Alteration of brain glycogen turnover in the conscious rat after 5h of prolonged wakefulness.

Although glycogen (Glyc) is the main carbohydrate storage component, the role of Glyc in the brain during prolonged wakefulness is not clear. The aim of this study was to determine brain Glyc concentration ([]) and turnover time (tau) in euglycemic conscious and undisturbed rats, compared to rats maintained awake for 5h. To measure the metabolism of [1-(13)C]-labeled Glc into Glyc, 23 rats received a [1-(13)C]-labeled Glc solution as drink (10% weight per volume in tap water) ad libitum as their sole source of exogenous carbon for a "labeling period" of either 5h (n=13), 24h (n=5) or 48 h (n=5). Six of the rats labeled for 5h were continuously maintained awake by acoustic, tactile and olfactory stimuli during the labeling period, which resulted in slightly elevated corticosterone levels. Brain [Glyc] measured biochemically after focused microwave fixation in the rats maintained awake (3.9+/-0.2 micromol/g, n=6) was not significantly different from that of the control group (4.0+/-0.1 micromol/g, n=7; t-test, P>0.5). To account for potential variations in plasma Glc isotopic enrichment (IE), Glyc IE was normalized by N-acetyl-aspartate (NAA) IE. A simple mathematical model was developed to derive brain Glyc turnover time as 5.3h with a fit error of 3.2h and NAA turnover time as 15.6h with a fit error of 6.5h, in the control rats. A faster tau(Glyc) (2.9h with a fit error of 1.2h) was estimated in the rats maintained awake for 5h. In conclusion, 5h of prolonged wakefulness mainly activates glycogen metabolism, but has minimal effect on brain [Glyc].

Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy.

Central serous chorioretinopathy (CSCR) is a vision-threatening eye disease with no validated treatment and unknown pathogeny. In CSCR, dilation and leakage of choroid vessels underneath the retina cause subretinal fluid accumulation and retinal detachment. Because glucocorticoids induce and aggravate CSCR and are known to bind to the mineralocorticoid receptor (MR), CSCR may be related to inappropriate MR activation. Our aim was to assess the effect of MR activation on rat choroidal vasculature and translate the results to CSCR patients. Intravitreous injection of the glucocorticoid corticosterone in rat eyes induced choroidal enlargement. Aldosterone, a specific MR activator, elicited the same effect, producing choroid vessel dilation -and leakage. We identified an underlying mechanism of this effect: aldosterone upregulated the endothelial vasodilatory K channel KCa2.3. Its blockade prevented aldosterone-induced thickening. To translate these findings, we treated 2 patients with chronic nonresolved CSCR with oral eplerenone, a specific MR antagonist, for 5 weeks, and observed impressive and rapid resolution of retinal detachment and choroidal vasodilation as well as improved visual acuity. The benefit was maintained 5 months after eplerenone withdrawal. Our results identify MR signaling as a pathway controlling choroidal vascular bed relaxation and provide a pathogenic link with human CSCR, which suggests that blockade of MR could be used therapeutically to reverse choroid vasculopathy.

DETERMINATION OF CELL-SPECIFIC NEUROTOXICITY OF MALONATE, METHYLMALONATE AND PROPIONATE IN A 3D RAT BRAIN CELL AGGREGATE SYSTEM

Malonate, methylmalonate and propionate are potentially neurotoxic metabolites in branched-chain organic acidurias. Their effects were tested on cultured 3D rat brain cell aggregates, using dosages of 0.1, 1.0 and 10.0 mM with a short but intense (twice a day over 3 days) and a longer but less intense treatment (every 3 rdday over 9 days). CNS cell-specific immunohistochemical stainings allowed the follow-up of neurons (axons, phosphorylated medium-weight neurofilament), astrocytes (glial fibrillary acidic protein) and oligodendrocytes (myelin basic protein). Methylmalonate and malonate were quantified by tandem mass spectrometry. Tandem mass spectrometry analysis of harvested brain cell aggregates revealed clear intracellular accumulation of methylmalonate and malonate. In immunohistochemical stainings oligodendrocytes appeared the most affected brain cells. The MBP signal disappeared already at 0.1 mM treatment with each metabolite. Mature astrocytes were not affected by propionate, while immature astrocytes on intense treatment with propionate developed cell swelling. 1 mM methylmalonate induced cell swelling of both immature and mature astrocytes , while 1 mM malonate only affected mature astrocytes. Neurons were not affected by methylmalonate, but 10.0 mM malonate on less intense treatment and 0.1, 1.0 and 10.0 mM propionate on intense treatment affected axonal growth. Our study shows significant uptake and deleterious effects of these metabolites on brain cells, principally on astrocytes and oligodendrocytes. This may be explained by the absence of the pathway in glial cells, which thus are not able to degrade these metabolites. Further studies are ongoing to elucidate the underlying mechanisms of the observed neurotoxic effects.

