Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHβ mRNA levels. Only GnIH-II decreased LHβ mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHβ and FSHβ synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.     

Imaging of desmoplastic infantile ganglioglioma: a spectroscopic viewpoint

Purpose  Desmoplastic infantile gangliogliomas (DIG) are rare benign intracranial neoplasms of early childhood with involvement of superficial cerebral cortex and leptomeninges. The purpose of the study was to determine the alterations in metabolite ratios occurring in the neoplasm and combine with magnetic resonance (MR) imaging features to narrow down the diagnosis. Methods  MR imaging sequences include T1- and T2-weighted sequences, fluid-attenuated inversion recovery sequences, and post-gadolinium-contrast T1-weighted imaging. Single-voxel short TE ¹H MR spectroscopy was used to study the changes in metabolite ratios in the tumor. Results  Comparison of metabolite ratios between normal brain tissue and tumor-affected region showed lower N-acetyl aspartate to creatine (Cr; 1.58 vs.1.28), higher choline to Cr (0.82 vs.2.03), and no significant change in myo-inositol to Cr (0.42 vs.0.39). Conclusion  MR spectroscopy and imaging provide valuable information in the diagnosis of DIG.     

Angiotensin AT2 receptor activates the cyclic-AMP signaling pathway in eel

A unique angiotensin type 2 receptor (AT2) that induces a cAMP signaling pathway was cloned and characterized for the first time in fish, Anguilla japonica. Phylogeny and synteny results showed that the AT2s among fishes and tetrapods share the same origin despite a sub-cluster formation among eel, salmon, and zebrafish. The eel AT2 was expressed abundantly in the spleen and localized at straight arterioles and ellipsoid regions prior to the sinusoid, suggesting a role in the regulation of microcirculation and/or immune response. Various angiotensin (Ang) peptides, including Ang II, Ang III, and Ang IV, were detected in the spleen by a radioimmunoassay coupled with HPLC separation, and these endogenous peptides stimulated a cAMP signaling, which has no crosstalk with cGMP pathway. The common and contrasting features of AT2 between fishes and mammals imply some ancestral characters of AT2, which are important information for receptor binding and evolutionary studies.     

速避凝对照小剂量普通肝素治疗急性损伤性DIC的临床观察

目的评估低相对分子质量肝素(LMWH)速避凝在急性损伤性弥散性血管内凝血(DIC)的临床疗效。方法应用速避凝对照小剂量普通肝素治疗40例急性损伤性DIC。20例采用速避凝治疗,0.1-0.4ml皮下注射,每日1-2次(75-150u/kg.d-1),对照组20例采用常规小剂量普通肝素治疗,剂量为12.5-50mg,每日分1-3次(100-250u/kg.d-1)注射。结果速避凝组有效率(75%)比小剂量普通肝素组(65%)高,但无统计学意义。速避凝组出血率(30%,6例)低于小剂量普通肝素组(65%,13例),速避凝组死亡率明显低于小剂量普通肝素组(45%,vs65%,9例vs13例)。结论LMWH速避凝治疗急性损伤性DIC安全有效。     

Assay of DNA-binding proteins with a dsDNA-coupled plate

OBJECTIVES: This paper fabricated a cost-effective dsDNA-coupled plate (dcPlate) and applied it to measure the abundance and DNA-binding activity of a DNA-binding protein (DBP). DESIGN AND METHODS: The dcPlate was manufactured by covalently immobilizing an amino-modified oligonucleotide in wells of the plate coated with N-oxysuccinimide esters. The dcPlate was applied to measure the abundance of DNA-binding activity of a DBP in the same four steps, including protein incubation, primary antibody binding, enzyme-linked secondary antibody binding, and colorimetric development. RESULTS: The detections of three purified DBPs including NF-kappaB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-kappaB p50 to five various DNA sequences demonstrated that dcPlate can be used to measure the abundance of DBPs quantitatively and assay DNA-binding activity of DBPs in high throughputs format. CONCLUSIONS: The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs.     

The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.     

Differential expression of microRNAs in shrimp Marsupenaeus japonicus in response to Vibrio alginolyticus infection

Till date numerous microRNAs (miRNAs) have been discovered from various organisms, including mammals, plants, insects, nematodes and viruses. They are known to have antiviral functions in crustaceans such as shrimp Marsupenaeus japonicas. However, little is known about the role of miRNAs against bacterial infection in this shrimp caused by Vibrio alginolyticus. We performed small RNA sequencing to characterize the differentially expressed microRNAs in V. alginolyticus challenged shrimp, in comparison to that in control uninfected shrimp, at 24 h and 48 h. In total, 55 host miRNAs were differentially expressed in response to the infection and most of these were downregulated at both the time-points. TargetScan and miRanda algorithms showed that the target genes of these down-regulated miRNAs were related to innate immune functions such as production of phenoloxidase enzyme, apoptosis and phagocytosis. Further, gene ontology analysis revealed that many immune signaling pathways were mediated by these miRNAs. This study is one of the earliest attempts at characterizing shrimp miRNAs that respond to V. alginolyticus infection, and will help unravel the miRNA pathways involved in antibacterial action in shrimp.