Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Mapping the economic costs and benefits of conservation

Resources for biodiversity conservation are severely limited, requiring strategic investment. Understanding both the economic benefits and costs of conserving ecosystems will help to allocate scarce dollars most efficiently. However, although cost-benefit analyses are common in many areas of policy, they are not typically used in conservation planning. We conducted a spatial evaluation of the costs and benefits of conservation for a landscape in the Atlantic forests of Paraguay. We considered five ecosystem services (i.e., sustainable bushmeat harvest, sustainable timber harvest, bioprospecting for pharmaceutical products, existence value, and carbon storage in aboveground biomass) and compared them to estimates of the opportunity costs of conservation. We found a high degree of spatial variability in both costs and benefits over this relatively small (~3,000 km²) landscape. Benefits exceeded costs in some areas, with carbon storage dominating the ecosystem service values and swamping opportunity costs. Other benefits associated with conservation were more modest and exceeded costs only in protected areas and indigenous reserves. We used this cost-benefit information to show that one potential corridor between two large forest patches had net benefits that were three times greater than two otherwise similar alternatives. Spatial cost-benefit analysis can powerfully inform conservation planning, even though the availability of relevant data may be limited, as was the case in our study area. It can help us understand the synergies between biodiversity conservation and economic development when the two are indeed aligned and to clearly understand the trade-offs when they are not.     

New insights into creatine function and synthesis

The text-book view of the role of the creatine/creatine phosphate system as an energy buffer has been expanded to include functions such as energy shuttling, proton buffering and regulating cytosolic ADP levels. There is continuous need for creatine replacement due to creatinine formation. Replacement involves a combination of diet and de novo synthesis. Creatine synthesis makes very significant demands on amino acid metabolism, in particular that of glycine, arginine and methionine. It uses about 40% of all methyl groups transferred from S-adenosylmethionine. Although the traditional view of the function of the creatine/creatine phosphate system is largely concerned with its role in skeletal and cardiac muscle, recent work obliges us to take a broader view. In particular, its role in the brain is brought into sharp focus by the neurological symptoms displayed by children suffering from inborn errors of creatine synthesis and transport, as well as by suggestions that brain creatine status may play a role in cognitive performance in adults.     

Significance of Overvaluation of Shape/Weight in Binge‐eating Disorder: Comparative Study With Overweight and Bulimia Nervosa

Increasing empirical evidence supports the validity of binge‐eating disorder (BED) and its inclusion as a formal diagnosis in the Diagnostic and Statistical Manual of Mental Disorders (DSM‐V). Contention exists regarding the criteria for BED, including whether, like bulimia nervosa (BN), it should be characterized by overvaluation of shape/weight. This study examined the significance of overvaluation for BED using two complementary comparisons groups. Participants were 324 women who completed self‐report instruments as part of an Internet study. Analyses compared BMI, eating disorder (ED) features, and depressive levels in four groups: 123 overweight participants without ED, 47 BED participants who do not overvalue shape/weight, 101 BED participants who overvalue shape/weight, and 53 BN participants. Both BED groups had significantly greater ED psychopathology than the overweight group. Within BED, the group with overvaluation had significantly greater ED psychopathology and depressive levels despite no differences in binge eating. BED with overvaluation and BN groups differed little from each other but had significantly higher ED psychopathology and depressive levels than the other groups. Group differences existed despite similar age and BMI across the groups, as well as when controlling for group differences in depressive levels. These findings provide further support for the validity of BED and suggest that overvaluation of shape/weight, which provides important information about BED severity, warrants consideration as either a diagnostic specifier or as a dimensional severity rating. Although inclusion of overvaluation of shape/weight could be considered as a required criterion for BED, this would exclude a substantial proportion of BED patients with clinically significant problems.     

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.     

Acute regulation of steady-state GABA levels following GABA-transaminase inhibition in rat cerebral cortex

