Soil carbon dioxide efflux determined from large undisturbed soil cores collected in different soil management systems

The aim of this study was to evaluate a measuring technique for determining soil CO₂ efflux from large soil samples having undisturbed structure under controlled laboratory conditions. Further objectives were to use the developed measuring method for comparing soil CO₂ efflux from samples, collected in three different soil management systems at various soil water content values. The experimental technique was tested and optimised for timing of sampling by taking air samples after 1, 3 and 6 hours of incubation. Based on the results, the incubation time was set to three hours. The CO₂ efflux measured for different soil management systems was the highest in the no-till and the lowest in the ploughing treatment, which was in accordance with measurements on accessible organic carbon for microbes. An increase in CO₂ efflux with increasing soil water content was found in the studied soil water content range. Our results indicate that soil respiration rates, measured directly after tillage operations, can highly differ from those measured long after.     

Crystal Structures and Enzyme Mechanisms of a Dual Fucose Mutarotase/Ribose Pyranase

Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to l-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward l-fucose but also for the pyranase activity toward d-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.     

Appearance of hyperostosis frontalis interna in some osteoarcheological series from Hungary

Hyperostosis frontalis interna (HFI) is a generalised pathological condition with an unknown etiology and variable clinical association. It is characterized by excess bone growth and manifested on the inner table of the frontal bone, occasionally extending onto the temporals, parietals and the occipital.The etiology of HFI is uncertain: it may be an unknown genetic predisposition, a common environmental exposure, or special metabolic diseases.The purpose of the present study is to report cases of HFI in some osteoarcheological series from Hungary and to emphasize the importance of the investigation of HFI in ancient populations.Twenty out of 803 adults with observable frontal bones exhibited HFI, ranging from early to mid-type, including 15 females and 5 males. Some overgrowths with edges were blending into the endocranial surface, and some were prominently protruding from the surface. Advanced cases of HFI (type C) were observed after age 40-60 years.     

Monoclonal Antibodies Recognize Distinct Conformational Epitopes Formed by Polyglutamine in a Mutant Huntingtin Fragment

