Effects of fluvoxamine on nerve growth factor-induced neurite outgrowth inhibition by dexamethasone in PC12 cells

In the present study, we examined the effects of fluvoxamine on nerve growth factor (NGF)-induced neurite outgrowth inhibition by dexamethasone (DEX) in PC12 cells. Fluvoxamine increased NGF-induced neurite outgrowth. Compared with co-treatment with NGF and fluvoxamine, p-Akt levels were higher than the values without fluvoxamine. The phosphorylated extracellular regulated kinase 1/2 levels were slightly increased by co-treatment with NGF and fluvoxamine. Fluvoxamine concentration-dependently improved NGF-induced neurite outgrowth inhibition by DEX. Fluvoxamine also improved the decrease in the NGF-induced p-Akt level caused by DEX. Interestingly, the sigma-1 receptor antagonist NE-100 blocked the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX. The selective sigma-1 receptor agonist PRE-084 also improved NGF-induced neurite outgrowth inhibition by DEX, which is blocked by NE-100. These results indicate that the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX may be attributable to the phosphorylation of Akt and the sigma-1 receptor.     

Wolfram Syndrome in the Japanese Population; Molecular Analysis of WFS1 Gene and Characterization of Clinical Features

Background

Wolfram syndrome (WFS) is a recessive neurologic and endocrinologic degenerative disorder, and is also known as DIDMOAD (Diabetes Insipidus, early-onset Diabetes Mellitus, progressive Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene (WFS1). However, the phenotypic pleiomorphism, rarity and molecular complexity of this disease complicate our efforts to understand WFS. To address this limitation, we aimed to describe complications and to elucidate the contributions of WFS1 mutations to clinical manifestations in Japanese patients with WFS.

Methodology

The minimal ascertainment criterion for diagnosing WFS was having both early onset diabetes mellitus and bilateral optic atrophy. Genetic analysis for WFS1 was performed by direct sequencing.

Principal Findings

Sixty-seven patients were identified nationally for a prevalence of one per 710,000, with 33 patients (49%) having all 4 components of DIDMOAD. In 40 subjects who agreed to participate in this investigation from 30 unrelated families, the earliest manifestation was DM at a median age of 8.7 years, followed by OA at a median age of 15.8 years. However, either OA or DI was the first diagnosed feature in 6 subjects. In 10, features other than DM predated OA. Twenty-seven patients (67.5%) had a broad spectrum of recessive mutations in WFS1. Two patients had mutations in only one allele. Eleven patients (27.5%) had intact WFS1 alleles. Ages at onset of both DM and OA in patients with recessive WFS1 mutations were indistinguishable from those in patients without WFS1 mutations. In the patients with predicted complete loss-of-function mutations, ages at the onsets of both DM and OA were significantly earlier than those in patients with predicted partial-loss-of function mutations.

Conclusion/Significance

This study emphasizes the clinical and genetic heterogeneity in patients with WFS. Genotype-phenotype correlations may exist in patients with WFS1 mutations, as demonstrated by the disease onset.     

Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells

Embryonic stem (ES) cells have the potential to differentiate into various progenitor cells. Here we investigated the capacity of mouse ES cells to differentiate into renal tubular cells both in vitro and in vivo. After stably transfecting Wnt4 cDNA to mouse ES cells (Wnt4-ES cells), undifferentiated ES cells were incubated by the hanging drop culture method to induce differentiation to embryoid bodies (EBs). During culturing of the EBs derived from the Wnt4-ES cells, aquaporin-2 (AQP2) mRNA and protein were expressed within 15-20 days. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor (HGF) and activin A. We next performed in vivo experiments by transplanting the Wnt4-EBs into the mouse renal cortex. Four weeks after transplantation, some portions of the EB-derived cells expressing AQP2 in the kidney assembled into tubular-like formations. In conclusion, our in vitro and in vivo experiments revealed two new findings: first, that cultured Wnt4-EBs have an ability to differentiate into renal tubular cells; and second, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular cells.     

Genes Encoding the Group I Intron-containing tRNALeu and Subunit L of NADH Dehydrogenase from the Cyanobacterium Synechococcus PCC 6301

A part of the tRNA^Leu (UAA) gene containing a 240-nucleotidegroup I intron was amplified by PCR from cyanobacterium SynechococcusPCC 6301 genomic DNA. The pre-tRNA synthesized from the clonedPCR product was efficiently self-spliced in vitro under physiologicalconditions. The gene encoding the tRNA^Leu (UAA), trnL-UAA, wasisolated from a Synechococcus PCC 6301 genomic library and thenucleotide sequence of a 2,167-bp portion was determined. ThetrnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intronand a 50-bp 3' exon. In addition, three open reading frames(ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regionsof trnL-UAA. The predicted protein sequence of ORF3, which islocated 74-bp upstream from trnL-UAA on the opposite strand,shows 66.2% amino acid identity to that of the SynechocystisPCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).     

