Upregulation of CYP2e1 and CYP3a activities in histamine-deficient histidine decarboxylase gene targeted mice

Histamine is a biogenic amine with multiple physiological functions. Its importance in allergic inflammation is well characterized; moreover, it plays a role in the regulation of gastric acid production, various hypothalamic functions, such as food uptake, and enhancing TH2 balance during immune responses. Using histidine decarboxylase gene targeted (HDC(-/-)) BALB/c mice, we studied the effect of the absence of histamine on four cytochrome p450 enzyme activities. Their selective substrates were measured: ethoxyresorufin O-dealkylase activity of CYP1A, pentoxyresorufin O-dealkylase activity of CYP2B, chlorzoxazone 6-hydroxylase activity of CYP2E1 and ethylmorphine N-demethylase activity of CYP3A.The results indicate a significant elevation of CYP2E1 and CYP3A activities, however, no change in CYP1A and CYP2B activities was seen in HDC targeted mice compared to wild type controls with identical genetic backgrounds.     

Growth and developmental responses of potato to osmotic stress under in vitro conditions

The effect of mannitol on different genotypes of potato was studied in callus and plantlet culture. In vitro responses of five potato genotypes with well-known field behaviour to water deficit were analysed. After a 4-week-long cultivation on media containing mannitol up to 0.8 M, different morpho-physiological parameters were determined and statistically analysed. The useful concentration of mannitol for in vitro screening the osmotic tolerance of different genotypes depended on the type of culture; it was 0.4 M in plantlet-test and 0.8 M in callus-test. In callus-test the relative increase of callus mass was a useful parameter for determination of osmotic tolerance of genotypes at cellular level. In plantlet culture, stress index calculated from the rate of surviving in vitro shoots, number and length of roots per surviving explant and the rate of rooted explants were applicable to determine three groups according to the tolerant, medium tolerant and sensitive categories in agreement with the field behaviour of these genotypes. Under in vitro stress conditions we were able to distinguish the examined genotypes with different drought tolerance.     

Kappa-opioid receptor in the rodent hippocampus: a comparative immunocytochemical study in the rat,guinea pig,hamster and gerbil

Pre-embedding light microscopic immunocytochemistry, using a monoclonal antibody (mAb-KA8) raised against a frog brain kappa receptor preparation, recognising selectively the kappa-opioid receptor, was used for studying the occurrence, distribution, and species-specificity of the kappa-opioid receptor in the hippocampal formation of four rodent species (rat, guinea pig, hamster and gerbil). MAb-KA8 immunoreactivity was detectable in the rat, hamster and gerbil hippocampus, however the distribution of the labelled structures was heterogeneous. In the rat and hamster the hilus of dentate gyrus and the stratum oriens of the CA1 area contained immunoreactive cell bodies and proximal dendrites. In the gerbil mAb-KA8 immunopositive cell bodies were recognisable in the stratum radiatum of the CA1 and CA3 areas and in the subiculum. In the hamster varicose axon-like elements were also detected in the CA3 pyramidal layer. With the mAb-KA8 antibody there was no detectable kappa opioid receptor labelling in the hippocampus of the guinea pig. The results confirm the high degree of species-specific heterogeneity characterising the distribution of opioid peptides and their receptors in the hippocampal formation. The receptor was found in most cases postsynaptically, however in the hamster the immunopositive axons may refer to a presynaptic localisation.     

Inactivation of the glycine transporter 1 gene discloses vital role of glial glycine uptake in glycinergic inhibition

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.     

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.     

Enhanced NMDA Receptor-Mediated Modulation of Excitatory Neurotransmission in the Dorsal Vagal Complex of Streptozotocin-Treated,Chronically Hyperglycemic Mice

A variety of metabolic disorders, including complications experienced by diabetic patients, have been linked to altered neural activity in the dorsal vagal complex. This study tested the hypothesis that augmentation of N-Methyl-D-Aspartate (NMDA) receptor-mediated responses in the vagal complex contributes to increased glutamate release in the dorsal motor nucleus of the vagus nerve (DMV) in mice with streptozotocin-induced chronic hyperglycemia (i.e., hyperglycemic mice), a model of type 1 diabetes. Antagonism of NMDA receptors with AP-5 (100 μM) suppressed sEPSC frequency in vagal motor neurons recorded in vitro, confirming that constitutively active NMDA receptors regulate glutamate release in the DMV. There was a greater relative effect of NMDA receptor antagonism in hyperglycemic mice, suggesting that augmented NMDA effects occur in neurons presynaptic to the DMV. Effects of NMDA receptor blockade on mEPSC frequency were equivalent in control and diabetic mice, suggesting that differential effects on glutamate release were due to altered NMDA function in the soma-dendritic membrane of intact afferent neurons. Application of NMDA (300 μM) resulted in greater inward current and current density in NTS neurons recorded from hyperglycemic than control mice, particularly in glutamatergic NTS neurons identified by single-cell RT-PCR for VGLUT2. Overall expression of NR1 protein and message in the dorsal vagal complex were not different between the two groups. Enhanced postsynaptic NMDA responsiveness of glutamatergic NTS neurons is consistent with tonically-increased glutamate release in the DMV in mice with chronic hyperglycemia. Functional augmentation of NMDA-mediated responses may serve as a physiological counter-regulatory mechanism to control pathological disturbances of homeostatic autonomic function in type 1 diabetes.     

Thymic Atrophy and Apoptosis of CD4+CD8+ Thymocytes in the Cuprizone Model of Multiple Sclerosis

Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone’s deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes.     

Mycorrhiza Symbiosis Increases the Surface for Sunlight Capture in Medicago truncatula for Better Photosynthetic Production

Arbuscular mycorrhizal (AM) fungi play a prominent role in plant nutrition by supplying mineral nutrients, particularly inorganic phosphate (P_i), and also constitute an important carbon sink. AM stimulates plant growth and development, but the underlying mechanisms are not well understood. In this study, Medicago truncatula plants were grown with Rhizophagus irregularis BEG141 inoculum (AM), mock inoculum (control) or with P_i fertilization. We hypothesized that AM stimulates plant growth through either modifications of leaf anatomy or photosynthetic activity per leaf area. We investigated whether these effects are shared with P_i fertilization, and also assessed the relationship between levels of AM colonization and these effects. We found that increased P_i supply by either mycorrhization or fertilization led to improved shoot growth associated with increased nitrogen uptake and carbon assimilation. Both mycorrhized and P_i-fertilized plants had more and longer branches with larger and thicker leaves than the control plants, resulting in an increased photosynthetically active area. AM-specific effects were earlier appearance of the first growth axes and increased number of chloroplasts per cell section, since they were not induced by P_i fertilization. Photosynthetic activity per leaf area remained the same regardless of type of treatment. In conclusion, the increase in growth of mycorrhized and P_i-fertilized Medicago truncatula plants is linked to an increase in the surface for sunlight capture, hence increasing their photosynthetic production, rather than to an increase in the photosynthetic activity per leaf area.