In addition to the acute pathology produced by traumatic brain injury, there are chronic alterations that occur after the trauma, including a depressed state of neuronal activity (Feeney, 1991). This study included a preclinical testing of a novel treatment strategy focusing on increasing neuronal activity during the chronic hypofunctional posttraumatic stage. The present investigation tested the effects of repeated post-injury aniracetam administration on cognitive performance in the Morris water maze (MWM) and on the GluR2 - immunoreactivity and protein expression by Western blot analysis in the hippocampus. The first study examined the optimal dose of aniracetam in the MWM task. Animals received aniracetam (25 mg/kg, 50 mg/kg) or vehicle once daily for fifteen days and on days 11-15 were tested in the MWM. The results indicated that injured aniracetam-treated rats had a significant improvement in MWM performance compared to injured saline-treated animals. When the drug was delayed for 11 days post-injury in the second experiment, its beneficial effects were still present, as injured aniracetam-treated rats performed significantly better that injured saline treated rats on the MWM task. In the third experiment, chronic daily aniracetam administration was terminated after 15 days immediately before MWM testing on days 16-20. The results indicated that termination of aniracetam did not enhance MWM performance as injured terminated aniracetam-treated rats did not have significant improvement over injured saline-treated rats. In the fourth study we investigated the mechanism of aniracetam's effects by examining the expression of the AMPA receptor GluR2 subunit, the only AMPA receptor subunit that is Ca++ impermeable. Using a monoclonal antibody selective for the GluR2 subunit, immunohistochemical results indicated that injured rats treated with aniracetam (50mg/kg for 15 days post-injury) had a slight reduction in the GluR2- IR. The fifth study investigated a change in the GluR2 protein expression in the hippocampus with a Western blot analysis. The results were consistent with the immunohistochemical study outcome as the injured vehicle and injured aniracetam treated animals showed a reduced protein expression in the hippocampus. The changes were not significantly different from the controls. The results of these experiments suggested that chronic aniracetam treatment significantly attenuated injury induced spatial memory deficits when administered continually during the hypofunctional posttraumatic stage and when the treatment was delayed for 11 days, but not when the treatment was terminated before the MWM testing. These effects suggest that the compound does not induce chronic receptor changes and has to be biologically active in an organism for it to exert its beneficial properties. Results from the present studies suggest that aniracetam may become a potential treatment option for brain injury induced cognitive deficits.

The ankyrins comprise a family of proteins serving as components of the membrane cytoskeleton, and participate in a diverse set of associations with multiple binding partners including the cytoplasmic domains of transporters, ion channels, some classes of receptors, and cell adhesion proteins. Moreover, evidence is accumulating that ankyrin participates in defining functionally distinct subcellular regions. The complex functional and structural roles of ankyrins indicate they are likely to play essential roles in the pathology of traumatic axonal injury. The current study examined changes in ankyrin-G expression following a moderate central fluid percussion injury administered to adult rats. At 1d, 3d, and 7d postinjury (or following a sham control injury), protein levels of ankyrin-G in the corpus callosum and cerebral cortex were assessed using Western Blot analysis. Three immunopositive bands were identified in both brain regions as 220,212, and 75 kD forms of ankyrin-G. Time-dependent changes in ankyrin-G were observed in the corpus callosum. At 1d injury-induced elevations were observed in the callosal 220 kD (+147% relative to sham levels) and in the 212 kD (+73%) forms of ankyrin-G, but in both cases the expression decreased to control levels by 3d and 7d. In contrast, the 75 kD form showed moderate increases at 1d postinjury, but was significantly below control levels at 3d (-54%) and at 7d (-41%). Ankyrin-G expression in the cerebral cortex was only slightly affected by the injury, with a significant decrease in the `220 kD form occurring between 1d and 3d. These data suggest that the 220 and 212 kD changes probably represent postinjury proteolytic fragments derived from intact ankyrin-G isoforms of 480 andor 270 kD, while the 75 kD effects are likely breakdown products of intact 190 kD ankyrin-G. These results were discussed as they relate to prior findings of differential vulnerabilities of callosal myelinated and unmyelinated axons to injury. In this context, the 220,212 kD changes may reflect pathology within myelinated axons, and alterations to the 75 kD form may reflect more persistent pathology affecting unmyelinated callosal fibers.

