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GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.
BACKGROUND - MicroRNA regulate mRNA levels in a tissue specific way, either by inducing degradation of the transcript or by inhibiting translation or transcription. Putative mRNA targets of microRNA identified from seed sequence matches are available in many databases. However, such matches have a high false positive rate and cannot identify tissue specificity of regulation.
RESULTS - We describe a simple method to identify direct mRNA targets of microRNA dysregulated in cancers from expression level measurements in patient matched tumor/normal samples. The word "direct" is used here in a strict sense to: a) represent mRNA which have an exact seed sequence match to the microRNA in their 3'UTR, b) the seed sequence match is strictly conserved across mouse, human, rat and dog genomes, c) the mRNA and microRNA expression levels can distinguish tumor from normal with high significance and d) the microRNA/mRNA expression levels are strongly and significantly anti-correlated in tumor and/or normal samples. We apply and validate the method using clear cell Renal Cell Carcinoma (ccRCC) and matched normal kidney samples, limiting our analysis to mRNA targets which undergo degradation of the mRNA transcript because of a perfect seed sequence match. Dysregulated microRNA and mRNA are first identified by comparing their expression levels in tumor vs normal samples. Putative dysregulated microRNA/mRNA pairs are identified from these using seed sequence matches, requiring that the seed sequence be conserved in human/dog/rat/mouse genomes. These are further pruned by requiring a strong anti-correlation signature in tumor and/or normal samples. The method revealed many new regulations in ccRCC. For instance, loss of miR-149, miR-200c and mir-141 causes gain of function of oncogenes (KCNMA1, LOX), VEGFA and SEMA6A respectively and increased levels of miR-142-3p, miR-185, mir-34a, miR-224, miR-21 cause loss of function of tumor suppressors LRRC2, PTPN13, SFRP1, ERBB4, and (SLC12A1, TCF21) respectively. We also found strong anti-correlation between VEGFA and the miR-200 family of microRNA: miR-200a*, 200b, 200c and miR-141. Several identified microRNA/mRNA pairs were validated on an independent set of matched ccRCC/normal samples. The regulation of SEMA6A by miR-141 was verified by a transfection assay.
CONCLUSIONS - We describe a simple and reliable method to identify direct gene targets of microRNA in any cancer. The constraints we impose (strong dysregulation signature for microRNA and mRNA levels between tumor/normal samples, evolutionary conservation of seed sequence and strong anti-correlation of expression levels) remove spurious matches and identify a subset of robust, tissue specific, functional mRNA targets of dysregulated microRNA.
RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (adenosine-to-inosine editing). Commonly used methodologies to quantify RNA editing levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations of these methods lead to a small number of clones characterized in comparison to the number of mRNA molecules in the original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method for quantifying RNA editing patterns that increases sequence analysis to an average of more than 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5-hydroxytryptamine(2C); 5HT(2C)) transcript, an RNA editing substrate in which up to five adenosines are modified. Using a high-throughput multiplexed transcript analysis, we were able to quantify accurately the expression of twenty 5HT(2C) isoforms, each representing at least 0.25% of the total 5HT(2C) transcripts. Furthermore, this approach allowed the detection of previously unobserved changes in 5HT(2C) editing in RNA samples isolated from different inbred mouse strains and dissected brain regions, as well as editing differences in alternatively spliced 5HT(2C) variants. This approach provides a novel and efficient strategy for large-scale analyses of RNA editing and may prove to be a valuable tool for uncovering new information regarding editing patterns in specific disease states and in response to pharmacological and physiological perturbation, further elucidating the impact of 5HT(2C) RNA editing on central nervous system function.
Increasing the breakdown of stored fat in adipose tissue leads to reducing fat content, enhancing feed efficiency and, consequently, decreasing the production cost of poultry. The processes of lipolysis are not completely understood, and the proteins involved in this process need to be identified. An adipose triglyceride lipase (ATGL), recently identified in several species, has not been studied in avian species. We have cloned the full-length coding sequences of ATGL cDNA for the chicken, turkey, and quail. Sequence comparisons among mammals and these avian species showed that the avian ATGL have 2 conserved domains, the patatin domain and the hydrophobic domain. The patatin domain contains lipase activity, and the hydrophobic domain exhibits lipid droplet binding. The high levels of chicken, turkey, and quail ATGL mRNA and protein are exclusively found in subcutaneous and abdominal adipose tissues. In addition, chicken ATGL (gATGL) is mainly expressed in the fractionated adipocytes compared with stromal-vascular cells that mostly contain preadipocytes (P < 0.001). Furthermore, ontogeny of gATGL mRNA and protein expression in adipose tissue showed induction of gATGL immediately after hatching before access to food (P < 0.05), suggesting that an energy deficit due to posthatching starvation may increase breakdown of stored fat via increasing gATGL expression in adipose tissue. Our studies showed that expression of the chicken ATGL is adipose specific and regulated developmentally, suggesting that a possible modulation of ATGL expression would regulate fat deposition in avian species.
