NT-702 (parogrelil hydrochloride, NM-702), a novel and potent phosphodiesterase inhibitor, improves reduced walking distance and lowered hindlimb plantar surface temperature in a rat experimental intermittent claudication model

NT-702 (parogrelil hydrochloride, NM-702), 4-bromo-6-[3-(4-chlorophenyl)propoxy]-5-[(pyridin-3-ylmethyl)amino]pyridazin-3(2H)-one hydrochloride, a novel phosphodiesterase (PDE) inhibitor synthesized as a potent vasodilatory and antiplatelet agent, is being developed for the treatment of intermittent claudication (IC) in patients with peripheral arterial disease. We assessed the efficacy of NT-702 in an experimental IC model as compared with cilostazol and additionally investigated the pharmacological property in vitro and ex vivo. NT-702 selectively inhibited PDE3 (IC(50)=0.179 and 0.260 nM for PDE3A and 3B) more potently than cilostazol (IC(50)=231 and 237 nM for PDE3A and 3B) among recombinant human PDE1 to PDE6. NT-702 inhibited in vitro human platelet aggregation induced by various agonists (IC(50)=11 to 67 nM) and phenylephrine-induced rat aortic contraction (IC(50)=24 nM). Corresponding results for cilostazol were 4.1 to 17 microM and 1.0 microM, respectively. NT-702 (3 mg/kg or more) significantly inhibited ex vivo rat platelet aggregation after a single oral dose. For cilostazol, 300 mg/kg was effective. In a rat femoral artery ligation model, NT-702 at 5 and 10 mg/kg repeated oral doses twice a day (BID) for 13 days significantly improved the reduced walking distance while the lowered plantar surface temperature was improved at 2.5 mg/kg and more. Cilostazol also improved the walking distance and surface temperature at 300 mg/kg BID but significant difference was only observed for surface temperature on day 8. These results suggest that NT-702 can be expected to have therapeutic advantage for IC.     

Interactions responsible for secondary structure formation during folding of equine beta-lactoglobulin

Equine beta-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A state was cooperatively transformed into the C state at low temperatures. This suggests the importance of hydrophobic interactions for stabilizing the A state. Peptides encompassing native-like and non-native alpha-helices were synthesized to investigate the interactions responsible for helix formation in the A and C states. These did not form stable helices, indicating that not only the helices in the A state but also the helices in the C state are stabilized by long-range interactions. A longer fragment, CHIBL, which encompasses the structured region in the A and C states, showed a helical structure. Proline-substituted mutants of CHIBL showed CD spectral changes similar to the corresponding mutants of the full-length protein in the C state. Therefore, CHIBL has a structure similar to the corresponding region of the full-length protein in the C state. This result indicates that interactions responsible for helix formation in the C state reside in the sequence of CHIBL, and that the sequences outside CHIBL are essential for secondary structure formation in the A state.     

Lack of promoting effects of chronic exposure to 1.95-GHz W-CDMA signals for IMT-2000 cellular system on development of N-ethylnitrosourea-induced central nervous system tumors in F344 rats

The present study was performed to evaluate effects of a 2-year exposure to an electromagnetic near-field (EMF) equivalent to that generated by cellular phones on tumor development in the central nervous system (CNS) of rats. For this purpose, pregnant F344 rats were given a single administration of N-ethylnitrosourea (ENU) on gestational day 18. A total of 500 pups were divided into five groups, each composed of 50 males and 50 females: Group 1, untreated controls; Group 2, ENU alone; Groups 3 to 5, ENU + EMF (sham exposure and two exposure levels). A 1.95-GHz wide-band code division multiple access (W-CDMA) signal, which is a feature of the International Mobile Telecommunication 2000 (IMT-2000) cellular system was employed for exposure of the rat head starting from 5 weeks of age, 90 min a day, 5 days a week, for 104 weeks. Brain average specific absorption rates (SARs) were designed to be .67 and 2.0 W/kg for low and high exposures, respectively. The incidence and numbers of brain tumors in female rats exposed to 1.95-GHz W-CDMA signals showed tendencies to increase but without statistical significance. Overall, no significant increase in incidences or numbers, either in the males or females, was detected in the EMF-exposed groups. In addition, no clear changes in tumor types in the brain were evident. Thus, under the present experimental conditions, exposure of heads of rats to 1.95-GHz W-CDMA signals for IMT-2000 for a 2-year period was not demonstrated to accelerate or otherwise affect ENU-initiated brain tumorigenesis.     

Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D

Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic “types” than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (d_S). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments “universally conserved segments” (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics.     

Conserved function of Rho-related Rop/RAC GTPase signaling in regulation of cell polarity in Physcomitrella patens

Cell polarity is fundamentally important to growth and development in higher plants, from pollen tubes to root hairs. Basal land plants (mosses and ferns) also have cell polarity, developing protonemal apical cells that show polar tip growth. Flowering plants have a distinct group of Rho GTPases that regulate polarity in polarized cell growth. Rop/RAC signaling module components have been identified in non-flowering plants, but their roles remain unclear. To understand the importance and evolution of Rop/RAC signaling in polarity regulation in land plants, we examined the functions of PpRop and PpRopGEF in protonemal apical cells of the moss Physcomitrella patens. Inducible overexpression of PpRop2 or PpRopGEF3 caused depolarized growth of tip-growing apical cells. PpRop2 overexpression also caused aberrant cross wall formation. Fluorescent protein-tagged PpRop2 localized to the plasma membrane, including the cross wall membrane, and fluorescent-tagged PpRopGEF3 showed polarized localization to the tip region in apical cells. Thus, our results suggest common functions of PpRop and PpRopGEF in the tip-growing apical cells and the importance of a conserved Rop/RAC signaling module in the control of cell polarity in land plants.     

Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors

During the wound-healing process, macrophages, fibroblasts, and myofibroblasts play a leading role in shifting from the inflammation phase to the proliferation phase, although little is known about the cell differentiation and molecular control mechanisms underlying these processes. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions as a key extracellular coordinator of multiple signalling networks. In this study, we investigated the contribution of TSK to wound healing. Analysis of wound tissue in heterozygous TSK-lacZ knock-in mice revealed a pattern of sequential TSK expression from macrophages to myofibroblasts. Quantitative PCR and in vitro cell induction experiments showed that TSK controls macrophage function and myofibroblast differentiation by inhibiting TGF-β1 secreted from macrophages. Our results suggest TSK facilitates wound healing by maintaining inflammatory cell quiescence.     

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C₁₈ reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 °C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.