Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

Crystal Structures of Human Dipeptidyl Peptidase Ⅳ in its Apo and Diprotin B-complexed Forms

Dipeptidyl peptidase Ⅳ (DPPIV), which belongs to the prolyl oligopeptidase family of serine proteases, is known to have a variety of regulatory biological functions and has been shown to be implicated in type 2 diabetes. It is therefore important to develop selective human DPPIV (hDPPIV) inhibitors. In this study, we determined the crystal structure of apo hDPPIV at 1.9 A resolution. Our high-resolution crystal structure of apo hDPPIV revealed the presence of sodium ion and glycerol molecules at the active site. In order to elucidate the hDPPIV binding mode and substrate specificity, we determined the crystal structure of hDPPIV-diprotin B (Val-Pro-Leu) complex at 2.1 A resolution, and clarified the difference in binding mode between diprotin B and diprotin A (Ile-Pro-Ile) into the active site of hDPPIV. Comparison between our crystal structures and the reported apo hDPPIV structures revealed that positively charged functional groups and conserved water molecules contributed to the interaction of ligands with hDPPIV. These results are useful for the design of potent hDPPIV inhibitors.     

Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome

We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.     

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.     

Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

RhoGDIβ affects HeLa cell spindle orientation following UVC irradiation

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor β (RhoGDIβ), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIβ significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIβ, but not a noncleavable mutant, RhoGDIβ (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIβ impacts spindle orientation in response to DNA damage.     

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H₂O₂ exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H₂O₂ exposure. The present work shows that this tick cell line could tolerate high H₂O₂ concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.     

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.     

Effects of systemic pancreastatin on memory retention

Pancreastatin, a peptide isolated from the pancreas, was shown to enhance memory retention after peripheral administration in mice when administration following T-maze footshock avoidance training. The effect of pancreastatin on memory retention, one week after training, was time dependent showing enhancement of retention when pancreastatin was administered 0 and 30 min but not 60 min after training. Pancreastatin reversed the amnesia produced by scopolamine. The pancreastatin fragment (33-49) also enhanced memory. Pancreastatin did not increase glucose in vivo. We conclude that peripherally administered pancreastatin modulates memory processing.     

Analysis of zona pellucida modifications due to cortical granule exocytosis in single porcine oocytes, using enhanced chemiluminescence

The present study was carried out to determine whether modification of zona pellucida (ZP) of a single oocyte following the cortical granule (CG) exocytosis induced by electrical stimulation could be analyzed using enhanced chemiluminescent (ECL) detection of the biotinylated ZP in a porcine oocyte. When a biotinylated ZP derived from a single oocyte matured in vitro was subjected to SDS-PAGE, 3 major bands (ZP1, ZP2 and ZP3) were observed following ECL detection. In these oocytes, CGs staining with fluorescein isothiocyanate (FITC)-labeled peanut agglutinin (FITC-PNA) had formed a monolayer underlying the plasma membrane. Electrical stimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs with a concomitant decrease in the amounts of ZP1 and ZP2 bands. However, the mobility changes of ZP1 and ZP2 on SDS-PAGE were not found under the inhibitory condition of the CG exocytosis in which oocytes were treated with ethylene glycol-bis(beta-aminoethyl ether) N, N, N',N'-tetraacetic acid (EGTA) or 1, 2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA/AM). In addition, when a time-dependent decrease in amounts of ZP1 and ZP2 bands on SDS-PAGE was observed in a single oocyte during activation, a maximum decrease in these bands was detected in oocytes incubated for at least 3.5 h after electrical stimulation. These results show that the method employed, ECL detection of the biotinylated ZP of a single oocyte, is a valuable tool for the analysis of ZP modification resulting from a decrease in amounts of ZP1 and ZP2 glycoproteins in combination with exocytosis of CGs, and that the prolonged period after activation is required for complete ZP modification in porcine oocytes.