Strain-dependent effects of cognitive enhancers in the mouse.

A series of cognitive enhancers (CEs) have been reported to increase spatial memory in rodents, information on behavioral effects, however, is limited. The aim of the study was therefore to examine the behavioral effects of three CEs in two well-documented inbred mouse strains. C57BL/6J and DBA/2 mice were administered intraperitonial. D-cycloserine (DCS; NMDA receptor agonist), 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4-piperidinyl]-1-propanone hydrochloride (RS67333; 5HT4-receptor agonist), and (R)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol hydrochloride (SIB-1553A; beta-4-nicotinic receptor agonist) and tested in the open field (OF), elevated plus maze (EPM), neurological observational battery and rota-rod. Cognitive performance was tested in the Morris water maze. All compounds modified behavioral performance in the OF, DCS showed an anxiolytic effect in the EPM, and differences in the observational battery were observed i.e. vestibular drop was decreased by SIB-1553A and RS67333 treatment in C57BL/6J and increased with DCS treatment in DBA/2 mice. In the rota rod SIB-1553A improved motor performance. DCS effects on learning and memory was comparable to controls whereas the other compounds impaired performance in the Morris water maze. In conclusion, behavioral testing of CEs in the mouse revealed significant changes that may have to be taken into account for evaluation of CEs, interpretation of cognitive studies and warrant further neurotoxicological studies. Moreover, strain-dependent differences were observed that in turn may confound results obtained from behavioral and cognitive testing.

Long-term effect of moderate and profound hypothermia on morphology, neurological, cognitive and behavioural functions in a rat model of perinatal asphyxia.

BACKGROUND: Perinatal asphyxia is a frequent cause of neurological handicap with no known therapy. However, hypothermic therapy has recently attracted attention owing to its neuroprotective property in brain of immature organisms. OBJECTIVES: Hypothermia appears to be promising in reversing the immediate effect of perinatal asphyxia, but data on long-term neuroprotection is still lacking. We therefore intended to test the long-term effect of moderate and profound hypothermia on brain morphology and functions using a well established rat model of perinatal asphyxia. METHODS: Rat pups delivered by caesarean section were placed into a water bath, still in patent membranes, at 37 degrees C and variable hypothermic conditions to induce asphyxia and thereafter given to surrogate mothers. Examinations were performed at the age of three months, consisting of a battery of motor, behavioural, cognition and reflex tests including rota-rod, Morris water maze, multiple T-maze, elevated plus maze and open field studies. Morphological alterations were evaluated by Nissl staining of brain areas known to be hypoxia sensitive. Neurotransmission system markers, including tyrosine hydroxylase, vesicular monoamine transporter, vesicular acetylcholine transporter and excitatory amino acid carrier1 were analyzed by immunohistochemistry. RESULTS: Survival increased with hypothermia. The Nissl stain revealed neuronal loss in hippocampus and hypothalamus of normothermic asphyxiated group (20/37) compared to controls (0/37), but no neuroprotective patterns emerged from hypothermia. An overall inconsistent protection of the neural systems was noted by variable periods of hypothermia. Motor function was significantly impaired in 20/37 as compared to 0/37. In the Morris water maze and multiple T-maze, results were comparable between the groups. In the elevated plus maze, time spent in the closed arm was reduced and in the open field, vertical behaviour was altered in the 20/37 group with horizontal motor behaviour being unaffected. Hypothermia reversed all abnormalities seen in 20/37, with short-term moderate and profound hypothermia being superior to long-term hypothermia. CONCLUSION: Hypothermia not only significantly increased survival, but also resulted in unimpaired motor as well as improved cognitive functions. Those findings are in contrast to altered brain morphology. As neuronal loss was present in various brain regions, we conclude that deficits may be compensated in the maturing animal. Intrahypoxic hypothermia was able to protect the rat from the devastating effect of perinatal asphyxia not in morphological, but in functional terms.

Strain-dependent regulation of neurotransmission and actin-remodelling proteins in the mouse hippocampus.

