The mammalian retina as a clock

Many physiological, cellular, and biochemical parameters in the retina of vertebrates show daily rhythms that, in many cases, also persist under constant conditions. This demonstrates that they are driven by a circadian pacemaker. The presence of an autonomous circadian clock in the retina of vertebrates was first demonstrated in Xenopus laevis and then, several years later, in mammals. In X. laevis and in chicken, the retinal circadian pacemaker has been localized in the photoreceptor layer, whereas in mammals, such information is not yet available. Recent advances in molecular techniques have led to the identification of a group of genes that are believed to constitute the molecular core of the circadian clock. These genes are expressed in the retina, although with a slightly different 24-h profile from that observed in the central circadian pacemaker. This result suggests that some difference (at the molecular level) may exist between the retinal clock and the clock located in the suprachiasmatic nuclei of hypothalamus. The present review will focus on the current knowledge of the retinal rhythmicity and the mechanisms responsible for its control.     

Functional vanilloid receptors in cultured normal human epidermal keratinocytes

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.     

Dimer structure of magainin 2 bound to phospholipid vesicles

Magainin 2 from African clawed frog Xenopus laevis is an antimicrobial peptide with broad spectra and action mechanisms considered to permeabilize bacterial membranes. CD, vibration, and solid-state NMR spectroscopies indicate the peptide adopts an alpha-helical conformation on binding to phospholipid bilayers, and its micelle-bound conformation, being monomeric and alpha-helical, is well detailed. We showed, however, that the peptide dimerizes on binding to phospholipid bilayers. This difference in the conformation and aggregation state between micelle- and bilayer-bound states prompted us to analyze the conformation of an equipotent analog of magainin 2 (F5Y,F16W magainin 2) bound to phosphatidylcholine vesicles using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. While observed medium-range TRNOE cross peaks were characteristic of alpha-helix, many long-range cross peaks were not compatible with the peptide's monomeric state. Simulated annealing calculations generated dimer structures indicating (1) two peptide molecules have a largely helical conformation in antiparallel orientation forming a short coiled-coil structure, (2) residues 4-20 are well converged and residues 9-20 are in an alpha-helical conformation, and (3) the interface of the two peptide molecules is formed by well-defined side chains of hydrophobic residues. Finally, determined structures are compatible with numerous investigations examining magainin-phospholipid interactions.     

Intraspecific variation in the status of ant symbiosis on a myrmecophyte,<Emphasis Type="Italic"> Macaranga bancana,</Emphasis> between primary and secondary forests in Borneo

A tree species, Macaranga bancana , distributed in South East Asian tropics has a mutualistic relationship with specific symbiotic ant species, which defend the plant from herbivores. To examine the intraspecific variation in the status of the ant-plant symbiosis among microhabitats of different light conditions, we investigated the species composition of nesting ants and the herbivory damage on M. bancana saplings by field observations and sampling in primary and secondary forests in Sarawak. In addition, the effectiveness of non-ant (physical and chemical) defenses were estimated by feeding the larvae of a polyphagous lepidopteran with M. bancana leaves from saplings in the two types of forests. All saplings in the primary forest were colonized by two Crematogaster ant species that had been known to be the obligate symbionts of M. bancana, while in the secondary forest, about half of the saplings were occupied by several ant species that were not obligate symbionts. There was little herbivory damage on saplings colonized by the two Crematogaster symbiont ants in both forest types, while the saplings colonized by the other ant species suffered a 10–60% loss of leaf area. Larval mortality of the polyphagous lepidopteran Spodoptera litura was significantly higher when larvae fed on leaves of M. bancana saplings in the secondary forest than when fed on leaves of M. bancana saplings in the primary forest. These results suggest that the symbiosis between ants and M. bancana is looser and the non-ant-defenses are stronger in secondary forests, where light is more intense, than in primary forests.     

Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

The PAR-1-activating peptide facilitates pepsinogen secretion in rats

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.     

The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules resulted in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity caused loss of the leading process, whereas repression of JNK activity did not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. Our data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration.     

Tissue distribution,molecular cloning,and gene expression of cytosolic glutathione peroxidase in Japanese monkey

Cytosolic glutathione peroxidase (GPX-1) is an important antioxidant enzyme that scavange hydrogen peroxide in mammalian cells. The level of GPX-1 activity in Japanese monkey (Macaca fuscata) tissues was determined and it was found to be high in the liver, kidney, and adrenal gland followed by the small intestine. We also cloned the GPX-1 cDNA that included the whole protein-coding region. The active-site selenocysteine was assumed to be encoded by a TGA codon. Compared to the GPX-1s of other mammalian species, essential residues in catalysis were well conserved in monkey GPX-1. Amino acid substitutions were frequent in the N- and C-terminal regions which are less essential in catalysis. Expression of GPX-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in consistence with the tissue distribution of GPX-1 activity.