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OBJECTIVE: Neuroblastoma is a common malignancy in children. Despite the occurrence of diverse therapies in recent years, the survival rate of patients with high-risk NB is still unpredictable due to the high metastatic potential and poor prognosis. Therefore, it is urgent to study the molecular mechanism of NB metastasis. SNHG1 has been reported to be closely related to the development, metastasis, and prognosis of many cancers. The purpose of this study was to clarify the molecular mechanism of the role of SNHG1 in NB tumors. PATIENTS AND METHODS: The expression levels of SNHG1, miR-338-3p, and PLK4 were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. The functional targets between miR-338-3p and SNHG1 or PLK4 were predicted by online software Diana tools and observed by Luciferase reporter assay and RIP assay. Cell proliferation was measured by MTT assay. Cell migration and invasion were operated through flow cytometry. The expression of p-AKT was quantified by Western blot. Xenograft tumor model was established to confirm the biological role of SNHG1 in NB in vivo. RESULTS: The expression levels of SNHG1 and PLK4 were increased in NB tissues and cells, and miR-338-3p expression was on the contrary. PLK4 was verified as a direct target of miR-338-3p and miR-338-3p could specially bind to SNHG1. The negative effect of SNHG1 down-regulation on cell proliferation, migration, and invasion could be rescued by miR-338-3p inhibition. The suppression of miR-338-3p mimics on cell proliferation, migration, and invasion could be reversed by PLK4 overexpression. In addition, SNHG1 knockdown weakened the volume and weight of tumor in vivo. CONCLUSIONS: SNHG1 conduced to tumorigenesis and mechanism by upregulating PLK4 and by acting as miR-338-3p sponge in neuroblastoma.

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Strata within the inner plexiform layer (IPL) of vertebrate retinas are suspected to be distinct signaling regions. Functions performed within adult zebrafish IPL strata were examined through microelectrode recording and staining of stratified amacrine types. The stimulus protocol and analysis discriminated the pattern of input from red, green, blue, and UV cones as well as the light-response waveforms in this tetrachromatic species. A total of 36 cells were analyzed. Transient depolarizing waveforms at ON and OFF originated with bistratified amacrine types, whose dendritic planes branched either in IPL sublaminas a & b, or only within sublamina a. Monophasic-sustained depolarizing waveforms originated with types monostratified in IPL s4 (sublamina b). OFF responses hyperpolarized at onset, depolarized at offset, and in some cases depolarized during mid-stimulus. These signals originated with types monostratified in s1 or s2 (sublamina a). Bistratified amacrines received depolarizing signals only from red cones, at both ON and OFF, while s4 stratified ON cells combined red and green cone signals. The s1/s2 stratified OFF cells utilized hyperpolarizing signals from red, red and green, or red and blue cones at ON, but only depolarizing red cone signals at OFF. ON and OFF depolarizing transients from red cones appear widely distributed within IPL strata. "C-type" physiologies, depolarized by some wavelengths, hyperpolarized by others, in biphasic or triphasic spectral patterns, originated with amacrine cells monostratified in s5. Collectively, cells in this stratum processed signals from all cone types. J. Comp. Neurol. 525:1532-1557, 2017. © 2016 Wiley Periodicals, Inc.

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We have developed a complete system for discovery of lead compounds as inhibitors of creatine kinase B. In this article, we describe production and purification of the recombinant protein, conditions and features of an optimized high-throughput screening assay, and results of our implementation of the system using a diverse compound library.