Individual mouse strains differ significantly in terms of behaviour, cognitive function and long-term potentiation. Hippocampal gene expression profiling of eight different mouse strains points towards strain-specific regulation of genes involved in neuronal information storage. Protein expression with regard to strain- dependent expression of structures related to neuronal information storage has not been investigated yet. Herein, a proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) has been chosen to address this question by determining strain-dependent expression of proteins involved in neurotransmission and activity-induced actin remodelling in hippocampal tissue of five mouse strains. Of 31 spots representing 16 different gene products analysed and quantified, N-ethylmaleimide-sensitive fusion protein, N-ethylmaleimide-sensitive factor attachment protein-alpha, actin-like protein 3, profilin and cofilin were expressed in a strain-dependent manner. By treating protein expression as a phenotype, we have shown significant genetic variation in brain protein expression. Further experiments in this direction may provide an indication of the genetic elements that contribute to the phenotypic differences between the selected strains through the expressional level of the translated protein. In view of this, we propose that proteomic analysis enabling to concomitantly survey the expression of a large number of proteins could serve as a valuable tool for genetic and physiological studies of central nervous system function.

Strain-dependent expression of signaling proteins in the mouse hippocampus.

Individual mouse strains may differ significantly in terms of behavior and cognitive function. Hippocampal gene expression profiling on several mouse strains has been carried out and points toward substantial strain-specific variation of more than 200 genes including components of major signaling pathways involved in neuronal information storage. Strain-specific hippocampal protein expression, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry has been chosen to address this question by determining strain-dependent expression of signaling proteins in hippocampi of four inbred and one outbred mouse strain. Forty-six spots corresponding to 37 different signaling proteins have been analyzed and quantified. Statistical analysis revealed strain-dependent expression of serine/threonine protein phosphatase 1, serine/threonine protein phosphatase 2A, large GTP binding protein OPA1, guanine nucleotide-binding protein beta, putative GTP-binding protein Ran, receptor of activated protein kinase C1, WASP-family protein member 1, voltage-dependent anion channel 2 and 14-3-3 protein gamma. Differential expression of signaling proteins in the hippocampus may contribute to the molecular understanding of strain-dependent behavioral and cognitive performance. Moreover, these data highlight the importance of the genetic background for the analysis of signaling pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.

Pipecolic acid concentrations in brain tissue of nutritionally pyridoxine-deficient rats.

Elevated concentrations of pipecolic acid have been reported in plasma and CSF of patients with pyridoxine-dependent epilepsy, but its molecular background is unclear. To investigate any further association of pyridoxine and pipecolic acid metabolism, we have performed an animal trial and have measured the concentration of pipecolic acid in brain tissue of rats with nutritional pyridoxine deficiency and in control littermates. Concentrations of pyridoxal phosphate were significantly reduced in brain tissue of pyridoxine-deficient rats (p < 0.001), while concentrations of pipecolic acid were not significantly different from the normally nourished control group (p = 0.3). These data indicate that a direct association of pyridoxine and pipecolic acid metabolism is unlikely. We therefore assume that the characteristic elevation of pipecolic acid in pyridoxine-dependent epilepsy could rather be a secondary phenomenon with the primary defect of pyridoxine-dependent epilepsy being located outside the pipecolic acid pathway.

Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor gamma2 F77I point mutation.

Zolpidem is a hypnotic benzodiazepine site agonist with some gamma-aminobutyric acid (GABA)(A) receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of gamma2 subunit (gamma2F77I) point mutant mice. Analysis of forebrain GABA(A) receptor expression with immunocytochemistry, quantitative [(3)H]muscimol and [(35)S] t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with [(3)H]flunitrazepam and [(3)H]muscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous gamma2I77/I77 and gamma2F77/F77 mice. However, quantitative immunoblot analysis of gamma2I77/I77 hippocampi showed some increased levels of gamma2, alpha1, alpha4 and delta subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 microm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, gamma2F77/F77) mice by approximately 60%, and peak amplitude by approximately 20% at 33-34 degrees C in vitro. The actions of zolpidem (100 nm or 1 microm) were substantially reduced in gamma2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 microm) on carbachol-induced oscillations in the CA3 area of gamma2I77/I77 mice was significantly different compared with controls. Thus, the gamma2F77I point mutation virtually abolished the actions of zolpidem on GABA(A) receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the gamma2 subunit.

Genetic functional inactivation of neuronal nitric oxide synthase affects stress-related Fos expression in specific brain regions.

