (mpPVN) corticotropin-releasing factor cells at the apex of the HPA axis, as well as in most extra-hypothalamic brain regions examined in response to interleukin-1 beta. By contrast, SCH23390 failed to affect Fos expression in response to air puff in any brain region examined, while sulpiride resulted in an attenuation of the air puff-induced response in only the mpPVN and the bed nucleus of the stria terminalis. These results indicate that the mPFC differentially processes the response to different stressors and that the two types of dopamine receptor may have different roles.

Medial prefrontal cortex suppression of the hypothalamic-pituitary-adrenal axis response to a physical stressor, systemic delivery of interleukin-1beta.

Previous studies have shown that the medial prefrontal cortex can suppress the hypothalamic-pituitary-adrenal axis response to stress. However, this effect appears to vary with the type of stressor. Furthermore, the absence of direct projections between the medial prefrontal cortex and corticotropin-releasing factor cells at the apex of the hypothalamic-pituitary-adrenal axis suggest that other brain regions must act as a relay when this inhibitory mechanism is activated. In the present study, we first established that electrolytic lesions involving the prelimbic and infralimbic medial prefrontal cortex increased plasma adrenocorticotropic hormone levels seen in response to a physical stressor, the systemic delivery of interleukin-1beta. However, medial prefrontal cortex lesions did not alter plasma adrenocorticotropic hormone levels seen in response to a psychological stressor, noise. To identify brain regions that might mediate the effect of medial prefrontal cortex lesions on hypothalamic-pituitary-adrenal axis responses to systemic interleukin-1beta, we next mapped the effects of similar lesions on interleukin-1beta-induced Fos expression in regions previously shown to regulate the hypothalamic-pituitary-adrenal axis response to this stressor. It was found that medial prefrontal cortex lesions reduced the number of Fos-positive cells in the ventral aspect of the bed nucleus of the stria terminalis. However, the final experiment, which involved combining retrograde tracing with Fos immunolabelling, revealed that bed nucleus of the stria terminalis-projecting medial prefrontal cortex neurons were largely separate from medial prefrontal cortex neurons recruited by systemic interleukin-1beta, an outcome that is difficult to reconcile with a simple medial prefrontal cortex-bed nucleus of the stria terminalis-corticotropin-releasing factor cell control circuit.

Taurine selectively modulates the secretory activity of vasopressin neurons in conscious rats.

Previous experiments have shown that a 10-min forced swimming session triggers the release of vasopressin from somata and dendrites, but not axon terminals, of neurons of the hypothalamic-neurohypophysial system. To further investigate regulatory mechanisms underlying this dissociated release, we forced male Wistar rats to swim in warm (20 degrees C) water and monitored release of the potentially inhibitory amino acids gamma amino butyric acid (GABA) and taurine into the hypothalamic supraoptic nucleus using microdialysis. Forced swimming caused a significant increase in the release of taurine (up to 350%; P < 0.05 vs. prestress release), but not GABA. To reveal the physiological significance of centrally released taurine, the specific taurine antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide was administered into the supraoptic nucleus via retrodialysis. Administration of this antagonist caused a significant increase in the release of vasopressin within the supraoptic nucleus and into the blood both under basal conditions and during stress (up to 800%; P < 0.05 vs. basal values), without affecting hypothalamic or plasma oxytocin. Local administration of the GABA(A) receptor antagonist bicuculline, in contrast, failed to influence vasopressin secretion at either time point. In a separate series of in vivo electrophysiological experiments, administration of the same dosage of the taurine antagonist into the supraoptic nucleus via microdialysis resulted in an increased electrical activity of identified vasopressinergic, but not oxytocinergic, neurons. Taken together our data demonstrate that taurine is released within the supraoptic nucleus during physical/emotional stress. Furthermore, at the level of the supraoptic nucleus, taurine inhibits not only the electrical activity of vasopressin neurons but also acts as an inhibitor of both central and peripheral vasopressin secretion during different physiological states.

