Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

Cerebro-costo-mandibular syndrome

Summary A 7-month-old boy with the cerebro-costomandibular syndrome is presented. This is the first case report in an Oriental population.15 reported cases in the literature are reviewed.     

Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K⁺ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K⁺ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using ⁸⁶radioactive rubidium ion (⁸⁶Rb⁺) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.     

A 3D-Video-Based Computerized Analysis of Social and Sexual Interactions in Rats

A large number of studies have analyzed social and sexual interactions between rodents in relation to neural activity. Computerized video analysis has been successfully used to detect numerous behaviors quickly and objectively; however, to date only 2D video recording has been used, which cannot determine the 3D locations of animals and encounters difficulties in tracking animals when they are overlapping, e.g., when mounting. To overcome these limitations, we developed a novel 3D video analysis system for examining social and sexual interactions in rats. A 3D image was reconstructed by integrating images captured by multiple depth cameras at different viewpoints. The 3D positions of body parts of the rats were then estimated by fitting skeleton models of the rats to the 3D images using a physics-based fitting algorithm, and various behaviors were recognized based on the spatio-temporal patterns of the 3D movements of the body parts. Comparisons between the data collected by the 3D system and those by visual inspection indicated that this system could precisely estimate the 3D positions of body parts for 2 rats during social and sexual interactions with few manual interventions, and could compute the traces of the 2 animals even during mounting. We then analyzed the effects of AM-251 (a cannabinoid CB1 receptor antagonist) on male rat sexual behavior, and found that AM-251 decreased movements and trunk height before sexual behavior, but increased the duration of head-head contact during sexual behavior. These results demonstrate that the use of this 3D system in behavioral studies could open the door to new approaches for investigating the neuroscience of social and sexual behavior.     

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me₂SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0–2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO, and that recovered PGCs retained ability to differentiate into functional gametes.     

The Formation of Glucose 1,6-Diphosphate from Fructose 1,6-Diphosphate by Yeast Phosphoglucomutase

It was found that fructose 1,6-diphosphate, the main intermediate of glycolysis, was able to act as a coenzyme of yeast phosphoglucomutase reaction. The mechanism of the coenzymatic activity of fructose 1,6-diphosphate was studied. It was indicated in the fructose 1,6-diphosphate dependent reaction that glucose 1,6-diphosphate was formed by the phosphate-transfer of fructose 1,6-diphosphate to glucose 1-phosphate in the first step, and in the second step the conversion of glucose 1-phosphate to glucose 6-phosphate, the original mutase reaction, occurred in the presence of glucose 1,6-diphosphate. The kinetic constants in the reaction of the first step were determined from the time courses of the fructose 1,6-diphosphate dependent reaction.     

Subunits and Sulfhydryl Groups of Beef Liver d-Glycerate Dehydrogenase

The amino acid composition of beef liver d-glycerate dehydrogenase (EC 1.1.1.29) was determined. Results of sodium dodecyl sulfate gel electrophoresis and measurements of the number of NADH bound by the enzyme and the number of the essential sulfhydryl groups suggested that the enzyme was composed of two identical subunits with the molecular weight of 36,000. Close relation between the essential sulfhydryl groups and the coenzyme binding site was also suggested. Effect of an alkylating agent (bromopyruvate) with the structure similar to the substrate was studied. Effects of iodoacetate and iodoacetamide were also studied. It was suggested that these reagents behaved as active-site-directed irreversible inhibitors of the enzyme. Bromopyruvate exhibited a high affinity to the enzyme. Iodoacetate (anionic reagent) had a higher affinity than iodoacetamide (neutral reagent).     

Effect of 1-Anilino-8-naphthalenesulfonate on Properties and Associations of αs-, β- and κ-Caseins

It was indicated from fluorescence spectra and fluorescence titration that a hydrophobic probe, 1-anilino-8-naphthalenesulfonate (ANS), binds to casein components (α_s-, β- and κ-caseins). Fluorescence intensity and affinity of ANS-κ-casein complex were larger than that of ANS-α_s- and ANS-β-casein complexes. Enhancements of fluorescence intensity of complexes of casein components were observed by the addition of KCI or CaCl₂. Reason for the enhancement was postulated to be the increase of the quantum yield of the ANS fluorescence caused by the environmental change of ANS binding region of the casein components.

Marked increase of sedimentation coefficient of β-casein in the presence of KCl or CaCl₂ at 10°C was caused by the addition of ANS. This may be responsible for the stimulation of the Ca-dependent precipitation of β-casein by the addition of ANS.

It was found that α_s · κ-association was prevented by ANS and that hydrophobic interaction have an important role for α_s · κ-association.