To identify neuronal substrates involved in NO/stress interactions we used Fos expression as a marker and examined the pattern of neuronal activation in response to swim stress in nNOS knock-out (nNOS-/-) and wild-type (WT) mice. Forced swimming enhanced Fos expression in WT and nNOS-/- mice in several brain regions, including cortical, limbic and hypothalamic regions. Differences in the Fos response between the two groups were observed in a limited set (6 out of 42) of these brain areas only: nNOS-/- mice displayed increased stressor-induced Fos expression in the medial amygdala, periventricular hypothalamic nucleus, supraoptic nucleus, CA1 field of the hippocampus, dentate gyrus and infralimbic cortex. No differences were observed in regions including the septum, central amygdala, periaqueductal grey and locus coeruleus. During forced swimming, nNOS-/- mice displayed reduced immobility duration, while no differences in general locomotor activity were observed between the groups in the home cage and during the open field test. The findings indicate that deletion of nNOS alters stress-coping ability during forced swimming and leads to an altered pattern of neuronal activation in response to this stressor in specific parts of the limbic system, hypothalamus and the medial prefrontal cortex.

Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor gamma2 subunit.

Agonists of the allosteric benzodiazepine site of GABAA receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma2 subunit to the actions of zolpidem, an alpha1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit. The gamma2F77I mutation has no major effect on the expression of GABAA receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of [3H]flunitrazepam binding to cerebellar membranes is greatly reduced in gamma2I77/I77 mice. Zolpidem (1 microM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma2F77/F77 (20% and 84%) mice, but not in those of gamma2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma2F77I point mutation.

Expression of transcription factors in the brain of rats with perinatal asphyxia.

No information is available on transcription factors (TF), the main regulators of gene expression, in perinatal asphyxia (PA), and as pathomechanisms in PA are different, data on TFs from ischemia or hypoxia cannot be simply extrapolated to PA, and no studies have been reported to show an expressional pattern or the concerted action of TFs. We, therefore, used a gene-hunting technique, subtractive hybridization, to show sequences different in brains of normoxic and perinatally asphyxiated (10 and 20 min of asphyxia) rats. These subtracted sequences were identified by gene bank and assigned to individual genes. At 10 min of PA the TFs NFI/CAAT-binding protein, NF-kappa-B p65, N-myc, basic helix loop helix protein D82868, and c-myc intron binding protein were upregulated. At 20 min of PA the TFs SOX4 and neuronal death factor were upregulated, whereas the TFs c-maf, PEBP major transcription factor, brn-2, homeodomain protein Af004431, and zinc finger transcriptional factor M65008 were downregulated. The biological meaning of our findings is the demonstration of a pathophysiological pattern of TFs including POU, zinc finger, homeodomain, and basic helix-loop helix motifs in PA, proposing pathomechanisms for brain damage from PA, explaining transcriptional changes in general (as, e.g., NF-kappa-B p65, etc.) or in specific terms (as, e.g., neuronal death factor).

Long-term sequelae of perinatal asphyxia in the aging rat.

Information on the consequences of perinatal asphyxia (PA) on brain morphology and function in the aging rat is missing although several groups have hypothesized that PA may be responsible for neurological and psychiatric deficits in the adult. We therefore decided to study the effects of PA on the central nervous system (CNS) in terms of morphology, immunohistochemistry, neurology and behavior in the aging animal. Hippocampus and cerebellum were evaluated morphologically by histological, immunohistochemical and magnetic resonance imaging and cerebellum also by stereological tests. Neurological function was tested by an observational test battery and rota rod test. Cognitive functions were examined by multiple-T-maze and the Morris water maze (MWM). Increased serotonin transporter (SERT) immunoreactivity in the CA2 region of the hippocampus and a significant difference in the escape latency, when the platform of the MWM was moved to a new location, were observed in asphyxiated rats. We showed that deteriorated cognitive functions accompanied by aberrant expression of hippocampal SERT and impaired relearning are long-term sequelae of perinatal asphyxia, a finding that may form the basis for understanding CNS pathology in the aging subject, animal or human.

Brain fatty acids in perinatal asphyxia.

