The Retinal TNAP

Accumulating evidence from recent literature underline the important roles of tissue non specific alkaline phosphatase (TNAP) in diverse functions as well as diseases of the nervous system. Exploration of TNAP in well characterized neural circuits such as the retina, might significantly advance our understanding regarding neural TNAP’s roles. This chapter reviews the scarce literature as well as our findings on retinal TNAP. We found that retinal TNAP activity was preserved and followed diverse patterns throughout vertebrate evolution. We have consistently observed TNAP activity (1) in retinal vessels, (2) in photoreceptors and (3) in the majority of the studied species in the outer (OPL) and inner plexiform layers (IPL), where synaptic transmission occurs. Importantly, in some species the IPL exhibits several TNAP positive strata. These strata exactly corresponded those seen after quadruple immunohistochemistry with four canonical IPL markers (tyrosine hydroxylase, choline acetyltransferase, calretinin, protein kinase C α). Diabetes results in diminishing retinal TNAP activity before changes in canonical markers

Physicochemical Characterization and Cyclodextrin Complexation of the Anticancer Drug Lapatinib

Lapatinib (LAP), the tyrosine kinase inhibitor drug with moderate bioavailability, was characterized in terms of physicochemical properties: acid-base characteristics, lipophilicity, and solubility. The highly lipophilic nature of the drug and its extremely low water solubility (S0=0.82 nM) limit the development of a parenteral formulation. In order to enhance solubility and bioavailability, inclusion complex formation with cyclodextrins (CDs) is a promising method of choice. Therefore, LAP-CD interactions were also studied by a multianalytical approach. The stability constants of LAP with native cyclodextrins, determined by UV spectroscopy, identified the seven-membered β-CD as the most suitable host. Continuous variation method (Job’s plot) by 1H NMR showed a 1 : 1 stoichiometry for the complexes. The geometry of the complex was elucidated by 2D ROESY NMR measurements and molecular modeling, indicating that the partial molecular encapsulation includes the fluorophenyl ring of LAP. Phase-solubility studies with four CDs, β-CD, (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), randomly methylated-β- (RAMEB-) cyclodextrin, and sulfobutylether-β-cyclodextrin (SBE-β-CD), show an AL type diagram and highly increased solubility via CD complexation. The results are especially promising with SBE-β-CD, exerting more than 600-fold gain in solubility. The equilibrium and structural information presented herein can offer the molecular basis for an improved drug formulation with enhanced bioavailability

Strategic Positioning of Connexin36 Gap Junctions Across Human Retinal Ganglion Cell Dendritic Arbors

Connexin36 (Cx36) subunits form gap junctions (GJ) between neurons throughout the central nervous system. Such GJs of the mammalian retina serve the transmission, averaging and correlation of signals prior to conveying visual information to the brain. Retinal GJs have been exhaustively studied in various animal species, however, there is still a perplexing paucity of information regarding the presence and function of human retinal GJs. Particularly little is known about GJ formation of human retinal ganglion cells (hRGCs) due to the limited number of suitable experimental approaches. Compared to the neuronal coupling studies in animal models, where GJ permeable tracer injection is the gold standard method, the post-mortem nature of scarcely available human retinal samples leaves immunohistochemistry as a sole approach to obtain information on hRGC GJs. In this study Lucifer Yellow (LY) dye injections and Cx36 immunohistochemistry were performed in fixed short-post-mortem samples to stain hRGCs with complete dendritic arbors and locate dendritic Cx36 GJs. Subsequent neuronal reconstructions and morphometric analyses revealed that Cx36 plaques had a clear tendency to form clusters and particularly favored terminal dendritic segments

Brain protein expression changes in WAG/Rij rats, a genetic rat model of absence epilepsy after peripheral lipopolysaccharide treatment

