Accuracy evaluation of sleep-wake stage analysis with SleepSign Ver2.0

Sleep and Biological Rhythms -     

A lipophilic derivative of neocarzinostatin. A polymer conjugation of an antitumor protein antibiotic.

A lipophilic derivative of neocarzinostatin (NCS), an antitumor antibiotic, was prepared by reaction with a synthetic water-soluble polymer, [(styrene)1 approximately 3-(maleic acid 4 approximately 7/anhydride 1)]. The reaction was carried out at pH 8.6 for 3 h and aimed at modifying the two nonessential amino groups (alpha-amino of Ala-1, epsilon-amino of Lys-20). The NCS-polystyrene (SMANCS) was purified on a column of Sephadex G-100 in 0.05 M ammonium bicarbonate and the main product was obtained as a single peak. The elemental analysis showed an increased C and a decreased N content. U.v. and i.r. absorption spectra for SMANCS showed the presence of styrene. SDS-acrylamide gel electrophoresis at pH 8.5 and the decreased N content suggested a molecular weight of about 25 000, indicating the numbers of polymers conjugated to be about six units, two of which were found attached to the two amino groups. SMANCS was soluble in organic solvents, in contrast to NCS, and in water. SMANCS exhibited increased chemical and biological stability and appeared to possess similar in vitro biological activity.     

Diabetic modifier QTLs identified in F2 intercrosses between Akita and A/J mice

To identify novel genetic modifiers of type 2 diabetes (T2D), we performed quantitative trait loci (QTL) analysis on F₂ progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and nondiabetic A/J mice. We generated 625 heterozygous (F₂-Hetero) and 338 wild-type (F₂-Wild) mice with regard to the Ins2 mutation in F₂ intercross progeny. We measured quantitative traits, including plasma glucose and insulin concentrations during the intraperitoneal glucose tolerance test (IPGTT), and body weight (BW). We observed three significant QTLs in hypoinsulinemic hyperglycemic male F₂-Hetero mice, designated Dbm1, Dbm3, and Dbm4 on Chromosomes 6, 14, and 15, respectively. They showed linkage to plasma glucose concentrations, with significant maximum logarithm of odds (LOD) scores of 4.12, 4.17, and 6.17, respectively, all exceeding threshold values by permutation tests. In normoinsulinemic normoglycemic male F₂-Wild mice, Dbm1 on Chromosome 6 showed linkage to both plasma insulin concentrations and BW, and Dbm2 on Chromosome 11 showed linkage to plasma glucose concentrations only, with LOD scores of 4.52 and 6.32, and 5.78, respectively. Based on these results, we concluded that Dbm1, Dbm2, Dbm3, and Dbm4 represent four major modifier QTLs specifically affecting T2D-related traits and that these diabetic modifier QTLs are conditional on the heterozygous Ins2 gene mutation and sex to exert their modifier functions. Identification of the genes responsible for these QTLs would provide new drug development targets for human T2D. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.     

Protoplast-transfection of Streptomyces chartreusis SF1623 with Actinophage Φr5 DNA

To establish a procedure for high frequency transfection in streptomycetes, the conditions and factors affecting the polyethyleneglycol (PEG) mediated transfection of S. chartreusis SF1623 by actinophage Φr5 DNA were studied. Protoplasts of S. chartreusis SF1623 prepared by treatment with lysozyme and achromopeptidase were very stable. Protoplasts from 20 to 22hr culture cells were more competent for transfection. The optimal pH of the medium for transfection was pH 7.6. The presence of NaCl, thymidine, ATP, ADP or adenosine in the transfection medium enhanced the frequency of transfection. The optimal conditions determined for protoplast transfection were 12.5% PEG 4,000, 300 mm NaCl, 1 mm thymidine, final concentration, Φr5 DNA and protoplasts in P3 medium (pH 7.6). The frequency of transfection under the optimal conditions was 5 × 10⁵ per μg Φr5 DNA and was about 3 × 10^?3 per regenerated protoplasts.

Progenitively mature phages appeared 4hr after incubation in the regeneration solution and their number continued to increase for about 11 hr. The burst size was estimated to be about 400.     

Fungal Proteolytic Enzymes

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.     

Central and peripheral des-acyl ghrelin regulates body temperature in rats

In the present study using rats, we demonstrated that central and peripheral administration of des-acyl ghrelin induced a decrease in the surface temperature of the back, and an increase in the surface temperature of the tail, although the effect of peripheral administration was less marked than that of central administration. Furthermore, these effects of centrally administered des-acyl ghrelin could not be prevented by pretreatment with [D-Lys3]-GHRP-6 GH secretagogue receptor 1a (GHS-R1a) antagonists. Moreover, these actions of des-acyl ghrelin on body temperature were inhibited by the parasympathetic nerve blocker methylscopolamine but not by the sympathetic nerve blocker timolol. Using immunohistochemistry, we confirmed that des-acyl ghrelin induced an increase of cFos expression in the median preoptic nucleus (MnPO). Additionally, we found that des-acyl ghrelin dilated the aorta and tail artery in vitro. These results indicate that centrally administered des-acyl ghrelin regulates body temperature via the parasympathetic nervous system by activating neurons in the MnPO through interactions with a specific receptor distinct from the GHS-R1a, and that peripherally administered des-acyl ghrelin acts on the central nervous system by passing through the blood–brain barrier, whereas it exerts a direct action on the peripheral vascular system.     

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.