In hypoxic or ischemic states the release of fatty acids is proposed to have several harmful effects on brain structure and function. We therefore decided to study brain FFA in a simple, clinically related animal model resembling intrauterine perinatal asphyxia (PA). Cerebral blood flow (CBF), brain fatty acids (C14:0, C16:1, C16:0, C18:1, C1 8:0, sigma C), plasma glucose, lactate, beta-hydroxybutyrate (beta-OHB), non-esterified fatty acids (NEFA) and insulin were determined in PA and compared to the normoxic state. Brain C 14:0 FFA were not significantly different from normoxic rats. Brain FFA C 16:0 were comparable between groups but significantly decreased at 20 min of PA. C 18:0 FFA showed a trend to increase with the length of PA reaching significance at 10 min of asphyxia only and were declining at 20 min, however, not significantly. Brain C 16:1 and C 18:1 FFA concentrations were comparable between groups. The parameters cerebral blood flow, glucose and lactate showed a stepwise and significant increase with the length of PA, whereas beta-HOB, NEFA and insulin showed no changes. CBF, glucose and lactate showed a strong association whereas other parameters failed to correlate with each other. Only inconsistent trends of increased brain FFA were found and the association between brain glucose and brain FFA could be ruled out. Although CBF was manifold and significantly elevated in PA, brain FFA pattern suggests that the increase of CBF is obviously not mediated by brain FFA. We conclude that FFA may not be involved in the early phase-pathogenesis of PA.

Long term neurological and behavioral effects of graded perinatal asphyxia in the rat.

Perinatal hypoxic-ischemic states can cause irreversible damage to the brain, ranging from minimal brain dysfunction to death. Only few studies have been reported describing neurological, cognitive and behavioral deficits following perinatal asphyxia. We therefore decided to study long term effects of perinatal asphyxia in a well-documented animal model resembling the clinical situation. Caeserean section in rats was performed and the pups, still in the uterus horns, were placed into a water bath at 37 degrees C for periods of 5-20 min; pups were then given to surrogate mothers and examined at three month of age. Examinations consisted of a battery of motor and reflex tests, Morris water maze, multiple T-maze, elevated plus maze and open field studies. No abnormalities were found in rats even with long periods of perinatal asphyxia by neurological examination, in the open field and in mazes. Interestingly, in the elevated plus maze rats with long lasting exposure to hypoxia (15 and 20 min of asphyxia) showed reduced anxiety-related behavior. This finding may be relevant for the explanation of anxiety related disorders in adulthood with a tentative history in the perinatal period.

Brain RNA polymerase and nucleolar structure in perinatal asphyxia of the rat.

Ribosomes are integral constitutens of the protein synthesis machinery. Polymerase I (POL I) is located in the nucleolus and transcribes the large ribosomal genes. POL I activity is decreased in ischemia but nothing is known so far on POL I in perinatal asphyxia. We investigated the involvement of POL I in a well-documented model of graded systemic asphyxia at the level of activity, mRNA, protein, and morphology. Caeserean section was performed at the 21st day of gestation. Rat pups still in the uterus horns were immerged in a water bath for asphyctic periods from 5-20 min. Brain was taken for measurement of pH, nuclear POL I activity, and mRNA steady state, and protein levels of RPA40, an essential subunit of POL I and III. Silver staining and transmission electron microscopy with morphometry when appropriate were used to examine the nucleolus. Brain pH and nuclear POL I activity decreased with the length of the asphyctic period while POL-I mRNA and protein levels were unchanged. Accompanying the decrease in brain pH we found significant changes of nucleolar structure in the course of perinatal asphyxia at the light and electron microscopic level. As early as ten min following the asphyctic insult, morphological disintegration of the nucleolus was observed. The changes became more dramatic with longer duration of perinatal asphyxia. We conclude that severe acidosis may be responsible for decreased POL activity and for disintegration of nucleoli in neurons. This condition may lower the ribosome content in neonatal neurons and impair protein synthesis.

Glucose transporters, hexokinase, and phosphofructokinase in brain of rats with perinatal asphyxia.

Transport by glucose transporters from blood to the brain during hypoxic-ischemic conditions is well studied. However, the recent availability of a clinically related animal model of perinatal asphyxia and the fact that no concomitant determination of glucose transporters, parameters for glucose utilization, brain glucose, and cerebral blood flow (CBF) have been reported and the early phase of perinatal asphyxia has never been studied led us to perform the following study. Cesarean section was performed on full-term pregnant rats. The obtained pups within patent uterus horns were placed into a water bath at 37 degrees C from which they were subsequently removed after 5-20 min of graded asphyxia. Brain pH, brain tissue glucose, CBF, mRNA and activity of hexokinase and phosphofructokinase, and mRNA and protein of the glucose transporters GLUTI and GLUT3 were determined. Brain pH decreased and brain tissue glucose and CBF increased with the length of the asphyctic period; hexokinase and phosphofructokinase mRNA and activity were unchanged during the observation period. The mRNA and protein of both glucose transporters were comparable between normoxic and asphyctic groups. We show that glucose transport and utilization are unchanged in the early phase of perinatal asphyxia at a time point when CBF and brain glucose are already significantly increased and severe acidosis is present.