DIFFERENTIAL EXPRESSION OF GLUTARYL-CoA DEHYDROGENASE IN ADULT RAT CNS, PERIPHERAL TISSUES AND DURING EMBRYONIC DEVELOPMENT

Glutaryl-CoA dehydrogenase (GCDH, EC 1.3.99.7) deficiency, known as glutaric acidemia type I, is one of the more common organic acidurias. To investigate the role of this pathway in different organs we studied the tissue-specific expression pattern of rat Gcdh. The open reading frame cDNA of the rat Gcdh gene was cloned from rat brain mRNA by RT-PCR, allowing the synthesis of digoxigenin-labeled in situ hybridization (ISH) riboprobes. Gcdh mRNA expression was analyzed by ISH on cryosections of adult rat brain, kidney, liver, spleen and heart muscle, as well as on E15 and E18 rat embryos. Gcdh was found expressed in the whole rat brain, almost exclusively in neurons. Gcdh was absent from astrocytes but expressed in rare oligodendrocytes. Strong Gcdh expression was found in liver and spleen, where expression appears predominant to lymphatic nodules. In kidney, the highest Gcdh expression is found in the juxtamedullar cortex (but not in glomerula), and at lower levels in medulla. Heart muscle was negative. During embryonic development, Gcdh was found well expressed in liver, intestinal mucosa and skin, as well as at lower levels in CNS. Further studies are ongoing to provide evidence on the presence of the entire pathway in CNS in order to understand the mechanisms leading to neurotoxicity in glutaric aciduria. The high expression of Gcdh in kidney may explain why certain patients with residual enzyme activity are low excretors at the urine metabolite level.

A cognitive attempt to understanding female entrepreneurial potential: the role of social norms and culture

This article analyzes the different perceptions of both male and female potential entrepreneurs from three European regions differing in their respective level of economic development and entrepreneurial culture. We use an extended cognitive model of entrepreneurial intentions based on the theory of planned behaviour, the theory of normative social behaviour and social capital literature. Results show females have lower self-efficacy and entrepreneurial attraction than males, thus leading to lower entrepreneurial intention. Differences between the three subsamples are small when males are studied. However, female entrepreneurial intentions and perceptions are more affected by the cultural context.

Diagnostic Importance of Female External Genital Structure of Phlebotomine Sand Flies (Diptera:Psychodidae) as Observed by Scanning Electron Microscopy

Morphological description of sand flies has remained a neglected area. The different organs used in taxonomy have not yet been described adequately with the scanning electron microscopy (SEM). We have examined the external genital structures of females of three Old World phlebotomine sand flies under SEM and recorded the morphological variations of the organs. We have found the female external genital structures of the three species varied considerably in morphology. The importance of the female external genital structures in sand fly identification is indicated

Morphology and Histology of the Female Reproductive System of Abedus ovatus Stal (Belostomatidae: Hemiptera: Insecta)

In Abedus ovatus Stal, the female reproductive system consists of a pair of ovaries leading to oviduct and opens out by genital aperture. Each ovary is composed of five telotrophic ovarioles, with four different regions, terminal filament, germarium, vitellarium and pedicel. Germarium and vitellarium are the germ tubes in which the development of oocyte occurs. In the germarium, the differentiation of trophocytes into young oocytes was observed in three zones, ZI, ZII and ZIII. Further development of oocytes in the vitellarium could be divided into seven stages, STI STVII. The developed eggs are lodged in the pedicel

EIU in the rat promotes the potential of syngeneic retinal cells injected into the vitreous cavity to induce PVR.

