Dynamic changes in the gene expression of zebrafish Reelin receptors during embryogenesis and hatching period

The brain morphology of vertebrates exhibits huge evolutionary diversity, but one of the shared morphological features unique to vertebrate brain is laminar organization of neurons. Because the Reelin signal plays important roles in the development of the laminar structures in mammalian brain, investigation of Reelin signal in lower vertebrates will give some insights into evolution of vertebrate brain morphogenesis. Although zebrafish homologues of Reelin, the ligand, and Dab1, a cytoplasmic component of the signaling pathway, have been reported, the Reelin receptor molecules of zebrafish are not reported yet. Here, we sought cDNA sequence of zebrafish homologue of the receptors, vldlr and apoer2, and examined their expression patterns by in situ hybridization. Developmental gene expression pattern of reelin, dab1, vldlr, and apoer2 in the central nervous system of zebrafish was compared, and their remarkable expression was detected in the developing laminar structures, such as the tectum and the cerebellum, and also non-laminated structures, such as the pallium. The Reelin receptors exhibited different spatial and temporal gene expression. These results suggest a possibility that duplication and subsequent functional diversity of Reelin receptors contributed to the morphological and functional evolution of vertebrate brain.     

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

Background

Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.

Methodology/Principal Findings

Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca²⁺ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.

Conclusions/Significance

These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.     

Isolation and Characterization of a Hyphomicrobium Species and its Polysaccharide Formation from Methanol

A microorganism which could utilize methanol as the sole source of carbon and excreted a new polysaccharide was isolated from soil. This isolate was a stalked bacterium which reproduced by a budding process, and could grow on only methanol, formaldehyde or methylamines as the carbon source. The most suitable nitrogen source for growth was the ammonium ion. The optimum pH and temperature for growth were about 7.0 and 30°C, respectively. The cell growth was inhibited by blue light irradiation. Amino acid composition and fatty acid composition of the cells and electrophoretic behavior of methanol dehydrogenase were also studied. On the basis of these properties as well as taxonomical studies, the isolate (strain JTS-811) was identified as belonging to the genus Hyphomicrobium. This strain had different characteristics as compared to those described for other Hyphomicrobium isolates. At present, it is difficult to give a specific name to this strain, because classification of hyphomicrobia is not clear.     

Prominent Steatosis with Hypermetabolism of the Cell Line Permissive for Years of Infection with Hepatitis C Virus

Most of experiments for HCV infection have been done using lytic infection systems, in which HCV-infected cells inevitably die. Here, to elucidate metabolic alteration in HCV-infected cells in a more stable condition, we established an HCV-persistently-infected cell line, designated as HPI cells. This cell line has displayed prominent steatosis and supported HCV infection for more than 2 years, which is the longest ever reported. It enabled us to analyze metabolism in the HCV-infected cells integrally combining metabolomics and expression arrays. It revealed that rate-limiting enzymes for biosynthesis of cholesterol and fatty acids were up-regulated with actual increase in cholesterol, desmosterol (cholesterol precursor) and pool of fatty acids. Notably, the pentose phosphate pathway was facilitated with marked up-regulation of glucose-6-phosphate dehydrogenase, a rete-limiting enzyme, with actual increase in NADPH. In its downstream, enzymes for purine synthesis were also up-regulated resulting in increase of purine. Contrary to common cancers, the TCA cycle was preferentially facilitated comparing to glycolysis pathway with a marked increase of most of amino acids. Interestingly, some genes controlled by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of antioxidation and metabolism, were constitutively up-regulated in HPI cells. Knockdown of Nrf2 markedly reduced steatosis and HCV infection, indicating that Nrf2 and its target genes play important roles in metabolic alteration and HCV infection. In conclusion, HPI cell is a bona fide HCV-persistently-infected cell line supporting HCV infection for years. This cell line sustained prominent steatosis in a hypermetabolic status producing various metabolites. Therefore, HPI cell is a potent research tool not only for persistent HCV infection but also for liver metabolism, overcoming drawbacks of the lytic infection systems.     