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The relationship between SR Ca2+ ATPase (SERCA) activities, cell calcium level, SR calcium store and cell cycle events is not clearly understood. We studied SERCA overexpression in Cos cells using an adenovirus vector. Twofold increases in SERCA mRNA and in protein were correlated with a 2.3-fold and a 1.6-fold paralleled increase in SR calcium pump activity (R = 0.97 and R = 0.99 respectively). Dose-related apoptotic cell death was associated with SERCA overexpression (R = 0.92). When serum was reduced to 4%, cell apoptosis further increased from 20.7 +/- 4.8% to 47.5 +/- 12.9% (M+/-SD; P<0.05; n=3). Flow cytometry identified cell cycle arrest at the G2/M phase. The interleukin-1 converting enzyme (ICE) inhibitor z-VAD-fmk reduced apoptosis for low-, medium- and high-expressing constructs, whereas the CPP-32 inhibitor z-DEVD-fmk had no effect. Flow cytometry using Fluo-3 and Fura-Red revealed a 1.5-fold higher basal calcium and a 10-fold SR calcium overload. ICE inhibitor z-VAD-fmk did not alter calcium loading. An epitope-tagged SERCA mutant, which has no intrinsic Ca2+-pump activities, had a much smaller effect on the SR calcium. These findings suggest that SERCA2A overexpression has an intrinsic role in altering cell-cycle progression, augmenting cellular and SR calcium loading, and precipitating ICE protease-mediated apoptosis; this represents as a novel model for primary SR calcium overload and associated cell apoptosis.

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TNF alpha mRNA and protein biosynthesis were examined in the adult feline heart after stimulation with endotoxin. When freshly isolated hearts were stimulated with endotoxin in vitro, de novo TNF alpha mRNA expression occurred within 30 min, and TNF alpha protein production was detected within 60-75 min; however, TNF alpha mRNA and protein production were not detected in diluent-treated hearts. Immunohistochemical studies localized TNF alpha to endothelial cells, smooth muscle cells, and cardiac myocytes in the endotoxin-treated hearts, whereas TNF alpha immunostaining was absent in the diluent-treated hearts. To determine whether the cardiac myocyte was a source for TNF alpha production, two studies were performed. First, in situ hybridization studies, using highly specific biotinylated probes, demonstrated TNF alpha mRNA in cardiac myocytes from endotoxin-stimulated hearts; in contrast, TNF alpha mRNA was not expressed in myocytes from diluent-treated hearts. Second, TNF alpha protein production was observed when cultured cardiac myocytes were stimulated with endotoxin, whereas TNF alpha protein production was not detected in the diluent-treated cells. The functional significance of the intramyocardial production of TNF alpha was determined by examining cell motion in isolated cardiac myocytes treated with superfusates from endotoxin- and diluent-stimulated hearts. These studies showed that cell motion was depressed in myocytes treated with superfusates from the endotoxin-treated hearts, but was normal with the superfusates from the diluent-treated hearts; moreover, the negative inotropic effects of the superfusates from the endotoxin-treated hearts could be abrogated completely by pretreatment with an anti-TNF alpha antibody. Finally, endotoxin stimulation was also shown to result in the intramyocardial production of TNF alpha mRNA and protein in vivo. Thus, this study shows for the first time that the adult mammalian myocardium synthesizes biologically active TNF alpha.

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Site-directed mutagenesis was used to alter the amino-acid residues at the presumed catalytic site Cys-283 and ATP binding site Asp-340 of human creatine kinase B cDNA. In addition, a highly conserved arginine residue, Arg-292, was also mutated. Transfection of 0.1 to 1 microgram of recombinant plasmid into COS cells produced increasing creatine kinase activity in the cell lysate. The expression of mutant Cys283-Tyr and Cys283-Ser resulted in complete abolition of homodimer BB isoform enzymatic activity without alteration of the capacity for dimerization. Expression of mutants Arg292-His, Arg292-Leu, and Arg292-Gln produced non-functional homodimers, whereas expression of mutant Arg292-Lys produced a homodimer with enzymatic activity that was 42% of the enzymatic activity of the wild type. Expression of the Asp340-Glu mutant creatine kinase did not alter enzyme activity as compared to the wild type. Following heterodimerization, there was inhibition of the normal subunit by the mutant subunit, for both the BB and the MB dimer. The results showed residues Cys-283 and Arg-292 are essential for enzyme catalysis. The best fit model for the dimer is one in which there is close apposition of the two catalytic sites. The interaction of the individual subunits during dimerization provides a molecular approach for dominant negative modulation of the creatine kinase isozyme system in future genetic manipulative experiments.

