A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis

An Arabidopsis thaliana gene encoding a homologue of the potato alpha-glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.     

Periphyton-macroinvertebrate interactions in light and fish manipulated enclosures in a clear and a turbid shallow lake

In a clear and a turbid freshwater lake the biomasses of phytoplankton, periphytic algae and periphytonassociated macrograzers were followed in enclosures with and without fish (Rutilus rutilus) and four light levels (100%, 55%, 7% and < 1% of incoming light), respectively. Fish and light affected the biomass of primary producers and the benthic grazers in both lakes. The biomass of primary producers was generally higher in the turbid than the clear lake, and in both lakes fish positively affected the biomass, while shading reduced it. Total biomass of benthic grazing invertebrates was higher in the clear than in the turbid lake and the lakes were dominated by snails and chironomids + ostracods, respectively. While light had no effect on the biomass of grazers in the clear lake, snail breeding was delayed in the most shaded enclosures and presence of fish reduced the number of snails and the total biomass of grazers. In the turbid lake ostracod abundance was not influenced by light, but was higher in fish-free enclosures. Density of chironomids correlated positively with periphyton biomass in summer, while fish had no effect. Generally, light-mediated regulation of primary producers was stronger in the turbid than in the clear lake, but the regulation did not nambiguously influence the primary consumers. However, regulation by fish of the benthic grazer community was stronger in the clear than in the turbid lake, and in both lakes strong top-down effects on periphyton were seen. The results indicate that if present-day climate in Denmark in the future is found in coastal areas at higher latitudes, the effect of lower light during winter in such areas will be highest in clear lakes, with typically lower fish biomass and higher invertebrate grazer density.     

Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302

Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast two-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IR substrates, IRS-1. Using IR and IRS-1 as bait and prey, respectively, and c-Jun NH(2)-terminal kinase (JNK1) as the disruptor, we now show that phosphorylation of IRS-1 Ser-307, a previously identified site, is necessary but not sufficient for JNK1-mediated disruption of IR/IRS-1 binding. We further identify a new phosphorylation site, Ser-302, and show that this too is necessary for JNK1-mediated disruption. Seven additional kinases potentially linked to insulin resistance similarly block IR/IRS-1 binding in the disruptive Y3H, but through distinct Ser-302- and Ser-307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding Ser(P)-302 or Ser(P)-307 were used to determine whether the sites were phosphorylated under relevant conditions. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote Ser/Thr phosphorylation, including the phorbol ester phorbol 12-myristate 13-acetate, anisomycin, calyculin A, and insulin. The antibodies further showed that Ser(P)-302 and Ser(P)-307 are increased in animal models of obesity and insulin resistance, including genetically obese ob/ob mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings demonstrate that phosphorylation at both Ser-302 and Ser-307 is necessary for JNK1-mediated inhibition of the IR/IRS-1 interaction and that Ser-302 and Ser-307 are phosphorylated in parallel in cultured cells and in vivo under conditions that lead to insulin resistance.     

Using evolutionary information and ancestral sequences to understand the sequence-function relationship in GLP-1 agonists

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. A variety of GLP-1 sequences are known from amphibian species, and some of these have been tested here and found to be able to bind and activate the human GLP-1 receptor. While little difference was observed for the in vitro potency for the human GLP-1 receptor, larger differences were found in the enzymatic stability of these peptides. Two peptides showed increased enzymatic stability, and they group together phylogenetically, though they originate from Amphibia and Reptilia. We have used ancestral sequence reconstruction to analyze the evolution of these GLP-1 molecules, including the synthesis of new peptides. We find that the increased stability could not be observed in the resurrected peptides from the common ancestor of frogs, even though they maintain the ability to activate the human GLP-1 receptor. Another method, using residue mapping on evolutionary branches yielded peptides that had maintained potency towards the receptor and also showed increased stability. This represents a new approach using evolutionary data in protein engineering.     

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.     

Examining the Role of Collective Action in an Informal Seed System: A Case Study from the Central Valleys of Oaxaca, Mexico

This paper explores social arrangements associated with seed transactions among small-scale maize farmers in the Central Valleys of Oaxaca, Mexico, where no formal seed supply system exists. We test the hypothesis that individual farmers have strong incentives to participate in some form of collective action to ensure their access to seed. Six communities were studied, three of them in detail, using in-depth, semistructured interviews with key informants; focus group discussions; and a tracer study that followed seed flows among farmers. Farmers mostly saved seed and only occasionally acquired seed from outside sources. We found no evidence of a specialized social organization based on collective action to mediate seed flows. Seed transactions are infrequent, bilateral, and ad hoc, although trust is an important component, as it ensures reliable information about the seed is provided. Implications of these findings are discussed, especially for genetic diversity if the current supply system breaks down.     

Analysis of the collar-whisker structure of temperate lactococcal bacteriophage TP901-1

Proteins homologous to the protein NPS (neck passage structure) are widespread among lactococcal phages. We investigated the hypothesis that NPS is involved in the infection of phage TP901-1 by analysis of an NPS- mutant. NPS was determined to form a collar-whisker complex but was shown to be nonessential for infection, phage assembly, and stability.     

Identification of the lower baseplate protein as the antireceptor of the temperate lactococcal bacteriophages TP901-1 and Tuc2009

The first step in the infection process of tailed phages is recognition and binding to the host receptor. This interaction is mediated by the phage antireceptor located in the distal tail structure. The temperate Lactococcus lactis phage TP901-1 belongs to the P335 species of the Siphoviridae family, which also includes the related phage Tuc2009. The distal tail structure of TP901-1 is well characterized and contains a double-disk baseplate and a central tail fiber. The structural tail proteins of TP901-1 and Tuc2009 are highly similar, but the phages have different host ranges and must therefore encode different antireceptors. In order to identify the antireceptors of TP901-1 and Tuc2009, a chimeric phage was generated in which the gene encoding the TP901-1 lower baseplate protein (bppL(TP901-1)) was exchanged with the analogous gene (orf53(2009)) of phage Tuc2009. The chimeric phage (TP901-1C) infected the Tuc2009 host strain efficiently and thus displayed an altered host range compared to TP901-1. Genomic analysis and sequencing verified that TP901-1C is a TP901-1 derivative containing the orf53(2009) gene in exchange for bppL(TP901-1); however, a new sequence in the late promoter region was also discovered. Protein analysis confirmed that TP901-1C contains ORF53(2009) and not the lower baseplate protein BppL(TP901-1), and it was concluded that BppL(TP901-1) and ORF53(2009) constitute antireceptor proteins of TP901-1 and Tuc2009, respectively. Electron micrographs revealed altered baseplate morphology of TP901-1C compared to that of the parental phage.