Brain oxytocin inhibits the (re)activity of the hypothalamo-pituitary-adrenal axis in male rats: involvement of hypothalamic and limbic brain regions.

In response to various stressors, oxytocin is released not only into blood, but also within hypothalamic and extrahypothalamic limbic brain regions. Here, we describe the involvement of intracerebrally released oxytocin in the regulation of the activity of the hypothalamo-pituitary-adrenal (HPA) axis by infusion of the oxytocin receptor antagonist (des Gly-NH(2) d(CH(2))(5) [Tyr(Me)(2), Thr(4)] OVT; pH 7.4; Dr. M. Manning, Toledo, OH, USA) either into the lateral cerebral ventricle (icv[0.75 microg/5 microl,]) or via retrodialysis (10 microg/ml, 3.3 microl/min, 15 min) into the hypothalamic paraventricular nuclei (PVN), the medio-lateral septum or the amygdala. Male Wistar rats fitted with a chronic jugular vein catheter and an icv guide cannula or a microdialysis probe targeting the respective brain region 4 days prior to the experiment were blood sampled under basal as well as stressful conditions. Rats were exposed to the elevated platform (emotional stressor) and/or to forced swimming (combined physical and emotional stressor). Blockade of the receptor-mediated action of endogenous oxytocin within the PVN resulted in an enhanced basal secretion of ACTH whereas, in response to forced swimming, ACTH secretion was rather reduced, indicating a tonic inhibitory effect of OXT on basal HPA axis activity, but a potentiating action under conditions of stress. Within the medio-lateral septum, antagonist treatment did not alter basal ACTH secretion, but significantly disinhibited ACTH secretion in response to the elevated platform, but not to forced swimming. Within the amygdala, no significant effects either on basal or stress-induced HPA axis activity could be found. The results indicate a differential involvement of brain oxytocin in the regulation of the HPA axis activity which depends both on the site of intracerebral oxytocin release and the stressor the animals are exposed to.

Acute transcranial magnetic stimulation of frontal brain regions selectively modulates the release of vasopressin, biogenic amines and amino acids in the rat brain.

Using intracerebral microdialysis in urethane-anaesthetized adult male Wistar rats, we monitored the effects of acute repetitive transcranial magnetic stimulation (rTMS; 20 trains of 20 Hz, 2.5 s) on the intrahypothalamic release of arginine vasopressin (AVP) and selected amino acids (glutamate, glutamine, aspartate, serine, arginine, taurine, gamma-aminobutyric acid) and the intrahippocampal release of monoamines (dopamine, noradrenaline, serotonin) and their metabolites (homovanillic acid, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid). The stimulation parameters were adjusted according to the results of accurate computer reconstructions of the current density distributions induced by rTMS in the rat and human brains, ensuring similar stimulation patterns in both cases. There was a continuous reduction in AVP release of up to 50% within the hypothalamic paraventricular nucleus in response to rTMS. In contrast, the release of taurine, aspartate and serine was selectively stimulated within this nucleus by rTMS. Furthermore, in the dorsal hippocampus the extracellular concentration of dopamine was elevated in response to rTMS. Taken together, these data provide the first in vivo evidence that acute rTMS of frontal brain regions has a differentiated modulatory effect on selected neurotransmitter/neuromodulator systems in distinct brain areas.

A single social defeat experience selectively stimulates the release of oxytocin, but not vasopressin, within the septal brain area of male rats.

