Generation of knock-in mice carrying third cones with spectral sensitivity different from S and L cones

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.     

Analysis of expressed sequence tags in prothallia of Adiantum capillus-veneris

The analysis of expressed sequences from a diverse set of plant species has fueled the increase in understanding of the complex molecular mechanisms underlying plant growth regulation. While representative data sets can be found for the major branches of plant evolution, fern species data are lacking. To further the availability of genetic information in pteridophytes, a normalized cDNA library of Adiantum capillus-veneris was constructed from prothallia grown under white light. A total of 10,420 expressed sequence tags (ESTs) were obtained and clustering of these sequences resulted in 7,100 nonredundant clusters. Of these, 1,608 EST clusters were found to be similar to sequences of known function and 1,092 EST clusters showed similarity to sequences of unknown function. Given the usefulness of Adiantum for developmental studies, the sequence data represented in this report stand to make a significant contribution to the understanding of plant growth regulation, particularly for pteridophytes.     

Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin-degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin

A proteomic differential display technique was utilized to study cellular responses of Phanerochaete chrysosporium exposed to vanillin, one of the key intermediates found during lignin biodegradation. Intracellular proteins were resolved by 2-DE and target protein spots were identified using MALDI-MS after in-gel tryptic digestions. Upon addition of vanillin to P. chrysosporium, up-regulation of homogentisate 1,2-dioxygenase, 1,4-benzoquinone reductases, aldehyde dehydrogenase, and aryl-alcohol dehydrogenase, which seem to play roles in vanillin metabolism, was observed. Furthermore, enzymes involved in glycolysis, the tricarboxylic acid cycle, the pentose-phosphate cycle, and heme biosynthesis were also activated. Up-regulation of extracellular peroxidase was also observed. One of the most unique phenomena against exogenous vanillin was a switch from the glyoxylate cycle to the tricarboxylic acid cycle, where a drastic increase in isocitrate dehydrogenase activity was observed. The exogenous addition of other aromatic compounds also caused an increase in its activity, which in turn triggered NAD(P)H production via the action of dehydrogenases in the tricarboxylic acid cycle, heme biosynthesis via the action of aminolevulinic acid synthase on succinyl-CoA, and energy production via activation of the mitochondrial electron transfer system. These metabolic shifts seem to be required for activating a metabolic system for aromatic compounds.     

CD24 is expressed specifically in the nucleus pulposus of intervertebral discs

Intervertebral disc (IVD) consists of a soft gelatinous material in its center, the nucleus pulposus (NP), bounded peripherally by fibrocartilage, annulus fibrosus (AF). Despite the number of patients with IVD degeneration, gene expression analysis has not been undertaken in NP and therefore little is known about the molecular markers expressed in NP. Here, we undertook a microarray screen in NP with the other nine tissues to identify the specific cell surface markers for NP. Five membrane associating molecules out of 10,490 genes were identified as highly expressing genes in NP compared with the other tissues. Among them, we identified CD24, a glycosylphosphatidylinositol (GPI) anchor protein as a cell surface marker for NP. CD24 expression was also detected in the herniated NP and chordoma, a malignant primary tumor derived from notochordal cells, while it was absent in chondrosarcoma. Therefore, CD24 is a molecular marker for NP as well as the diseases of IVD.     

Isolation of the feline alpha1,3-galactosyltransferase gene, expression in transfected human cells and its phylogenetic analysis

The enzyme alpha 1,3-galactosyltransferase (alpha1,3-GT), which catalyzes synthesis of terminal alpha-galactosyl epitopes (Gal alpha1,3Gal beta1-4GlcNAc-R), is produced in non-primate mammals, prosimians and new-world monkeys, but not in old-world monkeys, apes and humans. We cloned and sequenced a cDNA that contains the coding sequence of the feline alpha1,3-GT gene. Flow cytometric analysis demonstrated that the alpha-galactosyl epitope was expressed on the surface of a human cell line transduced with an expression vector containing this cDNA, and this alpha-galactosyl epitope expression subsided by alpha-galactosidase treatment. The open reading frame of the feline alpha1,3-GT cDNA is 1,113 base pairs in length and encodes 371 amino acids. The nucleotide sequence and its deduced amino acid sequence of the feline alpha1,3-GT gene are 88-90% and 85-87%, respectively, similar to the reported sequences of the bovine, porcine, marmoset and cebus monkey alpha1,3-GT genes, while they are 88% and 82-83%, respectively, similar to those of the orangutan and human alpha1,3-GT pseudogenes, and 81% and 77%, respectively, similar to the murine alpha1,3-GT gene. Thus, the alpha1,3-GT genes and pseudogenes of mammals are highly similar. Ratios of non-synonymous nucleotide changes among the primate pseudogenes as well as the primate genes are still higher than the ratios of non-primates, suggesting that the primate alpha1,3-GT genes tend to be divergent.     

Regulation of Vascular Development by CLE Peptide-receptor Systems

Cell division and differentiation of stem cells are controlled by non-cell-autonomous signals in higher organisms. The plant vascular meristem is a stem-cell tissue comprising procambial cells that produce xylem cells on one side and phloem cells on the other side. Recent studies have revealed that TDIF (tracheary element differentiation inhibitory factor)/CLE41/CLE44 peptide signal controls the procambial cell fate in a non-cell-autonomous manner. TDIF produced in and secreted from phloem cells is perceive...     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.