Diverse substrate recognition and hydrolysis mechanisms of human NUDT5

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (α and β phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5–ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pβ, whereas ADPR is attacked at Pα. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site.     

Biochemical similarity of Ly-19, Ly-32, and Lyb-2 alloantigens encoded in the gene cluster on mouse chromosome 4

Mouse lymphocyte alloantigens Ly-19 and Ly-32 are controlled by the genes tightly linked to the Lyb-2 locus on chromosome 4. Despite the similarity in mouse strain distribution patterns, Ly-19 and Ly-32 antigens which have been detected on both B- and T-cell lineages are distinct from Lyb-2 antigen whose expression is restricted to the B cells. In this report, the close linkage of these three loci was confirmed by the typings of three sets of recombinant inbred mice including BXD, CXS, and OXA. Furthermore, the biochemical characterization of these Lyb-2-linked proteins, i. e., Ly-19, Ly-32, and Lyb-2, demonstrated their similarities on a molecular level. Two polypeptides of 45 000 and 95 000 were the components of these three alloantigens. Furthermore, sequential immunoprecipitation experiments indicated that the three alloantigenic determinants were located on the same molecular components. These findings may provide insight into the complexities and functional roles of Lyb-2 gene-cluster products.     

Optimal alignments of biological sequences on a microcomputer

An algorithm and a program have been developed which enableoptimal alignments of biological sequences on an 8–bitmicrocomputer. The compiled program can process sequences upto 1000 residues on a Commodore 64. Since this program was writtenoriginally in the BASIC language, it may readily be adaptedto other microcomputers with small changes. Received on March 11, 1985; accepted on March 14, 1985     

Identification and Characterization of CXCR4-Positive Gastric Cancer Stem Cells

Diffuse-type solid tumors are often composed of a high proportion of rarely proliferating (i.e., dormant) cancer cells, strongly indicating the involvement of cancer stem cells (CSCs) Although diffuse-type gastric cancer (GC) patients have a poor prognosis due to high-frequent development of peritoneal dissemination (PD), it is limited knowledge that the PD-associated CSCs and efficacy of CSC-targeting therapy in diffuse-type GC. In this study, we established highly metastatic GC cell lines by in vivo selection designed for the enrichment of PD-associated GC cells. By microarray analysis, we found C-X-C chemokine receptor type 4 (CXCR4) can be a novel marker for highly metastatic CSCs, since CXCR4-positive cells can grow anchorage-independently, initiate tumors in mice, be resistant to cytotoxic drug, and produce differentiated daughter cells. In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings). Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state. Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.     

Photosynthesis and growth of Ulva ohnoi and Ulva pertusa (Ulvophyceae) under high light and high temperature conditions,and implications for green tide in Japan

The macroalga Ulva ohnoi constitutes a considerable fraction of green tides in coastal areas of Japan, but little is known about the physiological characteristics of this species. To investigate the environmental factors that promote the formation of green tides, we tested the responses of U. ohnoi and another common Japanese species, Ulva pertusa, to various levels of irradiance at different water temperatures. Because the two species are morphologically similar, we identified them using the PCR‐restriction fragment length polymorphism method. Under laboratory conditions, we evaluated the photosynthetic, dark respiration, and relative growth rate at a range of water temperatures (5 to 35°C) and photosynthetically active radiation (0 to 1000 μmol photons m^?2 s^?1). The maximum gross photosynthetic rate of U. ohnoi was larger than that of U. pertusa. The dark respiration rates revealed no significant differences among the species and temperature conditions. At 500 μmol photons m^?2 s^?1, the relative growth rate of U. ohnoi was larger than that of U. pertusa in higher temperature and the difference was the largest at 20°C. The estimated compensation irradiance and estimated saturation irradiance of U. ohnoi and U. pertusa ranged from 0.709 to 5.510 and 40.530 to 58.674 μmol photons m^?2 s^?1, which were lower than those in other intertidal green macroalgae, from 6 to 11 and 50 to 82 μmol photons m^?2 s^?1, respectively. Thus, U. ohnoi which exists as free‐floating near the water surface and accumulating inside the green tide can survive extensively in the water column of the intertidal zone, furthermore, the species can maintain rapid growth in this situation. Therefore, as a result of this study, it is suggested that the ecological success of U. ohnoi in shallow waters such as the tidal flats, estuarine, and coasts of the inner bay in comparison with U. pertusa.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Reversible control of F1-ATPase rotational motion using a photochromic ATP analog at the single molecule level

Motor enzymes such as F₁-ATPase and kinesin utilize energy from ATP for their motion. Molecular motions of these enzymes are critical to their catalytic mechanisms and were analyzed thoroughly using a single molecule observation technique. As a tool to analyze and control the ATP-driven motor enzyme motion, we recently synthesized a photoresponsive ATP analog with a p-tert-butylazobenzene tethered to the 2′ position of the ribose ring. Using cis/trans isomerization of the azobenzene moiety, we achieved a successful reversible photochromic control over a kinesin-microtubule system in an in vitro motility assay. Here we succeeded to control the hydrolytic activity and rotation of the rotary motor enzyme, F₁-ATPase, using this photosensitive ATP analog. Subsequent single molecule observations indicated a unique pause occurring at the ATP binding angle position in the presence of cis form of the analog.     

Synthesis and antitumor activity of duocarmycin derivatives: modification at C-8 position of A-ring pyrrole compounds bearing the simplified DNA-binding groups

A series of the 8-O-substituted A-ring pyrrole derivatives of duocarmycin bearing the simplified DNA-binding moieties such as cinnamoyl or heteroarylacryloyl groups were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. In addition, the stability of the 8-O-substituted analogues in aqueous solution and the conversion to their active form (cyclopropane compound) from the 8-O-substituted analogues in mice or human serum were examined. The 8-O-substituted A-ring pyrrole derivatives bearing the simplified DNA-binding moieties showed remarkably potent in vivo antitumor activity and low peripheral blood toxicity compared with the 8-O-substituted A-ring pyrrole derivatives having the trimethoxyindole skeleton in segment-B (Seg-B), which were equal to 8-O-[(N-methylpiperazinyl)carbonyl] derivatives of 4'-methoxycinnamates and 4'-methoxy-beta-heteroarylacrylates. Moreover, among 8-O-substituted analogues, several compounds can be chemically or enzymatically converted to their active form in human serum. This result indicated that new 8-O-substituted derivatives were different prodrugs from KW-2189 and 8-O-substituted analogues being the same type of prodrug as KW-2189.