Novel, objective, multivariate biomarkers composed of plasma amino acid profiles for the diagnosis and assessment of inflammatory bowel disease

Background

Inflammatory bowel disease (IBD) is a chronic intestinal disorder that is associated with a limited number of clinical biomarkers. In order to facilitate the diagnosis of IBD and assess its disease activity, we investigated the potential of novel multivariate indexes using statistical modeling of plasma amino acid concentrations (aminogram).

Methodology and Principal Findings

We measured fasting plasma aminograms in 387 IBD patients (Crohn''s disease (CD), n = 165; ulcerative colitis (UC), n = 222) and 210 healthy controls. Based on Fisher linear classifiers, multivariate indexes were developed from the aminogram in discovery samples (CD, n = 102; UC, n = 102; age and sex-matched healthy controls, n = 102) and internally validated. The indexes were used to discriminate between CD or UC patients and healthy controls, as well as between patients with active disease and those in remission. We assessed index performances using the area under the curve of the receiver operating characteristic (ROC AUC). We observed significant alterations to the plasma aminogram, including histidine and tryptophan. The multivariate indexes established from plasma aminograms were able to distinguish CD or UC patients from healthy controls with ROC AUCs of 0.940 (95% confidence interval (CI): 0.898–0.983) and 0.894 (95%CI: 0.853–0.935), respectively in validation samples (CD, n = 63; UC, n = 120; healthy controls, n = 108). In addition, other indexes appeared to be a measure of disease activity. These indexes distinguished active CD or UC patients from each remission patients with ROC AUCs of 0.894 (95%CI: 0.853–0.935) and 0.849 (95%CI: 0.770–0.928), and correlated with clinical disease activity indexes for CD (r_s = 0.592, 95%CI: 0.385–0.742, p<0.001) or UC (r_s = 0.598, 95%CI: 0.452–0.713, p<0.001), respectively.

Conclusions and Significance

In this study, we demonstrated that established multivariate indexes composed of plasma amino acid profiles can serve as novel, non-invasive, objective biomarkers for the diagnosis and monitoring of IBD, providing us with new insights into the pathophysiology of the disease.     

Contribution of complement factor H Y402H polymorphism to sudden sensorineural hearing loss risk and possible interaction with diabetes

Sudden sensorineural hearing loss (SSNHL) is one of the most common diseases encountered by otolaryngologists; however, the etiology is unclear. The aim of this study was to assess the association between SSNHL and polymorphism of complement factor H (CFH) Y402H, which is implicated in age-related macular degeneration. We conducted a case-control study, in which the cases were 72 SSNHL patients and the controls were 2161 residents selected randomly from the resident register. The odds ratio (OR) for SSNHL risk was determined using the additive-genetic model of CFH Y402H polymorphism. The OR for SSNHL risk was 1.788 (95% confidence interval [CI]: 1.008-3.172) with no adjustments and 1.820 (CI: 1.025-3.232) after adjusting for age and sex. Of the three lifestyle-related diseases hypertension, dyslipidemia, and diabetes, only diabetes was significantly associated with SSNHL risk. We classified both the controls and SSNHL patients into those with or without diabetes, and the OR for SSNHL risk was 6.326 (CI: 1.885-21.225) in diabetic subjects and 1.214 (CI: 0.581-2.538) in nondiabetic subjects. We conclude that CFH Y402H polymorphism and SSNHL risk are significantly related, and that diabetic CFH Y402H minor allele carriers may be susceptible to SSNHL.     

A novel family of bacterial dioxygenases that catalyse the hydroxylation of free L-amino acids

L-isoleucine-4-hydroxylase (IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. L-methionine, L-leucine, L-isoleucine and L-threonine were found to be catalysed by the investigated enzymes, producing L-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free L-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed.