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Senile plaques, which are amorphous patches within the neuropil (a network of small fibers into which the nerve processes of the central nervous system divide), and neurofibrillary tangles, which are clumps and tangles of filaments within the neurons (nerve cells), are the two major neuropathologic signs of Alzheimer's disease. Since neurofibrillary tangles may be found in other brain diseases, the senile plaques are generally regarded as the more definitive sign. In an examination of the brains of 21 patients with Alzheimer's disease, the relative densities of plaques and tangles in various regions of the brain were determined, and this information was related the age of the patient at the onset of Alzheimer's, and to changes in two enzymes related to acetylcholine in the brain, choline acetyltransferase and acetylcholinesterase. Curiously, there seemed to be an inverse relationship between senile plaques and neurofibrillary tangles. The regions of the brain where the greatest number of plaques were seen, which were areas of the cerebral cortex, had relatively fewer tangles. The neurofibrillary tangles were more numerous in areas of allocortex, such as the entorhinal cortex and the prosubiculum of the hippocampus. An earlier age of onset was associated with a more general distribution of neurofibrillary tangles. All four brain areas analyzed for cholinergic enzymes, the middle frontal cortex, the superior temporal cortex, the entorhinal cortex, and the prosubiculum, had reduced levels of cholinergic enzymes, which is consistent with many previous reports of widespread cholinergic deficits in Alzheimer's disease. (Consumer Summary produced by Reliance Medical Information, Inc.)

Senile plaques, which are amorphous patches within the neuropil (a network of fibers into which nerve processes of the central nervous system divide), and neurofibrillary tangles, which are clumps and tangles of filaments within the neurons (nerve cells), are the two major neuropathologic signs of Alzheimer's disease. Since neurofibrillary tangles may be found in other brain diseases, the senile plaques are generally regarded as the more definitive sign. Histopathological specimens from 21 patients with Alzheimer's disease were examined to determine if there were any significant differences between the left and right hemispheres in the number of senile plaques, neurofibrillary tangles, or quantities of cholinergic enzymes. Although each brain showed some asymmetry, there were no consistent differences, and certainly no evidence to support the contention that the left hemisphere is preferentially affected. Within any brain, the observed asymmetry of senile plaques was likely to be inversely related to the severity of the disease. That is, in brains with greater severity of disease, plaques were more uniformly distributed. (Consumer Summary produced by Reliance Medical Information, Inc.)

A retrospective study was carried out on a group of 138 patients undergoing carotid endarterectomy for extracranial vascular disease. Risk factors of cerebrovascular disease and routine laboratory evaluations were assessed. Of the laboratory evaluations of blood lipids, only mean triglycerides were found to be significantly different from laboratory normals. Stroke as a clinical event has been suggested not to be correlated with blood lipids in a number of large studies, but the present investigation supports the notion that extracranial vascular disease may be associated with blood lipid concentrations. Previous studies of stroke and lipids have not separated out the anatomical site responsible for the cerebral infarction, and thus probably have underestimated the effect of lipids as a risk factor in cervical extracranial atherosclerosis and brain infarction. [ABSTRACT FROM AUTHOR]

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca 2+ -channel accomplishing store-operated Ca 2+ -entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca 2+ -sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca 2+ -concentration ([Ca 2+ ] i ) by Fura-2-fluorescence, and SOCE from increase of [Ca 2+ ] i following re-addition of extracellular Ca 2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca 2+ -entry.

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca 2+ channel accomplishing store-operated Ca 2+ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca 2+ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca 2+ signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca 2+ concentration ([Ca 2+ ] i ) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca 2+ ] i following readdition of extracellular Ca 2+ after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from α IIb β 3 integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca 2+ entry in megakaryocytes., (© FASEB.)

MicroRNAs (miRNAs) are short noncoding RNA sequences ~22 nucleotides in length that play an important role in gene regulation-transcription and translation. The processing of these miRNAs takes place in both the nucleus and the cytoplasm while the final maturation occurs in the cytoplasm. Some mature miRNAs with nuclear localisation signals (NLS) are transported back to the nucleus and some remain in the cytoplasm. The functional roles of these miRNAs are seen in both the nucleus and the cytoplasm. In the nucleus, miRNAs regulate gene expression by binding to the targeted promoter sequences and affect either the transcriptional gene silencing (TGS) or transcriptional gene activation (TGA). In the cytoplasm, targeted mRNAs are translationally repressed or cleaved based on the complementarity between the two sequences at the seed region of miRNA and mRNA. The selective transport of mature miRNAs to the nucleus follows the classical nuclear import mechanism. The classical nuclear import mechanism is a highly regulated process, involving exportins and importins. The nuclear pore complex (NPC) regulates all these transport events like a gate keeper. The half-life of miRNAs is rather low, so within a short time miRNAs perform their function. Temporal studies of miRNA biogenesis are, therefore, useful. We have carried out simulation studies for important miRNA biogenesis steps and also classical nuclear import mechanism using ordinary differential equation (ODE) solver in the Octave software., (© 2016 The Author(s). Published by Journal of Integrative Bioinformatics.)

Background: TGFβ1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFβ1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved., Methods: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX., Results: TGFβ1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFβ1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFβ1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM)., Conclusions: TGFβ1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Gonadotropin Regulated Testicular RNA Helicase (GRTH/Ddx25) is a testis-specific multifunctional RNA helicase and an essential post-transcriptional regulator of spermatogenesis. GRTH transports relevant mRNAs from nucleus to cytoplasmic sites of meiotic and haploid germ cells and associates with actively translating polyribosomes. It is also a negative regulator of steroidogenesis in Leydig cells. To obtain a genome-wide perspective of GRTH regulated genes, in particularly those associated with polyribosomes, microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice. 792 genes among the entire mouse genome were found to be polysomal GRTH-linked in WT. Among these 186 were down-regulated and 7 up-regulated genes in GRTH null mice. A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells. The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO underscore the relevance of GRTH in translation. Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. This study has revealed known and unexplored factors in the genome and regulatory pathways underlying GRTH action in male reproduction.

Multiple observations support the existence of developmental differences in megakaryocytopoiesis. We have previously shown that neonatal megakaryocyte (MK) progenitors are hyperproliferative and give rise to MKs smaller and of lower ploidy than adult MKs. Based on these characteristics, neonatal MKs have been considered immature. The molecular mechanisms underlying these differences are unclear, but contribute to the pathogenesis of disorders of neonatal megakaryocytopoiesis. In the present study, we demonstrate that low-ploidy neonatal MKs, contrary to traditional belief, are more mature than adult low-ploidy MKs. These mature MKs are generated at a 10-fold higher rate than adult MKs, and result from a developmental uncoupling of proliferation, polyploidization, and terminal differentiation. This pattern is associated with up-regulated thrombopoietin (TPO) signaling through mammalian target of rapamycin (mTOR) and elevated levels of full-length GATA-1 and its targets. Blocking of mTOR with rapamycin suppressed the maturation of neonatal MKs without affecting ploidy, in contrast to the synchronous inhibition of polyploidization and cytoplasmic maturation in adult MKs. We propose that these mechanisms allow fetuses/neonates to populate their rapidly expanding bone marrow and intravascular spaces while maintaining normal platelet counts, but also set the stage for disorders restricted to fetal/neonatal MK progenitors, including the Down syndrome-transient myeloproliferative disorder and the thrombocytopenia absent radius syndrome.

Thrombocytopenia is common among sick neonates, affecting 20% to 35% of all patients admitted to the neonatal intensive care unit (NICU). While most cases of neonatal thrombocytopenia are mild or moderate and resolve within 7 to 14 days with appropriate therapy, 2.5% to 5% of NICU patients develop severe thrombocytopenia, sometimes lasting for several weeks and requiring >20 platelet transfusions. The availability of thrombopoietic agents offers the possibility of decreasing the number of platelet transfusions and potentially improving the outcomes of these infants. However, adding thrombopoietin (TPO) mimetics to the therapeutic armamentarium of neonatologists will require careful attention to the substantial developmental differences between neonates and adults in the process of megakaryocytopoiesis and in their responses to TPO. Taken together, the available data suggest that TPO mimetics will stimulate platelet production in neonates, but might do so through different mechanisms and at different doses than those established for adults. In addition, the specific groups of thrombocytopenic neonates most likely to benefit from therapy with TPO mimetics need to be defined, and the potential nonhematological effects of these agents on the developing organism need to be considered. This review summarizes our current understanding of neonatal megakaryocytopoiesis, and examines in detail the developmental factors relevant to the potential use of TPO mimetics in neonates.

Gonadotropin-regulated testicular RNA helicase is a multifunctional enzyme present in both Leydig and germ cells that is essential for the progress of spermatogenesis. GRTH gene expression is transcriptionally upregulated by human chorionic gonadotropin (hCG) via second messenger (cAMP) and androgen in Leydig cells. The regulatory region(s) in the GRTH gene that is/are required for its cell-specific expression in the testis and hCG/androgen dependent expression were investigated in transgenic mice carrying sequential deletions of 5' flanking sequences of the GRTH gene. GFP-reporter gene expression directed by the GRTH 5' flanking sequences extending to -3.6 kb was specifically located in Leydig cells and the 205 bp minimal promoter domain was sufficient for this cell-specific expression. The 1 kb (5' to the ATG codon) transgene-directed expression was markedly increased by in vivo hCG treatment. Administration of the androgen receptor inhibitor Flutamide blocked the basal and hCG stimulated GFP expression in Leydig cells. We conclude that the expression of GRTH in testicular cells is differentially regulated by its 5' flanking sequence and that the 1 kb fragment of GRTH gene contains sequences for androgen regulation of its expression in Leydig cells.

Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is essential for completion of spermatogenesis. GRTH is present in the cytoplasm and nucleus of meiotic spermatocytes and round spermatids and functions as a component of mRNP particles, implicating its post-transcriptional regulatory roles in germ cells. In this study, GRTH antibodies specific to N- or C-terminal sequences showed differential subcellular expression of GRTH 56- and 61-kDa species in nucleus and cytoplasm, respectively, of rodent testis and transfected COS1 cells. The 56-kDa nuclear species interacted with CRM1 and participated in mRNA transport. The phosphorylated cytoplasmic 61-kDa species was associated with polyribosomes. Confocal studies on COS-1 cells showed that GRTH-GFP was retained in the nucleus by treatment with a RNA polymerase inhibitor or the nuclear protein export inhibitor. This indicated that GRTH is a shuttling protein associated with RNA export. The N-terminal leucine-rich region (61-74 amino acids) was identified as the nuclear export signal that participated in CRM1-dependent nuclear export pathway. Deletion analysis identified a 14-amino acid GRTH sequence (100-114 amino acids) as a nuclear localization signal. GRTH selectively regulated the translation of specific genes including histone 4 and HMG2 in germ cells. In addition, GRTH participated in the nuclear export of RNA messages (PGK2, tACE, and TP2) in a gene-specific manner. These studies strongly indicate that the mammalian GRTH/Ddx25 gene is a multifunctional RNA helicase that is an essential regulator of sperm maturation.