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt). PolyQ expansion above 35–40 results in disease associated with htt aggregation into inclusion bodies. It has been hypothesized that expanded polyQ domains adopt multiple potentially toxic conformations that belong to different aggregation pathways. Here, we used atomic force microscopy to analyze the effect of a panel of anti-htt antibodies (MW1–MW5, MW7, MW8, and 3B5H10) on aggregate formation and the stability of a mutant htt-exon1 fragment. Two antibodies, MW7 (polyproline-specific) and 3B5H10 (polyQ-specific), completely inhibited fibril formation and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These results suggest that expanded polyQ domains adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies, which has important implications for understanding the structural basis for polyQ toxicity and the development of intrabody-based therapeutics for HD.Huntington disease (HD)⁵ is a fatal neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein huntingtin (htt), which leads to its aggregation into fibrils (1). HD is part of a growing group of diseases that are classified as “conformational diseases,” which include Alzheimer disease (AD), Parkinson disease (PD), the prion encephalopathies, and many more (2–4). The length of polyQ expansion in HD is tightly correlated with disease onset, and a critical threshold of 35–40 glutamine residues is required for disease manifestation (5). Biochemical and electron microscopic studies with htt fragments demonstrated that expanded polyQ repeats (>39) form detergent-insoluble aggregates that share characteristics with amyloid fibrils (6–8), and the formation of amyloid-like fibrils by polyQ was confirmed by studies with synthetic polyQ peptides (9). Collectively, these studies demonstrated a correlation between polyQ length and the kinetics of aggregation. This phenomenon has been recapitulated in cell-culture models that express htt fragments (10–12). Although it is clear that proteins with expanded polyQ repeats assemble into fibrils in vitro, recent studies have reported that htt fragments can also assemble into spherical and annular oligomeric structures (13–16) similar to those formed by Aβ and α-synuclein, which are implicated in AD and PD, respectively.While the major hallmark of HD is the formation of intranuclear and cytoplasmic inclusion bodies of aggregated htt (17), the role of these structures in the etiology of HD remains controversial. For instance, the onset of symptoms in a transgenic mouse model of HD follows the appearance of inclusion bodies (18), while other studies indicate that inclusion body formation may protect against toxicity by sequestering diffuse, soluble forms of htt (10, 19, 20). Based on the direct correlation between polyQ length, htt aggregation propensity, and toxicity (6), it has been hypothesized that the aggregation of htt may mediate neurodegeneration in HD. However, there is no clear consensus on the aggregate form(s) that underlie toxicity, and there likely exist bioactive, oligomeric aggregates undetectable by traditional biochemical and electron microscopic approaches whose formation precedes disease symptoms. Although identification of the one or more toxic species of htt that trigger neurodegeneration in HD remains elusive, such species might exist in a diffuse, mobile fraction rather than in inclusion bodies (19). A thioredoxin-polyQ fusion protein was recently reported to exhibit toxicity in a meta-stable, β-sheet-rich, monomeric conformation (21), suggesting that polyQ can adopt multiple monomeric conformations, only some of which may be toxic. Consistent with such a scenario, molecular dynamic simulations and fluorescence correlation spectroscopy experiments with synthetic polyQ peptides indicate that polyQ domains can adopt a heterogeneous collection of collapsed conformations that are in equilibrium before aggregation (22–25).Although biochemical, biophysical, and computational approaches have yielded insight into the structures formed by polyQ in vitro, whether such structures form in vivo remains largely unknown. Indeed, determining the conformational state of any misfolded/aggregated protein in situ and/or in vivo remains a major technical challenge.Toward this goal, antibodies have been explored as a potentially powerful tool for detecting specific conformations or multimeric states of aggregated proteins in situ. Antibodies specific for amyloid fibrils often do not react with natively folded globular proteins from which they are derived, suggesting that such antibodies recognize a conformational epitope (26, 27). Several antibodies display conformation-dependent interactions with amyloids, aggregation intermediates, or natively folded precursor proteins. For example, there are antibodies specific for paired helical filaments of Tau (28–31), of aggregated forms of Aβ ranging from dimers to fibrils (32–34), and of native (35) or disease-related (36) forms of the prion protein. Antibodies have also been developed that are specific for common structural motifs associated with amyloid diseases, such as oligomers (37) and fibrils (38), independent of the peptide sequence of the amyloid forming protein from which they are derived, suggesting the potential for a common mechanism of aggregation and toxicity for these diseases.With regard to htt, several antibodies (MW1, MW2, MW3, MW4, MW5, IC2, and IF8), which are specific for polyQ repeats, stain Western blots of htt with expanded polyQ repeats much more strongly than htt with normal polyQ length (39, 40), suggesting that these antibodies may recognize abnormal polyQ conformations. Furthermore, these polyQ-specific antibodies have distinct staining patterns in immunohistochemical studies of brain tissue sections (39). In one study, the affinity and stoichiometry of MW1 binding to htt increased with polyQ length, suggesting a “linear lattice” model for polyQ (41). This model is supported by a crystal structure of polyQ bound to MW1, which showed that polyQ can adopt an extended, coil-like structure (42). However, an independent structural study showed that the anti-polyQ antibody 3B5H10 binds to a compact β-sheet-like structure of polyQ in a monomeric htt fragment.⁶ These results clearly indicate that polyQ domains can fold into at least two unique, stable, monomeric conformations and suggest that the “linear lattice” model is not generally applicable to all polyQ structures.Not only are antibodies useful for understanding what polyQ structures exist in situ, especially in the diffuse htt fraction of neurons, but antibodies and/or intrabodies may also have potential as therapeutic agents. For example, several studies showed that intrabodies reduce htt toxicity in cellular models (44–49). Moreover, one intrabody (C4) slows htt aggregation and prolongs lifespan in a Drosophila model of HD (50, 51), while another (mEM48) ameliorates neurological symptoms in a mouse model of HD (48).Three of the antibodies examined in this study (MW1, MW2, and MW7) modulate htt-induced cell death when co-transfected as single-chain variable region fragment antibodies (scFvs) in 293 cells with htt exon 1 containing an expanded polyQ domain (46). In these studies MW1 and MW2, which bind to the polyQ repeat in htt, increased htt-induced toxicity and aggregation (46). Conversely, MW7, which binds to the polyproline (polyP) regions adjacent to the polyQ repeat in htt, decreased its aggregation and toxicity (46). Interestingly, MW7 has also been shown to increase the turnover of mutant htt in cultured cells and reduce its toxicity in corticostriatal brain slice explants (49).Given the difficulty in understanding which specie(s) of htt exist and mediate pathogenesis in the putative toxic diffuse fraction of neurons, we sought to rigorously characterize the conformational specificity of a panel of anti-htt antibodies, the best in situ probes currently available for distinguishing specie(s) of htt. We reasoned that if htt can adopt multiple conformations that mediate different aggregation pathways, then anti-htt antibodies should differentially alter htt aggregation pathways by stabilizing or sequestering the specific conformers or aggregates they recognize. We therefore examined the effects of various antibodies on mutant htt fragment fibril formation and stability by atomic force microscopy (AFM). Our results are consistent with the hypothesis that monoclonal antibodies recognize distinct conformational epitopes formed by polyQ in a mutant htt fragment.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

