Ab initio molecular orbital study of the mispairing ability of a nucleotide base analogue, N4-aminocytosine

The intrinsic properties of N4-aminocytosine, a base analogue of cytosine, are analyzed by an ab initio molecular orbital method. Relative stabilities of four possible isomeric structures of N4-aminocytosine are shown. The more stable isomer has the smaller dipole moment, so the relative stabilities of the isomers in solutions are subject to solvent polarity. The mutagenicity of this base analogue must arise because it can behave like either cytosine or thymine. It can form a guanine-cytosine-like base pair more easily than cytosine, and an adenine-thymine-like base pair less easily than thymine.     

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin''s peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.     

Effects of photoperiod on pituitary gonadotropin levels in masu salmon

We examined the effects of photoperiod on pituitary levels of two types of gonadotropin (GTH), GTH I and GTH II, in masu salmon Oncorhynchus masou to study their mechanism of synthesis. In Experiment 1, the effects of long or short photoperiod combined with castration were examined using 8-month-old precocious males. Castration was carried out in early August and then the fish were reared under a short (8L16D) or long (16L8D) photoperiod for 60 days. In Experiment 2, the effects of photoperiod combined with testosterone treatment were examined using 12-month-old immature females. Silastic tubes containing testosterone (500 microg /fish) or vehicle were implanted intra-peritoneally in early October. Fish were reared under 16L8D for 60 days, and then half of the fish were transferred to 8L16D, while the remaining fish were kept under 16L8D until Day 90. In Experiment 1, GTH I contents were higher under 16L8D than under 8L16D in the castrated group on Day 30. Moreover, GTH I contents were higher in the castrated group than the control group under 16L8D on Day 30. GTH II contents increased with testicular maturation in the control groups, whereas they remained at low levels in the castrated groups regardless of photoperiodic treatment. In Experiment 2, GTH I contents did not change remarkably in all the groups, while GTH II contents were remarkably increased by testosterone treatment regardless of photoperiodic treatment. These results indicate that the synthesis of GTH I and GTH II are differently regulated by photoperiod and testosterone in masu salmon.     

An Mn2+-stimulated neutral-sphingomyelinase in seminiferous tubules of immature Wistar rats

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. The seminiferous tubules of immature (19 day-old) Wistar rats have at least three types of sphingomyelinases, a lysosomal one and two microsomal ones. One of the microsomal sphingomyelinases is active at pH 6.5 and is stimulated by Mn²⁺ > Co²⁺ > Mg²⁺, and the other is active at pH 7.4 and is stimulated by Mn²⁺ > Mg²⁺ and inhibited by Co²⁺. The two microsomal enzymes are only slightly inhibited by EDTA and at pH 7.4 the stimulatory effects of Mn²⁺ and Mg²⁺ are additive. These data characterize the existence of two different membrane-bound sphingomyelinases in the seminiferous tubules of the rat.     

Isolation and cDNA cloning of ovarian cortical rod protein in kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

Amphibians as a model system for the investigation of respiratory control development

Recent medical advances have made it possible for babies to survive premature birth at increasingly earlier developmental stages. This population requires costly and sophisticated medical care to address the problems associated with immaturity of the respiratory system. In addition to pulmonary complications, respiratory instability and apnea reflecting immaturity of the respiratory control system are major causes of hospitalization and morbidity in this highly vulnerable population. These medical concerns, combined with the curiosity of physiologists, have contributed to the expansion of research in respiratory neurobiology. While most researchers working in this field commonly use rodents as an animal model, recent research using in vitro brainstem preparation from bullfrogs (Rana catesbeiana) have revealed the technical advantages of this animal model, and shown that the basic principles underlying respiratory control and its ontogeny are very similar between these two groups of vertebrates. The present review highlights the recent advances in the area of research with a focus on intermittent (episodic) breathing and the role of serotonergic and GABAergic modulation of respiratory activity during development.     

A nonradioactive whole-mount in situ hybridization protocol for Porphyra (Rhodophyta) gametophytic germlings

The objective of this study was to develop a method for whole-mount in situ hybridization (WISH) by using elongation factor-1 (EF-1) riboprobes in the red alga Porphyra yezoensis Ueda. Several modifications to the general WISH protocol, such as use of a short-length probe, performing partial digestion of the cell wall, optimization of proteinase K concentration and additional washing steps after hybridization were essential to reduce non-specific staining and to obtain sufficient quality of data. This protocol made it possible to detect a specific signal as a positive control in WISH assays of P. yezoensis.     

Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharomyces pombe

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the S. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.