Cloning of cDNAs encoding cell-wall hydrolases from pear (Pyrus communis) fruit and their involvement in fruit softening and development of melting texture

'La France' pear ( Pyrus communis L.) fruit stored at 1°C for 1 month (short-term storage) before transfer to 20°C softened and developed a melting texture during ripening, whereas fruit stored for 5 months (long-term storage) before transfer to 20°C softened but did not develop a melting texture. To clarify the mechanisms involved in fruit softening and textural changes, the cDNAs encoding cell-wall hydrolases were isolated by RT-PCR, and their expression and localization were investigated in 'La France' pears. Genes encoding three polygalacturonases (PG; EC 3.2.1.15), four pectin methylesterases (PME; EC 3.1.1.11), one α -arabinofuranosidase (ARF; EC 3.2.1.55), three β -galactosidases (GAL; EC 3.2.1.23), and two endo-1,4- β - d -glucanases (Cel; EC 3.2.1.4) were isolated. Among these 13 isolated genes, PcPG1 was the only gene for which the mRNA expression levels increased in both the short- and long-term stored fruits. This suggested that PcPG1 is involved in fruit softening rather than in the development of the melting texture. In contrast, the expression levels of PcPG3 , PcPME1 , PcPME2 , PcPME3 , PcGAL1 , PcGAL2 , and PcCel2 increased during ripening only in the short-term stored fruit. These genes might thus be involved in the development of the melting texture.     

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.     

Crystal structure of the RUN domain of the RAP2-interacting protein x

Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 A of resolution by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel alpha-helices, which form an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.     

Magnetic characterization of Daphnia resting eggs

This study characterized the magnetic materials found within Daphnia resting eggs by measuring static magnetization with a superconducting quantum interference device (SQUID) magnetometer, after forming two types of conditions, each of which consists of zero-field cooling (ZFC) and field cooling (FC). Magnetic ions, such as Fe(3+), contained in Daphnia resting eggs existed as (1) paramagnetic and superparamagnetic particles, demonstrated by a magnetization and temperature dependence of the magnetic moments under an applied magnetic field after ZFC and FC, and (2) ferromagnetic particles with definite magnetic moments, the content of which was estimated to be very low, demonstrated by the Moskowitz test. Conventionally, biomagnets have been directly detected by transmission electron microscopes (TEM). As demonstrated in this study, it is possible to nondestructively detect small biomagnets by magnetization measurement, especially after two types of ZFC and FC. This nondestructive method can be applied in detecting biomagnets in complex biological organisms.     

Protection of atherogenesis in thromboxane A2 receptor-deficient mice is not associated with thromboxane A2 receptor in bone marrow-derived cells

In the previous study, we generated mice lacking thromboxane A2 receptor (TP) and apolipoprotein E, apoE(-/-)TP(-/-) mice, and reported that the double knockout mice developed markedly smaller atherosclerotic lesions than those in apoE(-/-) mice. To investigate the mechanism responsible for reduced atherosclerosis in apoE(-/-)TP(-/-) mice, we examined the role of TP in bone marrow (BM)-derived cells in the development of the atherosclerotic lesions. When we compared the function of macrophages in apoE(-/-) and in apoE(-/-)TP(-/-) mouse in vitro, there was no difference in the expression levels of cytokines and chemokines after stimulation with lipopolysaccharide. We then transplanted the BM from either apoE(-/-) or apoE(-/-)TP(-/-) mice to either apoE(-/-) or apoE(-/-)TP(-/-) mice after sublethal irradiation. After 12 weeks with high fat diet, we analyzed the atherosclerotic lesion of aortic sinus. When the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-) mice, the lesion size was almost the same as that of apoE(-/-) mice without BM transplantation. In contrast, when the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-)TP(-/-) mice, the lesion size was markedly reduced. These results indicate that the protection of atherogenesis in TP(-/-) mice is not associated with TP in BM-derived cells.     

Characterization and cDNA cloning of dipeptidyl peptidase IV from the venom of Gloydius blomhoffi brevicaudus

Dipeptidyl peptidase activity was investigated in snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis and Crotalus atrox. The strongest dipeptidyl peptidase IV (DPP IV) activity was found in venom from G. blomhoffi brevicaudus. The substrate specificity, susceptibility to inhibitors, and pH optimum of the partially purified enzyme were similar to those of known DPP IVs from bacteria and eukaryotes. The G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on amino acid sequences highly conserved in known DPP IVs. Two cDNA species encoding DPP IV were obtained, and designated as DPP IVa and DPP IVb. This is the first study to report the primary structure of DPP IV from a reptile. The deduced amino acid sequences for DPP IVa and DPP IVb both consist of 751amino acid residues and are highly homologous to each other. A putative catalytic triad for serine proteases, Ser-616, Asp-694, and His-726, is present. It is of particular interest that the deduced NH(2)-terminal sequence associated with the characteristic signal peptide is identical to that determined from the purified DPP IV. This indicates that the signal peptide of snake venom DPP IV is not cleaved off during biosynthesis, unlike those of other snake venom proteins.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.     

Bone morphogenetic protein (BMP)-4 and BMP-7 suppress granulosa cell apoptosis via different pathways: BMP-4 via PI3K/PDK-1/Akt and BMP-7 via PI3K/PDK-1/PKC

BMP-4 and BMP-7 are associated with the suppression of granulosa cell apoptosis. LY294002 (PI3K inhibitor) or UCN-01 (PDK-1 inhibitor) increased the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. The inhibitors of ERK and p38 (SB203580) did not increase the percentage of apoptotic cells in the granulosa cells treated with BMP-4 or BMP-7. Akt inhibitor did not induce apoptosis in the BMP-4-treated granulosa cells, whereas it did induce apoptosis of the BMP-7-treated granulosa cells. In the granulosa cells treated with BMP-4, the PKC inhibitor increased the percentage of apoptotic cells. Our data show that BMP-4 and BMP-7 are associated with granulosa cell survival via several non-Smad specific pathways: BMP-4 via the PI3K/PDK-1/PKC and BMP-7 via the PI3K/PDK-1/Akt.     

Structural Confirmation of a Unique Carotenoid Lactoside,P457, in Symbiodinium sp. Strain nbrc 104787 Isolated from a Sea Anemone and its Distribution in Dinoflagellates and Various Marine Organisms

The molecular structure of the carotenoid lactoside P457, (3S,5R,6R,3′S,5′R,6′S)‐13′‐cis‐5,6‐epoxy‐3′,5′‐dihydroxy‐3‐(β‐d ‐galactosyl‐(1→4)‐β‐d ‐glucosyl)oxy‐6′,7′‐didehydro‐5,6,7,8,5′,6′‐hexahydro‐β,β‐caroten‐20‐al, was confirmed by spectroscopic methods using Symbiodinium sp. strain NBRC 104787 cells isolated from a sea anemone. Among various algae, cyanobacteria, land plants, and marine invertebrates, the distribution of this unique diglycosyl carotenoid was restricted to free‐living peridinin‐containing dinoflagellates and marine invertebrates that harbor peridinin‐containing zooxanthellae. Neoxanthin appeared to be a common precursor for biosynthesis of peridinin and P457, although neoxanthin was not found in peridinin‐containing dinoflagellates. Fucoxanthin‐containing dinoflagellates did not possess peridinin or P457; green dinoflagellates, which contain chlorophyll a and b, did not contain peridinin, fucoxanthin, or P457; and no unicellular algae containing both peridinin and P457, other than peridinin‐containing dinoflagellates, have been observed. Therefore, the biosynthetic pathways for peridinin and P457 may have been coestablished during the evolution of dinoflagellates after the host heterotrophic eukaryotic microorganism formed a symbiotic association with red alga that does not contain peridinin or P457.