Peripheral injection of bacterial lipopolysaccharide (LPS) facilitates 8-10Hz spike-wave discharges (SWD) characterizing absence epilepsy in WAG/Rij rats. It is unknown however, whether peripherally administered LPS is able to alter the generator areas of epileptic activity at the molecular level. We injected 1mg/kg dose of LPS intraperitoneally into WAG/Rij rats, recorded the body temperature and EEG, and examined the protein expression changes of the proteome 12h after injection in the fronto-parietal cortex and thalamus. We used fluorescent two-dimensional differential gel electrophoresis to investigate the expression profile. We found 16 differentially expressed proteins in the fronto-parietal cortex and 35 proteins in the thalamus. It is known that SWD genesis correlates with the transitional state of sleep-wake cycle thus we performed meta-analysis of the altered proteins in relation to inflammation, epilepsy as well as sleep. The analysis revealed that all categories are highly represented by the altered proteins and these protein-sets have considerable overlap. Protein network modeling suggested that the alterations in the proteome were largely induced by the immune response, which invokes the NFkB signaling pathway. The proteomics and computational analysis verified the known functional interplay between inflammation, epilepsy and sleep and highlighted proteins that are involved in their common synaptic mechanisms. Our physiological findings support the phenomenon that high dose of peripheral LPS injection increases SWD-number, modifies its duration as well as the sleep-wake stages and decreases body temperature

Inclusion complexation of the anticancer drug pomalidomide with cyclodextrins: fast dissolution and improved solubility

Pomalidomide (POM), a potent anticancer thalidomide analogue was characterized in terms of cyclodextrin complexation to improve its aqueous solubility and maintain its anti-angiogenic activity. The most promising cyclodextrin derivatives were selected by phase-solubility studies. From the investigated nine cyclodextrins – differing in cavity size, nature of substituents, degree of substitution and charge – the highest solubility increase was observed with sulfobutylether-β-cyclodextrin (SBE-β-CD). The inclusion complexation between POM and SBE-β-CD was further characterized with a wide variety of state-of-the-art analytical techniques, such as nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), circular dichroism spectroscopy, fluorescence spectroscopy as well as X-ray powder diffraction method (XRD). Job plot titration by NMR and the AL-type phase-solubility diagram indicated 1:1 stoichiometry in a liquid state. Complementary analytical methods were employed for the determination of the stability constant of the complex; the advantages and disadvantages of the different approaches are also discussed. Inclusion complex formation was also assessed by molecular modelling study. Solid state complexation in a 1:1 M ratio was carried out by lyophilization and investigated by IR and XRD. The complex exhibited fast-dissolution with immediate release of POM, when compared to the pure drug at acidic and neutral pH. Kinetic analysis of POM release from lyophilized complex shows that Korsmeyer-Peppas and Weibull model described the best the dissolution kinetics. The cytotoxicity of the complex was tested against the LP-1 human myeloma cell line which revealed that supramolecular interactions did not significantly affect the anti-cancer activity of the drug. Overall, our results suggest that the inclusion complexation of POM with SBE-β-CD could be a promising approach for developing more effective POM formulations with increased solubility

Complex X chromosome rearrangement associated with multiorgan autoimmunity

BACKGROUND: Turner syndrome, a congenital condition that affects 1/2,500 births, results from absence or structural alteration of the second sex chromosome. Turner syndrome is usually associated with short stature, gonadal dysgenesis and variable dysmorphic features. The classical 45,X karyotype accounts approximately for half of all patients, the remainder exhibit mosaicism or structural abnormalities of the X chromosome. However, complex intra-X chromosomal rearrangements involving more than three breakpoints are extremely rare. RESULTS: We present a unique case of a novel complex X chromosome rearrangement in a young female patient presenting successively a wide range of autoimmune diseases including insulin dependent diabetes mellitus, Hashimoto's thyroiditis, celiac disease, anaemia perniciosa, possible inner ear disease and severe hair loss. For the genetic evaluation, conventional cytogenetic analysis and FISH with different X specific probes were initially performed. The complexity of these results and the variety of autoimmune problems of the patient prompted us to identify the exact composition and breakpoints of the rearranged X as well as methylation status of the X chromosomes. The high resolution array-CGH (assembly GRCh37/hg19) detected single copy for the whole chromosome X short arm. Two different sized segments of Xq arm were present in three copies: one large size of 80,3 Mb from Xq11.1 to Xq27.3 region and another smaller (11,1 Mb) from Xq27.3 to Xq28 region. An 1,6 Mb Xq27.3 region of the long arm was present in two copies. Southern blot analysis identified a skewed X inactivation with approximately 70:30 % ratios of methylated/unmethylated fragments. The G-band and FISH patterns of the rearranged X suggested the aspect of a restructured i(Xq) chromosome which was shattered and fortuitously repaired. The X-STR genotype analysis of the family detected that the patient inherited intact maternal X chromosome and a rearranged paternal X chromosome. The multiple Xq breakages and fusions as well as inverted duplication would have been expected to cause a severe Turner phenotype. However, the patient lacks many of the classic somatic features of Turner syndrome, instead she presented multiorgan autoimmune diseases. CONCLUSIONS: The clinical data of the presented patient suggest that fragmentation of the i(Xq) chromosome elevates the risk of autoimmune diseases

