Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

The effect of implantation on scaffoldless three-dimensional engineered bone constructs

Our laboratory has previously developed scaffoldless engineered bone constructs (EBC). Bone marrow stromal cells (BMSC) were harvested from rat femur and cultured in medium that induced osteogenic differentiation. After reaching confluence, the monolayer of cells contracted around two constraint points forming a cylinder. EBCs were placed in small diameter (0.5905 × 0.0625 in.) or large diameter (0.5905 × 0.125 in.) silicone tubing and implanted intramuscularly in the hind limb of a rat. Bone mineral content (BMC) of the EBC was analyzed before implantation and at 1 and 2 mo following implantation and compared to that of native femur bone at different stages of development. Negligible BMC was observed in E-20 femur or EBCs prior to implantation. One-month implantation in both small and large tubing increased BMC in the EBC. BMC of EBC from large tubing was greater than in 14 d rat neonatal femurs, but was 2% and 3% of BMC content in adult bone after 1 and 2 mo of implantation, respectively. Alizarine Red and osteopontin staining of the EBCs before and after implantation confirmed increased bone mineralization in the implanted EBCs. Implanted EBCs also had extensive vascularization. Our data suggest that BMSC can be successfully used for the generation of scaffoldless EBC, and this model can be potentially used for the generation of autologous bone transplants in humans.     

Isolation of the MurineNbr1Gene Adjacent to the MurineBrca1Gene

The humanNBR1cDNA has previously been identified using polyclonal sera to CA125, an ovarian tumor antigen used in monitoring ovarian cancer. The gene was mapped to theBRCA1region on chromosome 17q21 and subsequently found to lie in close proximity to the recently identifiedBRCA1gene. The NBR1 protein has a B-box motif but the function of the protein is as yet unknown. To investigate the function and importance of this gene, we have studied the conservation of this gene in other species and in particular in the mouse. We have isolated murineNbr1cDNA and genomic clones. Translation of the cDNA sequence indicates that the protein is highly conserved, being 89% similar and 84% identical to the human. Analysis of the murineNbr1genomic clones indicates that it maps less than 1 kb from theBrca1gene and that, unlike that in human, this region is not duplicated.     

Structure,function, aging and turnover of aggrecan in the intervertebral disc

Background

Aggrecan is the major non-collagenous component of the intervertebral disc. It is a large proteoglycan possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. Its abundance and unique molecular features provide the disc with its osmotic properties and ability to withstand compressive loads. Degradation and loss of aggrecan result in impairment of disc function and the onset of degeneration.

Scope of review

This review summarizes current knowledge concerning the structure and function of aggrecan in the normal intervertebral disc and how and why these change in aging and degenerative disc disease. It also outlines how supplementation with aggrecan or a biomimetic may be of therapeutic value in treating the degenerate disc.

Major conclusions

Aggrecan abundance reaches a plateau in the early twenties, declining thereafter due to proteolysis, mainly by matrix metalloproteinases and aggrecanases, though degradation of hyaluronan and non-enzymic glycation may also participate. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process as degradation products may accumulate in the disc for decades. The low turnover rate of the remaining aggrecan is an additional contributing factor, preventing protein renewal. It may be possible to retard the degenerative process by restoring the aggrecan content of the disc, or by supplementing with a bioimimetic possessing similar osmotic properties.

General significance

This review provides a basis for scientists and clinicians to understand and appreciate the central role of aggrecan in the function, degeneration and repair of the intervertebral disc.     

Histidine ligand protonation and redox potential in the rieske dioxygenases: role of a conserved aspartate in anthranilate 1,2-dioxygenase

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.     

A Human Health Risk Assessment of Pharmaceuticals in the Aquatic Environment

Analyses were conducted on four pharmaceutical compounds, representing different therapeutic classes, to evaluate the presence and potential adverse human health effects of trace levels of these substances in aqueous environmental media. Acetylsalicylic acid, clofibrate, cyclophosphamide, and indomethacin have been detected in aqueous environmental media including sewage treatment plant effluent, surface water, drinking water, and groundwater. An extensive literature search and chemical-specific risk assessments were performed to assess the potential human health significance of each compound's individual presence in environmental media. Safe water quality limits were estimated for each pharmaceutical by following the USEPA Methodology for Deriving Ambient Water Quality Criteria for the Protection of Human Health and were compared to the concentrations found in the environment. The calculation of the provisional ambient water quality criteria involved estimation of human exposure to contaminated water, including intake via bioaccumulation in fish, and calculation of cancer risk and non-cancer hazard indices. Parameters detailing the toxicological and pharmacological nature, exposure assessment, and environmental fate and transport of each pharmaceutical were also considered. The overall conclusion was that based on available data, no appreciable risk to humans exists, as the detected concentrations of each of these pharmaceutical compounds found in aqueous media were far below the derived safe limits     

Morphometry of Macaca mulatta forelimb. II. Fiber-type composition in shoulder and elbow muscles

The present study examined the fiber-type proportions of 22 muscles spanning the shoulder and/or elbow joints of three Macaca mulatta. Fibers were classified as one of three types: fast-glycolytic (FG), fast-oxidative-glycolytic (FOG), or slow-oxidative (SO). In most muscles, the FG fibers predominated, but proportions ranged from 25-67% in different muscles. SO fibers were less abundant except in a few deep, small muscles where they comprised as much as 56% of the fibers. Cross-sectional area (CSA) of the three fiber types was measured in six different muscles. FG fibers tended to be the largest, whereas SO fibers were the smallest. While fiber-type size was not always consistent between muscles, the relative size of FG fibers was generally larger than FOG and SO fibers within the same muscle. When fiber CSA was taken into consideration, FG fibers were found to comprise over 50% of the muscle's CSA in almost all muscles.     

Dynamic Scapular Movement Analysis: Is It Feasible and Reliable in Stroke Patients during Arm Elevation?

Knowledge of three-dimensional scapular movements is essential to understand post-stroke shoulder pain. The goal of the present work is to determine the feasibility and the within and between session reliability of a movement protocol for three-dimensional scapular movement analysis in stroke patients with mild to moderate impairment, using an optoelectronic measurement system. Scapular kinematics of 10 stroke patients and 10 healthy controls was recorded on two occasions during active anteflexion and abduction from 0° to 60° and from 0° to 120°. All tasks were executed unilaterally and bilaterally. The protocol’s feasibility was first assessed, followed by within and between session reliability of scapular total range of motion (ROM), joint angles at start position and of angular waveforms. Additionally, measurement errors were calculated for all parameters. Results indicated that the protocol was generally feasible for this group of patients and assessors. Within session reliability was very good for all tasks. Between sessions, scapular angles at start position were measured reliably for most tasks, while scapular ROM was more reliable during the 120° tasks. In general, scapular angles showed higher reliability during anteflexion compared to abduction, especially for protraction. Scapular lateral rotations resulted in smallest measurement errors. This study indicates that scapular kinematics can be measured reliably and with precision within one measurement session. In case of multiple test sessions, further methodological optimization is required for this protocol to be suitable for clinical decision-making and evaluation of treatment efficacy.