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OBJECTIVES: Quantitation by polymerase chain reaction (PCR) has not been validated by an independent assay and is not easily applicable to multiple samples. The aim of this study was therefore to attempt to quantify human heart creatine kinase M (CKM) and B (CKB) mRNA simultaneously and to validate the method with a quantitative S1 nuclease protection assay. METHODS: Conditions were optimised to achieve high efficiency of reverse transcription and PCR amplification of a short target sequence using total human heart RNA and an in vitro transcribed RNA standard as a substrate. A nested radiolabelled primer was used for specific detection and quantification of the amplified DNA sequence. RESULTS: The amplification efficiency for CKM and CKB mRNA were 0.98 (SD 0.07) and 1.11 (0.04) respectively. CKM and CKB mRNA levels were determined in 42 samples from 24 human hearts and found to be 156.7(36.2) and 19.9(5.2) amol.microgram-1 RNA, respectively. Parallel quantitative S1 nuclease protection assay yielded results of 131.2(64.2) and 9.4(5.2) amol.microgram-1 RNA. The cardiac CKB, but not CKM mRNA level, was twofold higher using the quantitative PCR method. However this discrepancy was abolished when compared to a higher stringency S1 nuclease protection assay. The cardiac CKB mRNA was 12.7% of the CKM level. This proportion remained the same from hearts with end stage cardiomyopathies of various aetiologies. CONCLUSIONS: This validated quantitative PCR method offers advantages over the S1 nuclease protection assay in that less RNA is required, the procedure is less dependent on RNA integrity and secondary structure, and multiple RNA species can be quantified simultaneously. The results also suggest that the abundance of the CKM and CKB mRNA level are coordinately regulated in the human heart.

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We have isolated, sequenced, and characterized a single-copy B creatine kinase pseudogene. The chromosomal assignment of this gene is 16p13 and a unique sequence probe from this locus detects EcoRI restriction fragment length polymorphisms of 7.8 and 5.4 kb. In 26 unrelated individuals, the frequencies for the 7.8- and 5.4-kb B creatine kinase pseudogene alleles were calculated to be 17.3 and 82.7%, respectively. The B creatine kinase pseudogene is interrupted by a 904-bp DNA insertion composed of three Alu repeat sequences in tandem flanked by an 18-bp direct repeat, derived from the pseudogene sequence. Nucleotide sequence analysis of the Alu elements suggests that the Alu sequences were incorporated into this locus in three separate integration events. Several complex clustered Alu repeat sequences without defined integration borders have been previously identified at different genomic loci. This is the first evidence that complex tandem Alu elements can integrate in an apparently serial manner in the human genome and supports the contention that Alu repeats integrate nonrandomly into the human genome.

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Previous studies have suggested that MM creatine kinase is a muscle-specific protein and is not present in adult brain tissue. We have isolated a protein from human brain with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis which is identical to the muscle M creatine kinase isoenzyme subunit at all 30 sequenced amino acid residues and possesses creatine kinase enzymatic activity following nondenaturing agarose-gel electrophoresis. Immunohistochemistry localizes M creatine kinase to discrete areas of adult human brain. Northern blot analysis of both total and poly(A)-selected RNA isolated from brain did not detect M creatine kinase mRNA. However, polymerase chain reaction amplification of cDNA synthesized from human placenta, heart, and brain mRNA detected M creatine kinase message in both heart and brain but not placenta which contains no detectable M creatine kinase protein. N1E115 and NS20Y, mouse neuroblastoma cell lines which have been used as models of neural cell differentiation, were found also to express MM creatine kinase. Moreover, a transiently transfected reporter gene with 4,800 base pairs of M creatine kinase upstream region fused to chloramphenicol acetyltransferase was expressed during differentiation of these neural cell lines. In summary, MM creatine kinase is present in human brain and we suggest the M creatine kinase upstream region is sufficient to modulate M creatine kinase expression in certain neuronal cells and may be regulated independently from other muscle genes.