Efficient selection of transgenic mouse embryos using EGFP as a marker gene

We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/β‐actin/EGFP fusion gene were cultured in vitro until they developed into the morulae‐ or blastocyst‐stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals. Mol. Reprod. Dev. 54:43–48, 1999. © 1999 Wiley‐Liss, Inc.     

Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage

Carbon tetrachloride (CCl4) induces liver damage, apparently through the formation of free-radical metabolites. Molecular chaperones such as heat shock protein (Hsp) of 70 kDa have been found to protect cells from various stresses. We previously found that cytosolic chaperone pairs of the Hsp70 family and their DnaJ homolog cochaperones prevent nitric oxide-mediated apoptosis and heat-induced cell death. Expression of cytosolic chaperones, including Hsp70; heat shock cognate (Hsc) 70; and DnaJ homologs dj1 (DjB1/Hsp40/hdj-1), dj2 (DjA1/HSDJ/hdj-2), dj3 (DjA2), and dj4 (DjA4), in the liver of CCl4-treated rats was analyzed. Messenger ribonucleic acids for all these chaperones were markedly induced 3-12 hours after CCl4 treatment with a maximum at 6 hours. Hsp70 and dj1 proteins were markedly induced at 6-24 hours with a maximum at 12 hours, whereas dj2 and dj4 were moderately induced at around 12 hours. Hsc70 was weakly induced after treatment, and dj3 was little induced. To better understand the significance of the induction of chaperones, the effect of preinduction of chaperones on CCl4-induced liver damage was analyzed. When chaperones were preinduced in the liver by heat treatment, increase in serum alanine aminotransferase activity after CCl4 treatment was significantly attenuated. Hsp90, another major cytosolic chaperone, also was induced by heat treatment. On the other hand, Mn- and Cu/Zn-superoxide dismutase were not induced by heat treatment or by CCl4 treatment. These results suggest that cytosolic chaperones of Hsp70 and DnaJ families or Hsp90 (or both) are induced in CCl4-treated rat liver to protect the hepatocytes from the damage being inflicted.     

Pig lens glutathione S-transferase belongs to class Pi enzyme

Class Pi glutathione S-transferase was purified to homogeneity from pig lens cytosol. This enzyme was composed of two identical 22 kDa subunits and had isoelectric point of 8.5 from the results of SDS gel electrophoresis, gel filtration, amino acid sequence analysis and isoelectric focusing. Amino acid sequence of N-terminal 15 residues was almost identical to class Pi enzymes from human, rat and mouse. Antibody against the pig enzyme crossreacted to human glutathione S-transferase-pi and anti-rat glutathione S-transferase-P antibody crossreacted to pig enzyme.     

Junction ribonuclease activity specified in RNases HII/2

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA-DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA-DNA/RNA heteroduplex in a manner similar to the RNA-DNA/DNA substrate. The cleavage site of the RNA-DNA/RNA substrate corresponds to the RNA/RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA-DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single-stranded RNA-DNA, it can recognize a single-stranded RNA-DNA junction if a double-stranded region is located adjacent to the junction.     

Microtubule-associated protein 2-positive cells derived from microglia possess properties of functional neurons

Microglia are believed to play an important role in the regulation of phagocytosis, neuronal survival, neuronal cell death, and inflammation. Recent studies have demonstrated that microglia are multipotential stem cells that give rise to neurons, astrocytes, and oligodendrocytes. However, the functional properties of neurons derived from microglia are poorly understood. In this study, we investigated the possibility that microglia differentiate into functional neurons. Immunocytochemical study demonstrated that microtubule-associated protein 2 (MAP2)-positive cells were derived from microglia under differentiation conditions. Intracellular Ca²⁺ imaging study demonstrated that KCl caused no significant changes in [Ca²⁺]_i in microglia, whereas it caused a remarkable increase in [Ca²⁺]_i in microglia-derived cells. Furthermore, electrophysiological study demonstrated that the spike waveform, firing rate, and tetrodotoxin sensitivity of extracellular action potentials evoked by 4-aminopyridine from microglia-derived MAP2-positive cells were nearly identical to those from cultured cortical neurons. These results suggest that microglia-derived MAP2-positive cells possess properties of functional neurons.