Cellular GABA levels are determined by the dynamic balance between synthesis and catabolism and are regulated at the level of glutamate decarboxylase, precursor availability (e.g., glutamate and glutamine), and possibly GABA degradation. GABA levels rise and stabilize within hours in human cortex following orally administered vigabatrin, an irreversible inhibitor of GABA-T, suggesting potential product inhibition of GABA synthesis or enhanced GABA degradation through the non-inhibited GABA-T fraction. In this study time courses of the rise in cortical GABA were measured in anesthetized rats in vivo after vigabatrin treatment using localized (1)H magnetic resonance spectroscopy and the times to reach steady-state for a given dose were determined. Rates of GABA synthesis were estimated for the period of constant GABA level from the accumulation of [2-(13)C]GABA following a short intravenous infusion (20 min) of either [1,6-(13)C(2)]glucose or [2-(13)C]acetate. No evidence of product inhibition of glutamate decarboxylase by the increased GABA concentration or reduced synthesis from [1,6-(13)C(2)]glucose (control, 0.031+/-0.010; vigabatrin-treated, 0.037+/-0.004 micromol/g/min, P=0.30) or [2-(13)C]acetate (control, 0.078+/-0.010; vigabatrin-treated, 0.084+/-0.006 micromol/g/min, P=0.42) was found. Fractional changes in steady-state GABA levels and GABA-T activities 5-6 h after vigabatrin treatment were approximately equal. The lack of change in GABA synthesis (and GABA catabolic flux for constant GABA levels) suggests that GABA-T has a near-zero flux control coefficient in vivo-capable of greatly altering the steady-state GABA concentration but exerting little or no control on GABA synthesis or GABA/glutamine cycling flux. The findings are consistent with a Michaelis-Menten kinetic model whereby cellular GABA levels increase until flux through the remaining (uninhibited) transaminase equals the rate of GABA synthesis. The findings suggest that astroglia may be the site of continuing GABA catabolism after acute vigabatrin treatment.     

Comparison of three fecal steroid metabolites for pregnancy detection used with single sampling in bighorn sheep (Ovis canadensis)

We conpared three fecal steroid metabolite assays for their usefulness in detecting pregnalcy among free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Bighorn Canyon National Recreation Area, Wyoming and Montana (USA) and captive bighorn ewes at ZooMontana in Billings, Montana. Fecal samples were collected from 11 free-ranging, radio-collared bighorn ewes in late January-May 2001 and from 20 free-ranging, radio-collared ewes in late March to mid-May 2002. Free-ranging ewes were monitored the following spring to determine whether or not they lambed. In addition, two captive ewes were studied at ZooMontana. With three exceptions, free-ranging bighorn ewes that produced lambs had nonspecific progesterone metabolite (iPdG) levels of >1800 ng/g feces and iPdG levels >7000 ng/gm feces when samples were collected between early March and mid-May. Samples collected earlier in the year were inconclusive. One false negative was suspected to be the result of sample collection error. Of the captive ewes, nonspecific pregnanediol-3alpha-glucuronide (PdG) and iPdG followed a predictable curve over the course of the 180-day pregnancies. We conclude that estrone conjugates are not useful in diagnosing pregnancy; however, fecal steroid analysis of PdG and iPdG can be used to accurately determine pregnancy and reproductive function in bighorn sheep. This holds great potential as a noninvasive technique for understanding the role of reproductive disease in wild bighom sheep.     

Metabolomics Analysis Identifies Intestinal Microbiota-Derived Biomarkers of Colonization Resistance in Clindamycin-Treated Mice

Background

The intestinal microbiota protect the host against enteric pathogens through a defense mechanism termed colonization resistance. Antibiotics excreted into the intestinal tract may disrupt colonization resistance and alter normal metabolic functions of the microbiota. We used a mouse model to test the hypothesis that alterations in levels of bacterial metabolites in fecal specimens could provide useful biomarkers indicating disrupted or intact colonization resistance after antibiotic treatment.

Methods

To assess in vivo colonization resistance, mice were challenged with oral vancomycin-resistant Enterococcus or Clostridium difficile spores at varying time points after treatment with the lincosamide antibiotic clindamycin. For concurrent groups of antibiotic-treated mice, stool samples were analyzed using quantitative real-time polymerase chain reaction to assess changes in the microbiota and using non-targeted metabolic profiling. To assess whether the findings were applicable to another antibiotic class that suppresses intestinal anaerobes, similar experiments were conducted with piperacillin/tazobactam.

Results

Colonization resistance began to recover within 5 days and was intact by 12 days after clindamycin treatment, coinciding with the recovery bacteria from the families Lachnospiraceae and Ruminococcaceae, both part of the phylum Firmicutes. Clindamycin treatment caused marked changes in metabolites present in fecal specimens. Of 484 compounds analyzed, 146 (30%) exhibited a significant increase or decrease in concentration during clindamycin treatment followed by recovery to baseline that coincided with restoration of in vivo colonization resistance. Identified as potential biomarkers of colonization resistance, these compounds included intermediates in carbohydrate or protein metabolism that increased (pentitols, gamma-glutamyl amino acids and inositol metabolites) or decreased (pentoses, dipeptides) with clindamycin treatment. Piperacillin/tazobactam treatment caused similar alterations in the intestinal microbiota and fecal metabolites.

Conclusions

Recovery of colonization resistance after antibiotic treatment coincided with restoration of several fecal bacterial metabolites. These metabolites could provide useful biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.     

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross‐braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra‐molecular assembly to specifically entrap massive baculoviruses. Inter‐subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re‐selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano‐particles.