Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′‐phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP‐binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT‐pyridoxamine 5′‐phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300‐ to 500‐fold decrease in both the rate constant of L‐alanine half‐transamination and the k_cat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc.     

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at − 5 °C, and then cooled slowly to − 30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation–dehydration, vitrification, encapsulation–vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants.     

New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7^T has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7^T that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).     

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.     

Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.     

Fate of Pathogenic Bacteria in Microcosms Mimicking Human Body Sites

During the infectious process, pathogens may reach anatomical sites where they are exposed to substances interfering with their growth. These substances can include molecules produced by the host, and his resident microbial population, as well as exogenous antibacterial drugs. Suboptimal concentrations of inhibitory molecules and stress conditions found in vivo (high or low temperatures, lack of oxygen, extreme pH) might induce in bacteria the activation of survival mechanisms blocking their division capability but allowing them to stay alive. These “dormant” bacteria can be reactivated in particular circumstances and would be able to express their virulence traits. In this study, it was evaluated the effect of some environmental conditions, such as optimal and suboptimal temperatures, direct light and antibiotic sub-inhibitory concentrations doses of antibiotic, on the human pathogens Escherichia coli and Enterococcus faecalis when incubated in fluids accumulated in the body of patients with different pathologies. It is shown that inoculation in a number of accumulated body fluids and the presence of gentamicin, reliable conditions encountered during pathological states, induce stress-responding strategies enabling bacteria to persist in microcosms mimicking the human body. Significant differences were detected in Gram-negative and Gram-positive species with E. faecalis surviving, as starved or viable but non-culturable forms, in any microcosm and condition tested and E. coli activating a viable but non-culturable state only in some clinical samples. The persistence of bacteria under these conditions, being non-culturable, might explain some recurrent infections without isolation of the causative agent after application of the standard microbiological methods.     

Effects of road salt deicers on sediment biogeochemistry

Road salt deicers, especially NaCl and CaCl₂, are increasingly applied to paved areas throughout the world. The goal of this study is to investigate the influence of high concentrations of these salts on wetland biogeochemistry. Sediment cores were collected in fall and spring from a freshwater wetland fringing an urban kettle lake (Asylum Lake, Kalamazoo, MI, USA), and incubated for 100 days in deionized water (control) or with treatments of 1 or 5 g/L CaCl₂·2H₂O or 5 g/L NaCl to simulate addition of road salt deciers. At monthly intervals, cores were sliced into three depths (0–5, 5–10, 10–15 cm) and pore waters extracted for analysis of pH, total alkalinity and dissolved Mn(II), Fe(II), PO ₄ ^?3 , NH₃, H₂S, SO₄ ^?2, Na, K, Mg, and Ca. Changes in solid phase geochemistry were assessed by measuring the percent organic matter and the distribution of Fe and Mn among four operationally defined sediment fractions (exchangeable, carbonate, reducible, oxidizable) in the control and treatment cores. Addition of NaCl, and especially CaCl₂, stimulated significant growth of microbial mats at the core sediment–water interface and led to decreased pH and increased concentrations of Mn(II), Fe(II) and exchangeable cations (Ca, Mg, K, Na) in the sediment pore waters. This study demonstrates that the influx of road salt deciers is likely to have a significant impact on biogeochemical cycling in wetland sediments.     

Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.     

From Dust to Ashes: The Challenges of Difference

By attending to ways in which middle-class wives in Yogyakarta, Java, describe negotiating sentiments among family members (including children, maids and husbands), this article argues that domestic relations in middle-class homes in Java have been importantly inflected by state rhetoric on gender propriety and market ideas of work. As a result, both middle-class women and maids have come to conceive of emotion work as part of an array of domestic obligations central to social reproduction.