PURPOSE: To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR). METHODS: Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed. RESULTS: The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye. CONCLUSIONS: In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.

Two monoclonal rat antibodies with specificity for the beta-chain variable region V beta 6 of the murine T-cell receptor.

Two rat monoclonal antibodies (mAbs), 44-22-1 and 46-6B5, which recognize an alloreactive cytotoxic clone, 3F9, have been further tested on a panel of T hybridomas and cytotoxic T-cell clones for binding and functional activities. The mAbs recognized only those cells sharing the expression of the T-cell receptor beta-chain variable region gene V beta 6 with 3F9. All V beta 6+ cells were activated by these mAbs under cross-linking conditions and their antigen-specific activation was blocked by soluble mAb. Furthermore, depletion of 46-6B5+ normal lymph node T cells eliminated all cells expressing the epitope recognized by 44-22-1 and V beta 6 mRNA.

Effect of selective phosphodiesterase inhibitors on the rat eosinophil chemotactic response in vitro

In the present study, we have performed a comparative analysis of the effect of selective inhibitors of phosphodiesterase (PDE) type III, IV and V on eosinophil chemotaxis triggered by platelet activating factor (PAF) and leukotriene B4 (LTB4) in vitro. The effect of the analogues N6-2'-O-dibutyryladenosine 3':5' cyclic monophosphate (Bt2 cyclic AMP) and N2-2'-O- dibutyrylguanosine 3':5' cyclic monophosphate (Bt2 cyclic GMP) has also been determined. The eosinophils were obtained from the peritoneal cavity of naive Wistar rats and purified in discontinuous Percoll gradients to 85-95% purity. We observed that pre-incubation of eosinophils with the PDE type IV inhibitor rolipram suppressed the chemotactic response triggered by PAF and LTB4, in association with an increase in the intracellular levels of cyclic AMP. In contrast, neither zaprinast (type V inhibitor) nor type III inhibitors milrinone and SK&F 94836 affected the eosinophil migration. Only at the highest concentration tested did the analogue Bt2 cyclic AMP suppress the eosinophil chemotaxis, under conditions where Bt2 cyclic GMP was ineffective. We have concluded that inhibition of PDE IV, but not PDE III or V, was able to block the eosinophil chemotaxis in vitro, suggesting that the suppressive activity of selective PDE IV inhibitors on tissue eosinophil accumulation may, at least, be partially dependent on their ability to directly inhibit the eosinophil migration.

Induction of calmodulin kinase IV by the thyroid hormone during the development of rat brain.

This communication reports the specific induction of calmodulin kinase IV by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3) in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system, which can grow and differentiate under chemically defined conditions. The induction of the enzyme that can be observed both on the mRNA and on the protein level is T3-specific, i.e. it cannot be induced by retinoic acid or reverse T3, and can be inhibited on both the transcriptional and the translational level by adding to the culture medium actinomycin D or cycloheximide, respectively. The earliest detection of calmodulin kinase IV in the fetal brain tissue of the rat is at days E16/E17, both on the mRNA as well as on the protein level. This is the first report in which a second messenger-dependent kinase involved in the control of cell regulatory processes is itself controlled by a primary messenger, the thyroid hormone.

Female-biased infection and transmission of the gastrointestinal nematode Trichuris arvicolae infecting the common vole, Microtus arvalis.

Previous studies addressing the importance of host gender in parasite transmission have shed light on males as the more important hosts, with the higher transmission potential of males being explained by the fact that they often harbour higher parasite loads than females. However, in some systems females are more heavily infected than males and may be responsible for driving infection under such circumstances. Using a wild population of common voles (Microtus arvalis), we showed that females were more frequently infected by the intestinal nematode Trichuris arvicolae than males (i.e. prevalence based on the presence of eggs in the faeces) and that females were shedding greater numbers of parasite eggs per gram of faeces (EPG) than males. By applying an anthelmintic treatment to either male or female voles, we demonstrated that treating females significantly reduced parasite burdens (i.e. prevalence and EPG) of both male and female hosts, while treating males only reduced parasite burden in males. These findings indicate that in this female-biased infection system females play a more important role than males in driving the dynamics of parasite transmission.