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cDNA clones for human B creatine kinase were isolated from human brain and placenta libraries. The entire coding and 3' untranslated regions, as well as 23 bp of the 5' untranslated region were sequenced. Complete sequence identity was found among the clones, with the exception of an area of heterogeneity among the 3' untranslated region of the brain and placenta clones. A 77.7% nucleotide sequence identity was found between the coding region of human B creatine kinase and our previously reported human M creatine kinase. In contrast, no homology was found in the 3' untranslated regions. Probes were constructed from the nonconserved 3' untranslated regions of human M and B creatine kinase and were shown to be highly specific. Southern transfers of total genomic DNA derived from human placenta and digested to completion with several restriction enzymes were probed with the MCK and BCK specific probes producing single hybridization bands. These results suggest that creatine kinase M and B are single copy genes in the human genome.

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Extracellular and surface bound Ca is essential to excitation-contraction (E-C) coupling in mammalian cardiac muscle. In intact hearts from cardiomyopathic hamster with congestive heart failure, a concomitant decrease in the Ca content of a superficial pool was associated with the reduced contractility. Ca binding to cardiac sarcolemmal ghosts prepared from these hearts revealed two binding sites by Scatchard plot. In normal hamsters, the low affinity site had a capacity of 114 nmol Ca.mg-1 protein, a KD of 1.5 mmol . litre-1 and was sensitive to neuraminidase treatment but not to 100 mmol . litre-1 Na, K, or Li, Ca binding in vitro approached a 1:1 relationship with the sialic acid content of the ghosts, 159 nmol . mg-1 protein. The activity of the enzyme responsible for glycosidically linking sialic acid to interstitial and sarcolemmal glycoproteins, sialyltransferase, was reduced from 1.80 to 0.41 pmol . mg-1 protein in the myopathic hearts. We suggest the functional defect in the hamster cardiomyopathy is a reduction in sialyltransferase activity leading to the deficiency in surface sialic acid residues. As a consequence, contractility is reduced, but Ca influx is increased. Reflex sympathetic activity increases Ca influx resulting in "Ca overload" and eventual cellular necrosis.

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The results of this study indicate that two min of Ca-free perfusion did not significantly alter the exchange kinetics of mannitol or Ca in isolated heart preparations. A numerical solution for series Ca exchange between a superficial Ca pool, Ca1, and an intracellular pool, Ca2, was developed in terms of the coefficients and constants determined from a parallel analysis. The series and parallel models for Ca efflux yielded a high correlation between contractile activity and the rate of exchange and Ca content of Ca1. The kinetics of Ca influx were best described by a series model of exchange and agreed closely with the Ca contents and exchange properties measured during Ca efflux. No differences were detected between normal and myopathic hamster hearts in the kinetics of Ca exchange or the role of Ca1 in the E-C coupling process.

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Myocardial contractility and Ca exchange in the pool responsible for E-C coupling was studied in cardiomyopathic (B10 14.6) and control (B10.RB) hamster hearts at three stages in the development of the disease. Contractility (dP/dt) was significantly reduced in the B10 14.6 strain at 26 to 30 days of age, and at all subsequent stages of the disease. Reduced contractility was related directly to a reduction in the Ca content of a superficial pool, Ca1, representing Ca contained in the interstitial space and bound to the sarcolemma and other structures in the interstitium. Increased entry of Ca into cellular pools was associated with the reduction of superficial Ca content. The results suggest that the reduction in surface Ca binding sites may be responsible for the reduction in contractility.

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The contraction of guinea-pig taenia coli due to high K+ could not be reversed by washing when Na+ was absent from the medium. Reintroduction of Na+ (7 mM or more) caused relaxation. Similar results were obtained with rat uterus. The effect of sodium replacement was not due to change in ionic strength because equal or higher osmoles of choline, Ca++, K+, Mn++, or Mg++ had little or no effect. Persistence of contraction in the Na+-free medium was not due to a "catch state" of the contractile apparatus. Impairment of Ca+ removal from the cytoplasm rather than persistent increase in Ca+ influx seemed to sustain the mechanical response. This was because D600 (a calcium influx blocker) failed to completely relax K+-induced contraction in the absence of Na+ and also because the ability of EGTA to produce relaxation was reduced in the absence of Na+. Measurement of tissue calcium content using the lanthanum method revealed coincident decrease in tissue calcium and tension to control level during Na+-mediated relaxation. The results suggest a role for transmembrane Na+-Ca++ exchange in causing the Na+-mediated relaxation of taenia undergoing Na+-free contracture.

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