Plastid DNA polymerases from higher plants, Arabidopsis thaliana

Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H(2)O(2). These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.     

Selective inhibitors of terminal deoxyribonucleotidyltransferase (TdT): baicalin and genistin

Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.     

Gpr1, a putative G-protein-coupled receptor, regulates morphogenesis and hypha formation in the pathogenic fungus Candida albicans

In response to various extracellular signals, the morphology of the human fungal pathogen Candida albicans switches from yeast to hypha form. Here, we report that GPR1 encoding a putative G-protein-coupled receptor and GPA2 encoding a Galpha subunit are required for hypha formation and morphogenesis in C. albicans. Mutants lacking Gpr1 (gpr1/gpr1) or Gpa2 (gpa2/gpa2) are defective in hypha formation and morphogenesis on solid hypha-inducing media. These phenotypic defects in solid cultures are suppressed by exogenously added dibutyryl-cyclic AMP (dibutyryl-cAMP). Biochemical studies also reveal that GPR1 and GPA2 are required for a glucose-dependent increase in cellular cAMP. An epistasis analysis indicates that Gpr1 functions upstream of Gpa2 in the same signaling pathway, and a two-hybrid assay reveals that the carboxyl-terminal tail of Gpr1 interacts with Gpa2. Moreover, expression levels of HWP1 and ECE1, which are cAMP-dependent hypha-specific genes, are reduced in both mutant strains. These findings support a model that Gpr1, as well as Gpa2, regulates hypha formation and morphogenesis in a cAMP-dependent manner. In contrast, GPR1 and GPA2 are not required for hypha formation in liquid fetal bovine serum (FBS) medium. Furthermore, the gpr1 and the gpa2 mutant strains are fully virulent in a mouse infection. These findings suggest that Gpr1 and Gpa2 are involved in the glucose-sensing machinery that regulates morphogenesis and hypha formation in solid media via a cAMP-dependent mechanism, but they are not required for hypha formation in liquid medium or during invasive candidiasis.     

A Comparative Study of the Shell Matrix Protein Aspein in Pterioid Bivalves

Aspein is one of the unusually acidic shell matrix proteins originally identified from the pearl oyster Pinctada fucata. Aspein is thought to play important roles in the shell formation, especially in calcite precipitation in the prismatic layer. In this study, we identified Aspein homologs from three closely related pterioid species: Pinctada maxima, Isognomon perna, and Pteria penguin. Our immunoassays showed that they are present in the calcitic prismatic layer but not in the aragonitic nacreous layer of the shells. Sequence comparison showed that the Ser-Glu-Pro and the Asp-Ala repeat motifs are conserved among these Aspein homologs, indicating that they are functionally important. All Aspein homologs examined share the Asp-rich D-domain, suggesting that this domain might have a very important function in calcium carbonate formation. However, sequence analyses showed a significantly high level of variation in the arrangement of Asp in the D-domain even among very closely related species. This observation suggests that specific arrangements of Asp are not required for the functions of the D-domain.     

GABA-synthesizing enzyme, GAD67, from dermal fibroblasts: evidence for a new skin function

Glutamate decarboxylase (GAD) catalyzes the synthesis of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, from glutamate. An expression of GAD protein has been reported for brain and pancreas, but not for skin. In this study, we present evidence that GAD67 mRNA and protein are expressed in mouse skin and in human dermal fibroblasts. The expression of GAD67 gene is weaker in aged mouse than the young one. To further explore the function of GAD in skin, we have examined a potential role(s) of GABA in human dermal fibroblasts. We have observed that GABA stimulates the synthesis of hyaluronic acid (HA) and enhances the survival rate of the dermal fibroblasts when fibroblasts are exposed to H(2)O(2) an oxidative stress agent. Also observed were lowering the levels of HA and collagen in the embryonic skin from GAD67 deficient mouse as compared to those from the wild-type (WT) mouse. In this study, we have presented the evidences that GAD67 is localized in the dermis and is potentially involved in variety of skin activities.     

Optogenetic Patterning of Whisker-Barrel Cortical System in Transgenic Rat Expressing Channelrhodopsin-2

The rodent whisker-barrel system has been an ideal model for studying somatosensory representations in the cortex. However, it remains a challenge to experimentally stimulate whiskers with a given pattern under spatiotemporal precision. Recently the optogenetic manipulation of neuronal activity has made possible the analysis of the neuronal network with precise spatiotemporal resolution. Here we identified the selective expression of channelrhodopsin-2 (ChR2), an algal light-driven cation channel, in the large mechanoreceptive neurons in the trigeminal ganglion (TG) as well as their peripheral nerve endings innervating the whisker follicles of a transgenic rat. The spatiotemporal pattern of whisker irradiation thus produced a barrel-cortical response with a specific spatiotemporal pattern as evidenced by electrophysiological and functional MRI (fMRI) studies. Our methods of generating an optogenetic tactile pattern (OTP) can be expected to facilitate studies on how the spatiotemporal pattern of touch is represented in the somatosensory cortex, as Hubel and Wiesel did in the visual cortex.     

Development of a broadly reactive nested reverse transcription-PCR assay to detect murine noroviruses, and investigation of the prevalence of murine noroviruses in laboratory mice in Japan

A broadly reactive nested RT-PCR assay to detect MNV was developed and subsequently used to investigate the prevalence of MNV in laboratory mice in Japan. MNV were detected in 8 (22%) of 37 murine stool specimens by second-round PCR, although no positive band was obtained from any specimen by first-round PCR. Genetic analysis of the second round PCR products showed that MNV sequences detected in this study were closely matched (97.2 ∼ 99.1%) to that of MNV-3 (DQ223042). This is the first report demonstrating the prevalence of MNV in Japan.     

Dihydrokaempferol Glucoside from Cotyledons Promotes Flowering in Pharbitis nil

An extract of cotyledons of Pharbitis nil, which had been exposedto short-day conditions, was tested for flower-promoting activityin a shoot-tip assay system in vitro. The crude extract hadno flower-promoting activity, however, after partitioning ofthe crude extract with dichloromethane, the resulting aqueousfraction had flower-promoting activity. This activity was separatedinto two fractions by column chromatography on Toyopearl HW-40.One active fraction was identified as dihydrokaempferol-7-O-rß-D-glucoside(DHK-glc). This compound exhibited flower-promoting activityat the extremely low concentration of 4.4x10^-9. (Received April 25, 1995; Accepted August 11, 1995)