Cholinergic, monoaminergic and glutamatergic changes following perinatal asphyxia in the rat.

Perinatal asphyxia (PA) is considered to lead to a variety of brain disorders including spasticity, epilepsy, mental retardation, and minimal brain disorder syndromes and may form the basis for psychiatric and neurodegenerative diseases later in life. We examined markers for neuronal transmission involved in the pathomechanisms of PA and candidates as mediators for long-term sequelae. We tested tyrosine hydroxylase (TH) and the vesicular monoamine transporter (VMAT) representing the monoaminergic system, the vesicular acetylcholine transporter (VAChT), and the excitatory amino acid carrier 1 (EAAC1), a neuronal subtype of the glutamate transporter, using immunohistochemistry on brain sections of rats subjected to graded PA. Three months following the asphyxiant insult immunoreactive (IR)-TH was decreased in striatum, hippocampus, thalamus, frontal cortex, and cerebellum; IR-VMAT was increased, and IR-VAChT was decreased in striatum. IR-EAAC1 glutamate transporter was increased in frontal cortex. The cholinergic, monoaminergic, and glutamatergic changes, still observed 3 months after the asphyxiant insult, may reflect their involvement in the pathomechanisms of PA and indicate mechanisms leading to long-term complications of PA. The variable consequences on the individual markers in several brain regions may be explained by specific susceptibility of cholinergic, monoaminergic, and glutamatergic neurons to the asphyxiant insult.

Nitric oxide and nitric oxide synthase in the early phase of perinatal asphyxia of the rat.

The role of nitric oxide, a compound involved in neurotransmission and regulation of cerebral blood flow, in cerebral ischemia is still not fully elucidated yet. Although well studied in adult systems of cerebral ischemia/hypoxia, information on nitric oxide in perinatal asphyxia is limited and, in particular, no direct evidence for its generation has been provided. We therefore decided to study nitric oxide generation in brain of asphyctic rat pups by biophysical and biochemical methods. We used a simple, non-invasive rat model resembling the clinical situation in perinatal asphyxia: rat pups delivered by Caesarean section were placed into a water bath at 37 degrees C still in patent membranes for various asphyctic periods (up to 20 min). Brain pH, cerebral blood flow, neuronal nitrix oxide synthase messenger RNA (by northern and dot blot analysis), immunoreactive protein (by western blot analysis) and nitric oxide synthase activity were determined; generation of nitric oxide was evaluated directly by electron paramagnetic resonance spectroscopy. Neuronal nitric oxide synthase messenger RNA activity and nitric oxide generation were unaffected, whereas neuronal nitric oxide synthase-immunoreactive protein of 150,000 mol. wt was decreased and of 136,000 mol. wt was increased with the length of the asphyctic period. This is the first report on direct evidence for the generation of nitric oxide in perinatal asphyxia and we demonstrate that nitric oxide production remains unaffected even by 20 min of asphyxia, at a time-point when cerebral blood flow was increased four-fold and severe acidosis was present. However, it was found that levels of immunoreactive neuronal nitric oxide synthase of 136,000 mol. wt were increased paralleling the length of asphyxia. Levels of the 150,000 mol. wt immunoreactive neuronal nitric oxide synthase protein decreased, suggesting a different regulation pattern. Thus, the present biochemical and biophysical results form the basis for further investigations on nitric oxide in perinatal asphyxia.

Histological changes and neurotransmitter levels three months following perinatal asphyxia in the rat.

The involvement of excitatory amino acids (EAA) in the pathogenesis of hypoxic-ischemic states is well-documented. Information on the role of overexcitation by EAA in perinatalasphyxia (PA), however, is limited and data from adult models cannot be directly extrapolated to immature systems. Moreover, most adult models of ischemia are representing stroke rather than PA. We decided to study long term effects in a non-invasive rat model of PA resembling the clinical situation three months following the asphyctic insult. Morphometry on Nissl - stained sections was used to determine neuronal death in frontal cortex, striatum, hippocampus CA1, hypothalamus and cerebellum L1, and the amino acids glutamate, glutamine, aspartate, GABA, taurine, arginine as well as histamine, serotonin and 5-hydroxy-indoleacetic acid were determined in several brain regions and areas. Morphometry revealed that neuronal loss was present in the hippocampal area CA1 in all groups with PA and that morphological alterations were significantly higher in the cerebellar granular layer. The prominent light microscopical finding in all areas of asphyctic rats studied was decreased Nissl-staining, suggesting decreased cellular RNA levels. Glutamate, aspartate and glutamine were significantly elevated in the hypothalamus of asphyctic rats probably indicating overstimulation by EAA. Excitotoxicity in this area would be compatible with findings of emotional / behavioral deficits observed in a parallel study in our model of PA. Our observations point to and may help to explain behavioral and emotional deficits in Man with a history of perinatal asphyxia.

