Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily

OsIpk and HvIpk, inositol phosphate kinases, were cloned from rice (Oryza sativa L. var. indica, IR64) and barley (Hordeum vulgare) respectively. Sequence alignment showed that they belong to the ATP-grasp family, which includes inositol 1,3,4-trisphosphate 5/6-kinase from humans and Arabidopsis. Residues that are binding sites for ATP and coordinate magnesium in absence or presence of inositol phosphate are conserved and in total 23 residues are invariant among the twelve aligned inositol phosphate kinases. The genes were heterologously expressed in Escherichia coli and kinase activity assays with 17 different isomers of inositol mono-/di-/tri-/tetra-/pentaphosphate as well as phytate were performed. The strongest activity for both kinases was observed with Ins(3,4,5,6)P(4), which candidates as the primary substrate for these kinases in plants. Several species-specific differences between the two recombinant Ipks were observed. Rice OsIpk showed detectable kinase activity towards eight different substrates, whereas barley HvIpk showed kinase activity with all the substrates including inositol mono- and bisphosphates. HvIpk showed 3-kinase activity towards the Ins(1,4,5)P(3) substrate and it also interconverted the two substrates Ins(1,3,4,5)P(4) and Ins(1,3,4,6)P(4) by isomerase activity, which was not observed for the rice homologue. Both OsIpk and HvIpk had no detectable 2-kinase activity. Furthermore, the two Ipks showed phosphatase activity towards several inositol phosphates. Expression analysis by RT-PCR demonstrated that the Ipk gene was equally expressed in different tissues and developmental stages. Taken together, these results show that the Ipk kinase plays a significant role in the inositol phosphate interacting network in plants.     

Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.     

Effect of four plant species on soil 15N-access and herbage yield in temporary agricultural grasslands

Background and aims

We carried out field experiments to investigate if an agricultural grassland mixture comprising shallow- (perennial ryegrass: Lolium perenne L.; white clover: Trifolium repens L.) and deep- (chicory: Cichorium intybus L.; Lucerne: Medicago sativa L.) rooting grassland species has greater herbage yields than a shallow-rooting two-species mixture and pure stands, if deep-rooting grassland species are superior in accessing soil ¹⁵N from 1.2 m soil depth compared with shallow-rooting plant species and vice versa, if a mixture of deep- and shallow-rooting plant species has access to greater amounts of soil ¹⁵N compared with a shallow-rooting binary mixture, and if leguminous plants affect herbage yield and soil ¹⁵N-access.

Methods

¹⁵N-enriched ammonium-sulphate was placed at three different soil depths (0.4, 0.8 and 1.2 m) to determine the depth dependent soil ¹⁵N-access of pure stands, two-species and four-species grassland communities.

Results

Herbage yield and soil ¹⁵N-access of the mixture including deep- and shallow-rooting grassland species were generally greater than the pure stands and the two-species mixture, except for herbage yield in pure stand lucerne. This positive plant diversity effect could not be explained by complementary soil ¹⁵N-access of the different plant species from 0.4, 0.8 and 1.2 m soil depths, even though deep-rooting chicory acquired relatively large amounts of deep soil ¹⁵N and shallow-rooting perennial ryegrass when grown in a mixture relatively large amounts of shallow soil ¹⁵N. Legumes fixed large amounts of N₂, added and spared N for non-leguminous plants, which especially stimulated the growth of perennial ryegrass.

Conclusions

Our study showed that increased plant diversity in agricultural grasslands can have positive effects on the environment (improved N use may lead to reduced N leaching) and agricultural production (increased herbage yield). A complementary effect between legumes and non-leguminous plants and increasing plant diversity had a greater positive impact on herbage yield compared with complementary vertical soil ¹⁵N-access.     

Carboxylesterase 1 Gene Duplication and mRNA Expression in Adipose Tissue Are Linked to Obesity and Metabolic Function

Context and Aims

Carboxylesterase 1 (CES1) appears to play an important role in the control of the metabolism of triglycerides and cholesterol in adipocytes and other cell types including hepatocytes. Therefore, it is relevant to gain insights into the genetic versus non-genetic mechanisms involved in the control of CES1 mRNA expression. Here, we investigated CES1 mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of CES1 mRNA expression level in adipose tissue and the effect of CES1 gene duplication were assessed.

Methodology

A total of 295 monozygotic and dizygotic twin subjects (62–83 years) with (n = 48) or without (n = 247) type 2 diabetes mellitus were enrolled in the study. They were subjected to a standard oral glucose tolerance test and excision of abdominal subcutaneous fat biopsies during the fasting state. Levels of CES1 mRNA and copy number of the gene were assessed by quantitative PCR.

Results

CES1 mRNA expression level in adipose tissue was positively associated with body-mass index (P<0.001), homeostasis model assessment-insulin resistance (P = 0.003) and level of fasting glucose (P = 0.002), insulin (P = 0.006), and triglycerides (P = 0.003). The heritability for the expression of CES1 mRNA in adipose tissue was high. CES1 gene duplication was positively associated with insulin sensitivity (P = 0.05) as well as glucose tolerance (P = 0.03) and negatively associated with homeostasis model assessment-insulin resistance (P = 0.02). Duplication of CES1 was not linked to mRNA level of this gene (P = 0.63).

Conclusion

CES1 mRNA in adipose tissue appears to be under strong genetic control and was associated with measures of metabolic function raising the possibility of a potential role of this enzyme in the development of type 2 diabetes mellitus. Further studies are needed to understand the potential effect of CES1 gene duplication on adipocyte and whole-body metabolic functions.     

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p_meta-Euro = 2.08 × 10⁻¹⁰), transmembrane protein 39A (TMEM39A; rs1132200; p_meta-all = 8.62 × 10⁻⁹), and 17q21 (rs1453560; p_meta-all = 3.48 × 10⁻¹⁰) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10⁻⁸ < p_meta-Euro < 9.99 × 10⁻⁵) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.     

The effect of age at introduction and number of milk-portions on the behaviour of group housed dairy calves fed by a computer controlled milk feeder

This study is an investigation of the effect of age at introduction (6 days versus 14 days) and number of milk-portions (four milk-portions a day versus eight milk-portions a day) on integration into a large dynamic group of calves, fed by a computer controlled milk feeder. Forty calves (Jerseys, Danish Reds and Holstein-Friesians) were allocated equally to the two age conditions (A6 and A14) and the two milk-portion conditions (M4 and M8) in a 2 × 2 factorial design, according to sex and breed, and introduced into the group in pairs (one A6 and one A14). The behaviour of each pair was video-recorded for 8 h from 8 a.m. to 4 p.m. and 1 h from 1 to 2 a.m. on days 1, 8 and 15 after introduction.

The A6 calves performed less licking and sniffing, changed posture more often and tended to spend less time standing than the A14 calves. In the course of time A14 calves lay closer to other calves than the A6 calves.

The M8 calves, which were offered the same daily milk allowance as the M4 calves stood for a longer time in the milk feeding station and the M8 calves also sucked the empty teat more frequently than the M4 calves. Finally, the M8 calves initiated more social play behaviour than the M4 calves. On the first day after introduction the M8 calves lay closer to other calves than the M4 calves.

The results suggest that calves integrate better into a group when introduced at the age of 14 days than at the age of 6 days. Distributing the milk into eight daily milk-portions, rather than four milk-portions in the first period after introduction, increased milk feeder occupancy, which may facilitate learning to use the milk feeder. Surprisingly, more milk-portions also stimulated play behaviour, which is suggested to be due to M8 calves encountering more calves in the milk feeder area.     

Structure and expression of elongation factor 1 alpha in tomato.

A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues.