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.     

Diversity and distribution of the bmp gene cluster and its Polybrominated products in the genus Pseudoalteromonas

The production of pentabromopseudilin and related brominated compounds by Pseudoalteromonas spp. has recently been linked to the bmp biosynthetic gene cluster. This study explored the distribution and evolutionary history of this gene cluster in the genus Pseudoalteromonas. A phylogeny of the genus revealed numerous clades that do not contain type strains, suggesting considerable species level diversity has yet to be described. Comparative genomics revealed four distinct versions of the gene cluster distributed among 19 of the 101 Pseudoalteromonas genomes examined. These were largely localized to the least inclusive clades containing the Pseudoalteromonas luteoviolacea and Pseudoalteromonas phenolica type strains and show clear evidence of gene and gene cluster loss in certain lineages. Bmp gene phylogeny is largely congruent with the Pseudoalteromonas species phylogeny, suggesting vertical inheritance within the genus. However, the gene cluster is found in three different genomic environments suggesting either chromosomal rearrangement or multiple acquisition events. Bmp conservation within certain lineages suggests the encoded products are highly relevant to the ecology of these bacteria.     

Time-Place Learning by Garden Warblers (Sylvia borin): Route or Map?

• 1 Garden warblers are able to learn to go to four different feeding places at different times of day (time-place learning). We investigated whether or not the birds rely on a fixed route by preventing them from entering one of the places at the normal time (blocking experiments). At the end of the blocking period the birds were allowed to visit any of the four feeding places.
• 2 In two such blocking experiments, one in which the blocking period was first thing in the morning (from 06.00–10.00 h), the other in which it was later in the day (from 12.00–16.00 h), there was no subsequent shift in the phase of visiting the places during the rest of the day, apart from a tendency to go to the first place of the day after the morning block. These results suggest that the birds do not rely on following a fixed route, but instead use a time-place map.

     

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.     

Der Einfluß von Objektart und früher Erfahrung auf die Diskriminationsleistungen von Haushühnern (Gallus domesticus)3

Main subject of the reported investigation is the question in which way the acquisition of a conditional discrimination is modified on the one hand by the kind of objects presented, on the other hand by early experience. 40 newly hatched chickens grew up either with all the wooden eggs or cubes used for training and test, or without these objects, or in a ‘natural’ (enriched) environment. The results prove an influence of the kind of objects — cubes are discriminated twice or three times as fast as wooden eggs — but no effects of the different environments on acquisition (number of trials for reaching learning criterion). Results are discussed under aspects of species-specific constraints on learning.     

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.     

Activity and intracellular distribution of enzymes of ketone-body metabolism in rat liver

1. The activities of hydroxymethylglutaryl-CoA synthase and lyase in rat liver were found to be two- to 15-fold greater than those reported by other authors under similar conditions. 2. When expressed on the basis of body weight, no appreciable differences were found between the activities of hydroxymethylglutaryl-CoA synthase in whole homogenates of livers from normal and starved rats. The synthase activity increased by 70% and 140% in livers of alloxan-diabetic rats and rats fed on a high-fat diet respectively. 3. Hydroxymethylglutaryl-CoA lyase activity showed no significant increases in starvation or alloxan-diabetes, but a 40% increase was found in fat-fed rats. 4. Less than 12% of the activities of both enzymes were found in the cytoplasmic fraction of normal liver. The cytoplasmic activity doubled in alloxan-diabetes and starvation; on feeding with a high-fat diet the increase, though significant, was less marked. 6. The intracellular distribution of glutamate dehydrogenase indicated that the changes in the cytoplasmic activities observed were not due to leakage from the mitochondria. 7. Feeding with a normal or high-fat diet after 48hr. starvation caused within 24hr. a decrease in the cytoplasmic activity of hydroxymethylglutaryl-CoA synthase to values lower than those found in rats fed on a corresponding diet for a longer period of time. 8. Acetoacetyl-CoA deacylase activity in liver was about 20% of that of hydroxymethylglutaryl-CoA synthase and was primarily located in the cytoplasm. Starvation or alloxan-diabetes did not alter the acetoacetyl-CoA deacylase activity. 9. It is concluded that variations in the concentrations of enzymes involved in acetoacetate synthesis play no major role in the regulation of ketone-body formation in starvation and alloxan-diabetes. The changes in the cytoplasmic activities of hydroxymethylglutaryl-CoA synthase and lyase suggest that acetoacetate synthesis can occur in the cytoplasm. This may play a role in the disposal of surplus acetyl-CoA arising in the cytoplasm when lipogenesis is inhibited.     

Temporal variation in habitat selection breaks the catch‐22 of spatially contrasting predation risk from multiple predators

Predator avoidance depends on prey being able to discern how risk varies in space and time, but this is made considerably more complicated if risk is simultaneously present from multiple predators. This is the situation for an increasing number of mammalian prey species, as large carnivores recover or are reintroduced in ecosystems on several continents. Roe deer Capreolus capreolus in southern Norway illustrate a case in which prey face two predators with contrasting patterns of predation risk. They face a catch‐22 situation: spatially avoiding the risk from one predator (lynx Lynx lynx in dense habitat) implies exposure to the other (hunters in open habitat). Using GPS‐data from 29 roe deer, we tested for daily and seasonal variation in roe deer selection for habitat with respect to the habitats’ year‐round average risk level. Generally, roe deer altered their habitat selection between night and day in a pattern consistent with being able to avoid predicted risk from the nocturnal lynx during night and predicted risk from human hunters during day. However, seasonal variation in habitat selection only partially corresponded with the predicted seasonal variation in risk. Whereas roe deer avoided areas with high risk from hunters more strongly during the hunting season than in other seasons, there was a lack of selection towards areas and time periods lowering the risk of lynx predation during winter. It seems likely that the risk of starvation and thermal stress constrain roe deer habitat selection during this energetically challenging season with cold temperatures, snow cover and limited natural forage. The habitat selection pattern of roe deer fits thus only partly with the two contrasting risk gradients they face. Adjusting risk‐avoidance behavior temporally can be an adaptive response in the case of several predators whose predation patterns differ in space and time.