Stature and body weight growth patterns from longitudinal data of Japanese children born during World War II

Stature and body weight data of 100 boys and 100 girls from 7 to 17 years of age in Shimodate City who were born during World War II were longitudinally analyzed. The children were significantly smaller and lighter throughout their growth period than those born 11 years after the end of the war. The correlation coefficient between statures at each age and at age 17 showed a gradual increase with increasing age, while that between statures at each age and at age 7 decreased with age. However, a drop in the correlation coefficient was found during puberty, at age 11 for girls and at age 13 for boys. Comparing the normalized distance from mean values of stature and body weight at age 7, at puberty, and at age 17, only 51% of the children continued to be in the same relative position for both height and weight, 6% of boys and 4% of girls showing a decreasing pattern for both and 4% of boys and 7% of girls showing an increasing pattern for both. Thus, about 60% of the children of either sex presented parallel stature and body weight growth patterns for ages from 7 to 17.     

In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate

The activity of pyruvate kinase, subtype M2 (PKM2), is known to be increased by fructose 1,6-bisphosphate (Fru-1,6-P2), one of the metabolites in the glycolytic pathway. Recently, we have shown that in vitro, Fru-1,6-P2 activated the association of monomer to form the tetrameric PKM2. To ascertain whether this mode of regulation also occurs in vivo, we prepared monomer-specific monoclonal antibody and quantified the monomer formation in situ in cultured cells by immunocytochemistry. The intracellular Fru-1,6-P2 was manipulated by the glucose concentration in the media. At the physiological concentration of glucose (4-6 mM), 30-35% of PK existed as a monomer. However, PKM2 was dissociated into monomer within minutes after cells were deprived of glucose. The maximal level of monomer was detected after 1 h at 37 degrees C. Monomer was rapidly (within minutes) converted to tetramer after addition of glucose. Furthermore, when cells cultured in 10 mM of glucose were treated with cytochalasin B, an inhibitor of the glucose transporter, a maximal level of monomer was detected within 20-30 min. Determination of Fru-1,6-P2 indicated that its intracellular concentration decreased concomitantly with the reduction in glucose concentration in the medium. These results indicate that monomer-tetramer inter-conversion is a major in vivo cellular regulatory mechanism in response to changes in the extracellular glucose concentration via Fru-1,6-P2.     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Development of microsatellite markers in a riparian shrub, Spiraea thunbergii (Rosaceae)

? Premise of the study: Microsatellite primers in the deciduous shrub Spiraea thunbergii were developed to investigate genetic diversity and population genetic structure. Cross-species transferability was assayed in four congeneric species. ? Methods and Results: Using a compound simple sequence repeat (SSR) marker method, 10 primer sets were identified in Japanese populations of S. thunbergii. The primers amplified compound SSRs with two to five alleles per locus. More than half of the primers were also amplified in S. prunifolia, S. nipponica var. nipponica, and S. japonica. ? Conclusions: These markers might be useful for future studies of population genetics of S. thunbergii and congeneric species.     

An Unusual Reaction Observed in Sulfonylation and Acylation of 2′,3′-O-Isopropylidenenebularine

Abstract

Treatment of 2′,3′-O-isopropylidenenebularine with p-toluenesulfonyl chloride in pyridine afforded 7,8-dihydro-2′,3′-O-isopropylidene-N⁷-(P-toluenesulfonyl)-8(R),5′-O-cclclonebularine as the major product, the structure of which was determined by X-ray crystallography. The reactions with other sulfonyl and acyl (aroyl) chlorides were also examined.     

Life history traits of white-spotted charr in an alpine environment: Implications for local adaptation along an altitude gradient

As a case study of local adaptation, we compared the life history traits of white-spotted charr Salvelinus leucomaenis between populations isolated in an alpine environment (altitude above 2000 m) and a normal environment (ca. 1000 m) in the Fuji River basin, near the southern limit of its distribution (35° N, 138° E). Our results showed that white-spotted charr in alpine streams had lower growth rate, longer lifespan, earlier spawning season, and were older and larger size at maturity, compared with those habiting a normal mountain stream. We concluded that charr in alpine streams were adapted to low water temperatures and long starvation periods by specialized life history traits.     

An acyl-CoA-binding protein from grape that is induced through ER stress confers morphological changes and disease resistance in Arabidopsis

We here report characterization of a grape (Vitis vinifera) acyl-CoA-binding protein (VvACBP). Expression of VvACBP was detected in grape leaves exposed to tunicamycin-induced endoplasmic reticulum (ER) stress as well as cold and heat shock treatments. In tendrils and peduncles, however, high-temperature treatment induced BiP (luminal binding protein) expression, a marker of ER stress in berry skin, but not VvACBP expression. We hypothesize that VvACBP may be sorted to the periphery of plant cells. Transgenic Arabidopsis plants, expressing VvACBP, exhibited slowed-down floral transition. The gene expression of proteins related to the photoperiodic pathway, CONSTANS, FLOWERING LOCUS T (FT), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), was down-regulated in transgenic seedlings. These results underscore the possibility that VvACBP may affect the regulation of floral transition in Arabidopsis by suppressing the photoperiodic pathway. The transgenic Arabidopsis plants also exhibited morphological changes such as thicker inflorescences and rosette leaves. In addition, the rosette leaves of the transgenic plants had higher anthocyanin, total phenol, and chlorophyll contents than those of the control plants. Finally, the transgenic plants showed disease resistance to Pseudomonas syringae and Colletotrichum higginsianum, suggesting that VvACBP may also enhance disease resistance in grapevine.     

Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli (E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2mg per 1L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 μM and 35.6 μmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.