The naturally occurring social conflict situation to be confronted with an aggressive dominant conspecific was used to study the effects of emotional stress on the release of oxytocin (OXT) and arginine vasopressin (AVP) within the mediolateral septum of the rat brain. Male rats were chronically implanted with a microdialysis probe into this brain area. Local release patterns of both, OXT and AVP were monitored in response to a 30 min social defeat. Social defeat caused a significant increase in the release of OXT (to 254%+/-43%, P<0.01). In contrast, the release of AVP was not affected. In a preliminary experiment, to assess the physiological significance of stress-induced intraseptal OXT release, a separate group of animals received the OXT receptor antagonist des-Gly-NH(2)d(CH(2))(5)[Tyr(Me)(2)Thr(4)]OVT into the mediolateral septum via inverse microdialysis prior to and during the social defeat procedure. However, no difference could be observed in submissive freezing (passive coping) or in exploratory behavior (active coping) when compared to vehicle-treated animals, neither acutely nor 24 h after antagonist administration. Taken together, our results demonstrate that emotional stress activates the septal oxytocinergic, but not vasopressinergic, system. The physiological significance of intraseptally released OXT remains unclear and has to be elucidated in future studies.

Behavioural impact of intraseptally released vasopressin and oxytocin in rats.

The two nonapeptides arginine vasopressin and oxytocin are not only secreted from the neurohypophysis into the general circulation but are also released intracerebrally. Our recent research has focused on the release patterns and effects of oxytocin and vasopressin in brain areas, such as the septum and hypothalamus, that are thought to be involved in the regulation of (1) behavioural responses and (2) responses of the hypothalamo-neurohypophysial system (HNS) to stressor exposure in rats. The results demonstrate that combined physical and emotional stress (induced by exposure to forced swimming) selectively triggers the release of vasopressin within all brain areas under study but not into the general circulation. Under emotional stress conditions (induced by exposure to the 'social defeat' procedure), however, oxytocin rather than vasopressin release increased within the hypothalamus and septum. Experiments aimed at revealing the neuroendocrine and behavioural relevance of the local nonapeptide release provided evidence for an involvement of vasopressin in the regulation of HNS activity (within the hypothalamus) and, moreover, in acute stress-coping strategies, anxiety-related behaviour and learning and memory processes (within the septum). The observed dissociation between central and peripheral nonapeptide release not only supports the hypothesis that plasma vasopressin and oxytocin concentrations do not necessarily reflect central release patterns but also suggests vasopressin and oxytocin neurones are able to independently release their nonapeptide from different parts of their neuronal surface (e.g. from somata/dendrites vs. axon terminals). This remarkable regulatory capacity provides the basis for an differential involvement of vasopressin, and probably also oxytocin, in the co-ordination of neuroendocrine activity, emotionality and cognition at different brain levels to ensure an appropriate behavioural response of the organism to stressful stimuli.

Oxytocin induces preservation of social recognition in male rats by activating alpha-adrenoceptors of the olfactory bulb.

In this report, a series of four experiments was performed to evaluate the relationship between the olfactory bulb norepinephrine system and intra-olfactory bulb infusion of oxytocin in the preservation of social memory responses. The present data indicate that oxytocin exerts this preservation of social recognition through a specific, receptor-mediated mechanism within the olfactory bulb (experiment 1). The involvement of the olfactory bulb norepinephrine system is revealed by the demonstration that retrodialysis of oxytocin into the olfactory bulb increases norepinephrine release (experiment 4). Our data suggest that the increased output of olfactory bulb norepinephrine resulting from oxytocin appears to activate alpha-adrenoceptors to produce this preservation in recognition because infusions of clonidine into the olfactory bulb preserve recognition responses in a manner similar to that observed with oxytocin (experiment 2). In addition, a co-infusion of oxytocin with phentolamine abolishes recognition responses (experiment 3). Accordingly, this model affords the opportunity to study neuropeptide-catecholamine interactions, link these interactions with a specific behavioural outcome and identify a novel function/site of action for oxytocin in the male.

Emotional stress triggers intrahypothalamic but not peripheral release of oxytocin in male rats.