A néma szívinfarktus = Silent myocardial infarction

A különböző betegségekben elhaltak szekciójakor nemritkán látható a szívizomban korábban lezajlott szívinfarktus, melyről sem a betegnek, sem kezelőorvosának nem volt tudomása (néma szívinfarktus), és csak mint szekciós melléklelet kerül megállapításra. Célkitűzés: a néma szívinfarktus gyakoriságának, az életkilátásokat meghatározó szerepének, felismerhetőségének és prognózisának vizsgálata a lokalizáció függvényében. Módszer: 10 év alatt végzett 1568 szekció boncolási anyagának retrospektív elemzése, az adatok statisztikai értékelése. Eredmények: 470 beteg (30%) élete során szívinfarktust szenvedett el. Közülük 177 (37%) volt akut eset, melynek 90%-a élőben felismerhető volt, és a halál oka az infarktus, illetve annak közvetlen szövődménye volt. 293 (63%) esetben találtak idült szívizomelhalást, melyek közül 109 (37%) volt élőben felismert, 184 (63%) pedig néma infarktus, a nők és a férfiak aránya közel azonos (90/94 fő). Idült szívizomelhalásban cardialis okból – döntő többségében szívelégtelenségben – 97 (32%), extracardialis megbetegedésben pedig 196 (68%) beteg hunyt el, többségük stroke következtében, elsősorban inferior infarktusban szenvedők. Következtetések: A szívinfarktus gyakoribb betegség, mint azt a szekció nélküli klinikai és epidemiológiai adatokból vélni lehet, prognózisa pedig jobb, mivel a betegek 42%-a extracardialis megbetegedésben hal meg, életkoruk nem rövidebb a szívinfarktus nélküli betegekénél. A néma szívinfarktusok ismerete teszi lehetővé a beteg terhelhetőségének megítélését, kezelését és a recidíva megelőzését. | Introduction: By autopsies of patients expired from different diseases not rarely chronic myocardial infarction is found, that was known before neither to patient nor to medical attendant (silent myocardial infarction) and is interpreted as incidental finding. Aim: Study of frequency, role in expectation of life, diagnosis and prognosis of silent myocardial infarction in relation to localisation. Methods: Retrospective study and statistical analysis of 1568 autopsies performed during 10 years. Results: Acute or chronic myocardial infarction was found in 470 cases (30%), acute infarction in 177 cases (37%), 90% of which was diagnosed in vivo and patients died of infarction and its direct complications. In 293 cases (63%) chronic myocardial infarction was found, 109 cases (37%) were known and 184 cases (63%) were silent myocardial necrosis, the ratio of female/male patients was nearly the same (90/94 persons). 97 patients (32%) with chronic myocardial infarction died of cardiac cause – mostly in cardiac failure –, 196 (68%) of extracardial cause, most of them of stroke, predominantly the patients with inferior infarction. Conclusion: Considering the silent causes, the myocardial infarction is more frequent and has better prognosis, than it is known from epidemiological data without autopsies, because 42% of these patients dies of extracardial diseases, and the continuity of life is not shorter, than by patients without myocardial infarction. Knowledge of silent infarction gives possibility to estimate the physical charge of patients, their treatment and to prevent the recidiva