Temperature adaptation of DNA ligases from psychrophilic organisms

DNA ligases operating at low temperatures have potential advantages for use in biotechnological applications. For this reason, we have characterized the temperature optima and thermal stabilities of three minimal Lig E-type ATP-dependent DNA ligase originating from Gram-negative obligate psychrophilic bacteria. The three ligases, denoted Vib-Lig, Psy-Lig, and Par-Lig, show a remarkable range of thermal stabilities and optima, with the first bearing all the hallmarks of a genuinely cold-adapted enzyme, while the latter two have activity and stability profiles more typical of mesophilic proteins. A comparative approach based on sequence comparison and homology modeling indicates that the cold-adapted features of Vib-Lig may be ascribed to differences in surface charge rather than increased local or global flexibility which is consistent with the contemporary emerging paradigm of the physical basis of cold adaptation of enzymes.

     

Population Genetic Structure and Conservation of the Lesser White-Fronted GooseAnser erythropus

The lesser white-fronted goose is a sub-Arctic species with a currently fragmented breeding range, which extends from Fennoscandia to easternmost Siberia. The population started to decline at the beginning of the last century and, with a current world population estimate of 25,000 individuals, it is the most threatened of the Palearctic goose species. Of these, only 30–50 pairs breed in Fennoscandia. A fragment of the control region of mtDNA was sequenced from 110 individuals from four breeding, one staging and two wintering areas to study geographic subdivisions and gene flow. Sequences defined 15 mtDNA haplotypes that were assigned to two mtDNA lineages. Both the mtDNA lineages were found from all sampled localities indicating a common ancestry and/or some level of gene flow. Analyses of molecular variance showed significant structuring among populations ( _ST 0.220, P < 0.001). The results presented here together with ecological data indicate that the lesser white-fronted goose is fragmented into three distinctive subpopulations, and thus, the conservation status of the species should be reconsidered.     

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).     

Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens (J. & C. Presl)

Thlaspi caerulescens (J. & C. Presl, "Prayon") is a heavy-metal hyperaccumulator that accumulates Zn and Cd to high concentrations (40,000 and 4,000 mg kg DW-1 respectively) without phytotoxicity. The mechanism of Cd tolerance has not been characterized but reportedly involves vacuolar sequestration. The role of phytochelatins (PCs) in metal tolerance in T. caerulescens and the related non-accumulator T. arvense was examined. Although PCs were produced by both species in response to Cd, these peptides do not appear to be involved in metal tolerance in the hyperaccumulator. Leaf and root PC levels for both species showed a similar positive correlation with tissue Cd, but total PC levels in the hyperaccumulator were generally lower, despite correspondingly higher metal concentrations. The lack of a role for PCs in the hyperaccumulator's response to metal stress suggests that other mechanisms are responsible Cd tolerance. The lower level of leaf PCs in T. caerulescens also implies that Cd in the shoot is sequestered in a compartment or form that does not elicit a PC response.     

A pilot study testing the feasibility of skin temperature monitoring to reduce recurrent foot ulcers in patients with diabetes – a randomized controlled trial

Background

Although monitoring foot skin temperatures has been associated with diabetic foot ulcer recurrence, no studies have been carried out to test the feasibility among European Caucasians. Moreover, the educational and/or motivational models that promote cognitive or psychosocial processes in these studies are lacking. Thus, we conducted a pilot randomized controlled trial to test the feasibility of monitoring foot skin temperatures in combination with theory-based counselling to standard foot care to reduce diabetic foot ulcer recurrence.

Methods

In a single-blinded nurse-led 1-year controlled trial, conducted at a hospital setting in Norway, 41 patients with diabetic neuropathy and previous foot ulcer were randomized to the intervention (n?=?21) or control groups (n?=?20). All participants were instructed in foot care and recording observations daily. Additionally, the intervention group was taught how to monitor and record skin temperature at baseline, and received counselling every third month supporting them to use the new treatment. Subjects observing temperature differences >2.0 °C between corresponding sites on the left and right foot on two consecutive days were asked to contact the study nurse and reduce physical activity. Fisher exact test was used to evaluate the effect of the intervention on the proportion of subjects with a foot ulcer. Kaplan-Meier survival analysis was performed to compare the two groups in regard to the time to development of a foot ulcer.

Results

In the intervention group, 67 % (n?=?14/21) monitored and recorded skin temperatures ≥80 % of the time while 70 % (n?=?14/20) of the controls recorded foot inspections. Foot ulcer incidence was 39 % (7/21) vs. 50 % (10/20) in the intervention and control groups, respectively (ns).

Conclusions

This feasibility study showed that the addition of counselling to promote self-monitoring of skin temperature to standard care to prevent recurrence of foot ulcer is feasible in patients with diabetes in Norway. Home skin temperature monitoring was performed as frequently by the intervention group as usual foot observations in the controls despite the extra effort required. We did not detect a difference in foot ulcer recurrence between groups, but our study may inform future full scale studies.

Trial registration

Clinicaltrials.gov NCT01269502

     

Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis

Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.     

Electronic ménages a trois: a molecular orbital perspective of protonated ferryl intermediates and synthetic models

Presented here is a molecular orbital perspective of various S=1 iron(IV)-hydroxo compound II intermediates as well as of synthetic heme and nonheme analogues. A key conceptual issue concerns how the iron(IV) center in these species coexists with highly reducing alkoxide, thiolate, phenolate, and hydroperoxide ligands. We suggest that a clue to this conundrum involves a three-way splitting of the spin density among the iron and two pi-basic ligands, which effectively delocalizes the high positive charge away from the iron.     

The crystal structure of Arabidopsis thaliana RAC7/ROP9: the first RAS superfamily GTPase from the plant kingdom

Arabidopsis thaliana RAC/ROP GTPases constitute a plant specific Rho GTPase family in the RAS superfamily, which has been implicated in numerous pivotal signalling cascades in plants. Research has shown that plants in some cases have evolved different modes of regulating Rho GTPase activity as compared to the equivalent systems in animals and yeast. In order to gain structural insight into plant signaling at the molecular level, we have determined the first crystal structure of a RAC-like GTPase belonging to the RAS superfamily from the plant kingdom. The structure of AtRAC7/ROP9 bound to GDP was solved at a resolution of 1.78 A. We have found that the structure of plant Rho GTPases is based upon a conserved G-domain architecture, but structural differences were found concerning the insert region and switch II region of the protein.     

The crystal structure of mismatch-specific uracil-DNA glycosylase (MUG) from Deinococcus radiodurans reveals a novel catalytic residue and broad substrate specificity

Deinococcus radiodurans is extremely resistant to the effects of ionizing radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch-specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 angstroms. Structural analyses suggest that drMUG possesses a novel catalytic residue, Asp-93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared with Escherichia coli MUG. The importance of Asp-93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other microorganisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all of the bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/thymidine-DNA glycosylase subfamily, here called the MUG2 family. We suggest that the novel catalytic residue (Asp-93) has evolved to provide drMUG with broad substrate specificity to increase the DNA repair repertoire of D. radiodurans.