Previous experiments have shown that an exposure to defined stressors activates not only the 'classical' endocrine stress response but also the intrahypothalamic and peripheral release of oxytocin. In the present study we investigated the effects of an acute social defeat experience on the release of oxytocin within the hypothalamic supraoptic nucleus, just outside of the supraoptic nucleus toward the midline within the anterior ventro-lateral part of the hypothalamus, and into plasma of adult male rats. Our results demonstrate that emotional stress triggers the release of oxytocin into the extracellular fluid of both the supraoptic nucleus and the anterior ventro-lateral part of the hypothalamus (up to approximately 320% and 170%, respectively). Interestingly, oxytocin release within the latter brain area, which is likely to originate from axons forming the hypothalamo-neurohypophysial tract, was higher in absolute terms than that within the supraoptic nucleus itself, both under basal conditions and in response to social defeat. In contrast to intrahypothalamic release patterns, plasma oxytocin levels remained virtually unchanged upon stressor exposure. This demonstrates that the release of oxytocin within the hypothalamus is triggered by emotional stress. Furthermore, it indicates that under physiological conditions the release of oxytocin from the dendrites and somata upon axon terminals in the neurohypophysis is differentially regulated. Although not yet studied in detail, it may be hypothesized that the spatial and temporal release pattern of oxytocin is controlled by integrative neuronal networks at different brain levels (including hypothalamus and posterior pituitary) to ensure the appropriate involvement of this peptide in the stress response of the animal.

Formal swallowing evaluation and therapy after traumatic brain injury improves dysphagia outcomes.

BACKGROUND: The incidence of swallowing dysfunction after brain injury is unknown. The efficacy of dysphagia therapy is also unknown. We reviewed our experience to define the incidence of swallowing dysfunction and efficacy of therapeutic intervention. METHODS: Patients with brain injury sustained between January of 1996 and December of 1997 were reviewed. All were screened with trials of oral intake. Abnormal findings were confirmed with a videofluoroscopic swallow study. Standard therapies included diet, posture, and behavior modifications. RESULTS: A total of 47 patients were evaluated. Bedside evaluations were normal in 14 patients, 2 patients had overt aspiration and underwent gastrostomy, and 31 patients were referred for a videofluoroscopic swallow study (66%). The videofluoroscopic swallow study was abnormal in 22 of 31 patients (71%). Of these, 4 additional patients required gastrostomy, 13 patients had laryngeal penetration or minor aspiration responsive to dysphagia therapy and were fed. Five other patients had silent aspiration and were fed by means of nasogastric tube; these five patients responded to dysphagia therapy and were able to resume oral intake. CONCLUSION: Dysphagia is common after severe head injury. With formal swallowing service intervention, aspiration is avoided. Therapeutic interventions can be used to restore oral intake.

Vasopressin released within the septal brain area during swim stress modulates the behavioural stress response in rats.

The aim of the present study was to investigate the physiological significance of the neuropeptide arginine vasopressin (AVP) released within the septum, in the behavioural response of rats to stress. In the first experiment, rats were chronically implanted with a microdialysis probe aimed at the mediolateral or ventral septum to monitor the local release of AVP in response to 10 min of forced swimming in 20 degrees C warm water. Exposure to this stressor caused a significant increase in AVP release in both the mediolateral (174 +/- 21%, P < 0.01) and ventral septum (220 +/- 33%, P < 0.01). In contrast, microdialysates collected outside the mediolateral septum or in the lateral ventricle remained at prestress levels throughout the dialysis period. Furthermore, unstressed control animals failed to show significant alterations in vasopressin release in the mediolateral septum. In a second experiment, the introduction of the V1 receptor antagonist d(CH2)5Tyr(Me)AVP into the mediolateral septum via inverse microdialysis concomitant with stressor exposure caused the rats to spend an increased time floating and a reduced time swimming compared to vehicle-treated rats. This effect was acute and also detected 24 h after antagonist administration. Taken together, these findings demonstrate a significant activation of the septal vasopressinergic system in response to swim stress. Furthermore, our data support the view that AVP released within this brain area is involved in the generation of active behavioural strategies aimed at coping with new and challenging situations.

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids.

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.