Simvastatin ameliorates established pulmonary hypertension through a heme oxygenase-1 dependent pathway in rats

Background

Simvastatin has been shown to ameliorate pulmonary hypertension by several mechanisms in experimental animal models. In this study, we hypothesized that the major benefits of simvastatin in pulmonary hypertension occur via the heme oxygenase-1 pathway.

Methods

Simvastatin (10 mg/kgw/day) was tested in two rat models of pulmonary hypertension (PH): monocrotaline administration and chronic hypoxia. The hemodynamic changes, right heart hypertrophy, HO-1 protein expression, and heme oxygenase (HO) activity in lungs were measured in both models with and without simvastatin treatment. Tin-protoporphyrin (SnPP, 20 μmol/kg w/day), a potent inhibitor of HO activity, was used to confirm the role of HO-1.

Results

Simvastatin significantly ameliorated pulmonary arterial hypertension from 38.0 ± 2.2 mm Hg to 22.1 ± 1.9 mm Hg in monocrotaline-induced PH (MCT-PH) and from 33.3 ± 0.8 mm Hg to 17.5 ± 2.9 mm Hg in chronic hypoxia-induced PH (CH-PH) rats. The severity of right ventricular hypertrophy was significantly reduced by simvastatin in MCT-PH and CH-PH rats. Co-administration with SnPP abolished the benefits of simvastatin. Simvastatin significantly increased HO-1 protein expression and HO activity in the lungs of rats with PH; however co-administration of SnPP reduced HO-1 activity only. These observations indicate that the simvastatin-induced amelioration of pulmonary hypertension was directly related to the activity of HO-1, rather than its expression.

Conclusion

This study demonstrated that simvastatin treatment ameliorates established pulmonary hypertension primarily through an HO-1-dependent pathway.     

Colonisation and mass rearing: learning from others

Mosquitoes, just as other insects produced for the sterile insect technique (SIT), are subjected to several unnatural processes including laboratory colonisation and large-scale factory production. After these processes, sterile male mosquitoes must perform the natural task of locating and mating with wild females. Therefore, the colonisation and production processes must preserve characters necessary for these functions. Fortunately, in contrast to natural selection which favours a suite of characteristics that improve overall fitness, colonisation and production practices for SIT strive to maximize only the few qualities that are necessary to effectively control populations.However, there is considerable uncertainty about some of the appropriate characteristics due to the lack of data. Development of biological products for other applications suggest that it is possible to identify and modify competitiveness characteristics in order to produce competitive mass produced sterile mosquitoes. This goal has been pursued - and sometimes achieved - by mosquito colonisation, production, and studies that have linked these characteristics to field performance. Parallels are drawn to studies in other insect SIT programmes and aquaculture which serve as vital technical reference points for mass-production of mosquitoes, most of whose development occurs - and characteristics of which are determined - in an aquatic environment. Poorly understood areas that require further study are numerous: diet, mass handling and genetic and physiological factors that influence mating competitiveness. Compromises in such traits due to demands to increase numbers or reduce costs, should be carefully considered in light of the desired field performance.     

Generation of red fluorescent protein transgenic dogs

Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene‐expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP‐fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. genesis 47:314–322, 2009. © 2009 Wiley‐Liss, Inc.     

Inference of gene pathways using mixture Bayesian networks

Background  

Inference of gene networks typically relies on measurements across a wide range of conditions or treatments. Although one network structure is predicted, the relationship between genes could vary across conditions. A comprehensive approach to infer general and condition-dependent gene networks was evaluated. This approach integrated Bayesian network and Gaussian mixture models to describe continuous microarray gene expression measurements, and three gene networks were predicted.