BACKGROUND - Small intestinal ulcers are frequent complications of therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine.
METHODS - Eicosanoids and metabolites were measured by isotope dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR.
RESULTS - We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45-year-old man who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B2 (TxB2) production was 4.6% of normal controls (P<0.006), and serum 12-HETE was 1.3% of controls (P<0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB4 biosynthesis by ionophore-activated leukocytes was only 3% of controls, and urinary LTE4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A2-alpha (cPLA2-alpha), which regulates cyclooxygenase- and lipoxygenase-mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA2-alpha cDNA demonstrated two heterozygous nonsynonymous single-base-pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known single nucleotide polymorphism (SNP), Lys651Arg (K651R).
CONCLUSIONS - Characterization of this cPLA2-alpha deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers.
Cytochrome P450 (P450) 4X1 is one of the so-called 'orphan' P450s without an assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5alpha cells (< 100 nmol P450.L(-1)). The highest level of expression (300-450 nmol P450.L(-1) culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b(5). Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala and basal ganglia), in addition to expression in human heart, liver, prostate and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain.
Cytochrome P450 (P450) 20A1 is one of the so-called "orphan" P450s without assigned biological function. mRNA expression was detected in human liver, and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3- to 4-fold higher in brain and heart than in liver. In situ hybridization of rat whole brain sections showed an mRNA expression pattern similar to that observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200-280 nmol of P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function.
Human Goodpasture antigen-binding protein (GPBP) is an atypical protein kinase that phosphorylates the Goodpasture auto-antigen, the alpha3 chain of collagen IV. The COL4A3BP gene is alternatively spliced producing two protein isoforms: GPBP and GPBPDelta26. The latter lacks a serine-rich domain composed of 26 amino acid residues. Both isoforms also function as ceramide transfer proteins (CERT). Here, we explored the function of Gpbp and GpbpDelta26/CERT during embryogenesis in zebrafish. We cloned both splice variants of the zebrafish gene and found that they are differentially expressed during development. We used antisense oligonucleotide-mediated loss-of-function and synthetic mRNA-based gain-of-function approaches. Our results show that the loss-of-function phenotype is linked to cell death, evident primarily in the muscle of the somites, extensive loss of myelinated tracks, and brain edema. These results indicate that disruption of the nonvesicular ceramide transport is detrimental to normal embryonic development of somites and brain because of increased apoptosis. Moreover, this phenotype is mediated by Gpbp but not GpbpDelta26/CERT, suggesting that Gpbp is an important factor for normal skeletal muscle and brain development.
LKB1, a serine/threonine kinase, regulates cell polarity, metabolism, and cell growth. The activity and cellular distribution of LKB1 are determined by cofactors, STRADalpha and MO25. STRADalpha induces relocalization of LKB1 from the nucleus to the cytoplasm and stimulates its catalytic activity. MO25 stabilizes the STRADalpha/LKB1 interaction. We investigated the mechanism of nucleocytoplasmic transport of LKB1 in response to its cofactors. Although LKB1 is imported into the nucleus by importin-alpha/beta, STRADalpha and MO25 passively diffuse between the nucleus and the cytoplasm. STRADalpha induces nucleocytoplasmic shuttling of LKB1. STRADalpha facilitates nuclear export of LKB1 by serving as an adaptor between LKB1 and exportins CRM1 and exportin7. STRADalpha inhibits import of LKB1 by competing with importin-alpha for binding to LKB1. MO25 stabilizes the LKB1-STRADalpha complex but it does not facilitate its nucleocytoplasmic shuttling. Strikingly, the STRADbeta, isoform which differs from STRADalpha in the N- and C-terminal domains that are responsible for interaction with export receptors, does not efficiently relocalize LKB1 from the nucleus to the cytoplasm. These results identify a multifactored mechanism to control LKB1 localization, and they suggest that the STRADbeta-LKB1 complex might possess unique functions in the nucleus.
Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.