Genes involved in the pathophysiology of perinatal asphyxia.

Mechanisms in the pathogenesis of perinatal asphyxia (PA) at the gene level are only beginning to be elucidated, although gene hunting using differential display has revealed differences in gene expression between hypoxic and normoxic cells in vitro. As no information on gene expression was available from in vivo studies, we decided to use a non-invasive and clinically relevant animal model of PA for mRNA hunting applying the subtractive hybridization method. mRNAs from normoxic rat brain and brain of rat pups with 20 min of asphyxia were isolated and compared by this technique. The resulting subtracted mRNAs were converted to cDNA, sequenced and identified by gene bank data. A series of transcripts representing transcription factors, transporters, metabolic factors, were found to be up- or downregulated providing insight into mechanisms of PA, and on the other hand, genes with unknown functions could be given a preliminary role i.e. in PA. Results obtained with this powerful tool are now challenging quantitative determination of these genes and gene products at the protein and activity level to confirm their role in PA.

Evidence that taurine modulates osmoregulation by modification of osmolarity sensor protein ENVZ--expression.

Although the involvement of taurine in osmoregulation is well-documented and widely accepted, no detailed mechanism for this function has been reported so far. We used subtractive hybridization to study mRNA steady state levels of genes up- or downregulated by taurine. Rats were fed taurine 100 mg/kg body weight per day for a period of three days and hearts (total ventricular tissue) of experimental animals and controls were pooled and used for mRNA extraction. mRNAs from two groups were used for subtractive hybridization. Clones of the subtractive library were sequenced and the obtained sequences were identified by gen bank assignment. Two clones were found to contain sequences which could be assigned to the osmolarity sensor protein envZ, showing homologies of 61 and 65%. EnvZ is an inner membrane protein in bacteria, important for osmosensing and required for porine gene regulation. It undergoes autophosphorylation and subsequently phosphorylates OmpR, which in turn binds to the porine (outer membrane protein) promoters to regulate the expression of OmpF and OmpC, major outer membrane porines. This is the first report of an osmosensing mechanism in the mammalian system, which was described in bacteria only. Furthermore, we are assigning a tentative role for taurine in the osmoregulatory process by modifying the expression of the osmoregulatory sensor protein ENVZ.

Aromatic hydroxylation in animal models of diabetes mellitus.

Although the involvement of oxidative stress is well documented in the diabetic state, the individual active oxygen species generated have not been demonstrated in animal models of diabetes currently used. Since streptozotocin-induced diabetes mellitus in animals still serves as an animal model of diabetes mellitus, but streptozotocin induces diabetes and generates oxidative stress per se, we decided to study whether aromatic hydroxylation reflecting hydroxyl radical attack was found in three animal models of diabetes mellitus without streptozotocin induction or in streptozotocin-induced diabetes only. For this purpose, we compared lipid peroxidation, aromatic hydroxylation of phenylalanine, glycoxidation in genetically determined diabetic mouse strains db/db and kk, and the diabetic BB rat to these parameters in the streptozotocin-treated rat. Kidney malondialdehyde concentrations, reflecting lipid peroxidation, pentosidine, and Nepsilon-caboxymethyllysine concentrations, reflecting glycoxidation, were significantly elevated in all diabetic groups as compared to their nondiabetic mates. Aromatic hydroxylation was significantly elevated in the streptozotocin-induced diabetic state exclusively. We conclude that biochemical, pathophysiological, and treatment studies in the streptozotocin model of diabetes mellitus may be confounded by the presence of products, reactions, and tissue damage generated by aromatic hydroxylation reflecting hydroxyl radical attack. We suggest it is not the diabetic state but streptozotocin that generates the hydroxyl radical, as reflected by aromatic hydroxylation in this model.