<p>The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific</p><p>in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery.</p><p>In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases.</p> / <p>Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.</p>

Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of Diploma Thesis: Effect of long-term exposure to soluble endoglin on the expression of adhesion molecules on endothelial cells Author of Diploma Thesis: Dominika Tuschlová Supervisor of Diploma Thesis: PharmDr. Iveta Najmanová, Ph.D. Background: The aim of this thesis was to find out the effect of long-term exposure to soluble endoglin (sEng) on the expression of adhesion molecules on endothelial cells (ICAM-1, VCAM-1, P-selectin). We have worked with a control and experimental group of mice on a standard diet, with different levels of sEng. Methods: Genetically modified female mice from the CBAxC57BL/6J strain with high production of human sEng were used for analysis. These mice formed an experimental group, while mice with a low level of sEng formed a control group. They were 12 months old females. We used biochemical analysis to determine the level of total cholesterol and triacylglycerols (TAG). Levels of sVCAM-1 markers in all mice studied were determined by ELISA. The expression of the adhesion molecules ICAM-1, VCAM-1 and P-selectin was monitored and evaluated by Western blotting, where the structural protein GAPDH was used as a control. Results: Biochemical analysis did not show a...

Objective: Reductions in sodium pump (SP) abundance can give rise to increases in contractile force in uterine and vascular smooth muscle as well as an increased secretion in secretory cells, including potentially those of the placenta. To determine whether the mouse might serve as a model for human pregnancy in terms of the SP and to determine whether changes in SP abundance anticipate or follow labor, we studied pregnant mice over the final trimester of their pregnancy. Study Design: C57Bl6 dams (n=46) were bred and studied during their pregnancy. Animals (n=4) were sacrificed at specific gestational time points. Other mice had labor induced with LPS on Gestational day 15 and were then studied at specific time points after induction. Specimens were studied for mRNA abundance as well as protein abundance using methods such as Real time RT-PCR and Western blot analysis. Data were analyzed by ANOVA with post hoc Duncan's pair-wise comparisons. Results: Levels of uterine SP α3 isoform mRNA were most abundant on day 14 near the beginning of the third trimester. There was a significant fall in SP &alpha3 mRNA abundance by day 18 with a slightly lower level on the day of birth but an increased SP α3 mRNA abundance by one day post partum. Contrary to the uterus, SP α3 mRNA levels in the placenta increased over the last trimester, from day 14 to the day of birth. Western blot analysis on the two tissues demonstrated a somewhat similar pattern. In the LPS studies of uterus and placenta, the SP α3 isoform protein abundance appeared to fall when compared to the 2 hour time point. Those animals which were injected with a vehicle control showed very little change in SP α3 abundance after injection. While protein levels were reduced, there was no significant reduction in mRNA for all specimens. Conclusion: Uterine SP α3 isoform protein expression fell late in mouse pregnancy but prior to labor and appeared to be mediated by reductions in its mRNA. These reductions paralleled changes observed in term pregnant women. Such reductions would increase the sensitivity of the uterus to agents causing contraction but may directly increase the force, duration and frequency of contractions. Placental SP α3 isoform protein expression had no significant change over the final trimester. However, unlike uterine protein, the placental protein may not be mediated by its mRNA. Reductions in SP α3 protein abundance were also seen in preterm labor produced by LPS induction. These changes may not be mediated by mRNA. Taken together, changes in the SP α3 isoform may represent a fundamental mechanism in the initiation and/ or progression of term labor and in preterm in mouse and potentially in human.

The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery. In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases. / Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.

Type II diabetes mellitus is associated with many detrimental health situations including heart complications. The purpose of this study was to identify a role for PKA-dependent Rap1a signaling in the AGE-RAGE cascade. My hypothesis was Rap1a GTPase increased the downstream effects of AGE-RAGE signaling in diabetes via a PKA-dependent pathway leading to elevated ECM remodeling in the heart. Cardiac fibroblasts were isolated from heterozygous (Het) and diabetic (db/db) mice. To test the hypothesis, gain-ofunction and loss-ofunction treatments were used. PKC-Zeta is known as a major signaling hub that potentially links PKA-dependent and AGE-RAGE signaling cascades so PKC-Zeta inhibition to downregulate PKA-dependent cascade at PKC-Zeta was also used. Results showed a downregulation of signaling markers in the AGE-RAGE cascade when disrupting Rap1a crosstalk at PKC-Zeta. By understanding where the PKA-dependent and AGE-RAGE signaling cascades crosstalk, a new molecular mechanism is understood possibly leading to decreasing remodeling in a diabetic heart.

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