Monte Carlo simulations of individual variability and their effects on simulated heat stress using thermoregulatory modeling

This paper addresses a variable-dependence (VD) MC method developed based on a previous attempt (VI-MC method) (J. Therm. Biol. 29 (2004), 515) to be incorporated in a thermoregulatory model. Simulated individuals with anthropometrics by VI- and VD-MC methods for US Army population were compared using principal component analysis and Fisher's exact tests. The results indicated that VD-MC data represented overall body size as the primary component and body shape as the secondary component that were more realistic and similar to the measured US Army data (p>0.05) rather than VI-MC data (p<0.05). Such differences consequently affected individual thermoregulatory responses to simulated heat stress. The VD-MC method provides a more realistic representation of individual variability and thus underpins more realistic predictions of individual thermoregulatory responses.     

Solution Conformation and Dynamics of the HIV-1 Integrase Core Domain

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3′ processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl₂ concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg²⁺. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. ¹⁵N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.     

Genetic Diversity and Core Collection Evaluations in Common Wheat Germplasm from the Northwestern Spring Wheat Region in China

Fluorescence microsatellite markers were employed to reveal genetic diversity of 340 wheat accessions consisting of 229 landraces and 111 modern varieties from the Northwest Spring Wheat Region in China. The 340 accessions were chosen as candidate core collections for wheat germplasm in this region. A core collection representing the genetic diversity of these accessions was identified based on a cluster dendrogram of 78 SSR loci. A total of 967 alleles were detected with a mean of 13.6 alleles (5–32) per locus. Mean PIC was 0.64, ranged from 0.05 to 0.91. All loci were distributed relatively evenly in the A, B and D wheat genomes. Mean genetic richness of A, B and D genomes for both landraces and modern varieties was B > A > D. However, mean genetic diversity indices of landraces changed to B > D > A. As a whole, genetic diversity of the landraces was considerably higher than that of the modern varieties. The big difference of genetic diversity indices in the three genomes suggested that breeding has exerted greater selection pressure in the D than the A or B genomes in this region. Changes of allelic proportions represented in the proposed core collection at different sampling scales suggested that the sampling percentage of the core collection in the Northwest Spring Wheat Region should be greater than 4% of the base collection to ensure that more than 70% of the variation is represented by the core collection. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Free N-linked oligosaccharide chains: Formation and degradation

There is growing evidence that N-linked glycans play pivotal roles in protein folding and intra- and/or intercellular trafficking of N-glycosylated proteins. It has been shown that during the N-glycosylation of proteins, significant amounts of free oligosaccharides (free OSs) are generated in the lumen of the endoplasmic reticulum (ER) by a mechanism which remains to be clarified. Free OSs are also formed in the cytosol by enzymatic deglycosylation of misfolded glycoproteins, which are subjected to destruction by a cellular system called “ER-associated degradation (ERAD).” While the precise functions of free OSs remain obscure, biochemical studies have revealed that a novel cellular process enables them to be catabolized in a specialized manner, that involves pumping free OSs in the lumen of the ER into the cytosol where further processing occurs. This process is followed by entry into the lysosomes. In this review we summarize current knowledge about the formation, processing and degradation of free OSs in eukaryotes and also discuss the potential biological significance of this pathway. Other evidence for the occurrence of free OSs in various cellular processes is also presented.     

Carbohydrate microarrays reveal sulphation as a modulator of siglec binding

Siglecs are receptors on cells of the immune, haemopoietic, and nervous systems that recognize sialyl-glycans with differing preferences for sialic acid linkage and oligosaccharide backbone sequence. We investigate here siglec binding using microarrays of Lewis(x) (Le(x))- and 3'-sialyl-Le(x)-related probes with different sulphation patterns. These include sulphation at position 3 of the terminal galactose of Le(x), position 6 of the galactose of Le(x) and sialyl-Le(x), position 6 of N-acetylglucosamine of Le(x) and sialyl-Le(x), or both positions of sialyl-Le(x). Recombinant soluble forms of five siglecs have been investigated: human Siglec-7, -8, -9, and murine Siglec-F and CD22 (Siglec-2). Each siglec has a different binding pattern. Unlike two C-type lectins of leukocytes, L-selectin and Langerin, which also bind to sulphated analogues of sialyl-Le(x), the siglecs do not give detectable binding signals with sulphated analogues that are lacking sialic acid. The sulphate groups modulate, however, positively or negatively the siglec binding intensities to the sialyl-Le(x) sequence.     

Structural analysis of the lipooligosaccharide-derived oligosaccharide of Histophilus somni (Haemophilus somnus) strain 8025

Previous structural studies in our laboratory on lipooligosaccharide (LOS) inner core oligosaccharide (OS) had identified structures from several strains of Histophilus (Haemophilus) somni (738, 2336, 1P, 129Pt). Recently a type strain 8025 was proposed for this species and we therefore sought to determine the core OS structure of this H. somni strain. Core OS was isolated by standard methods from Westphal purified LOS. Structural information was established by a combination of monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core OS was determined on the basis of the combined data from these experiments: [carbohydrates: see text]. The structure determined contains aspects of other Histophilus somni core OS structures, such as the beta-Gal attached at the 2-position of Hep II (2336), PEtn only at the 6-position of Hep II (738, 129Pt) and a lactose extension from Hep I (1P). Since genetic manipulation has been achieved with this strain, the identification of the core OS structure will enable experiments designed to identify the role of glycosyltransferases involved in LOS biosynthesis.     

Different transformation pathways of murine fibroblast NIH 3T3 cells by hepatitis C virus core and NS3 proteins

The oncogenic potential of both Hepatitis C virus (HCV) core and HCV NS3 proteins has been demonstrated, but these proteins induce transformation of immortal murine fibroblasts NIH 3T3 via different pathways. As long-term expression (50-100 passages) of HCV core triggers neoplastic transformation of NIH 3T3 through crisis of growth, HCV NS3 induces transformation shortly after transfection. We explain this distinction by different effects of core and NS3 on p53-mediated transactivation: inhibition by NS3 and activation by core protein.     

The spatial distribution of bacteria in Grana-cheese during ripening

The microbial composition and its spatial distribution of Grana Trentino, a hard Parmesan-like cheese, was determined, from vat milk to cheese. After cutting along the vertical axis of the cheese wheels, three layers were sampled diagonally across the cheese: under the cheese rind, an intermediate section and the cheese core. After two different ripening periods (9 and 18 months), the cheese samples were analysed using traditional culture dependent and culture independent methods. Milk samples were dominated by mesophilic and psychrophilic bacterial counts. Thermophilic bacteria (Lactobacillus helveticus) were found in high amounts in cooked whey and natural whey starter cultures. After 9 months of ripening, lactic acid bacteria (LAB) counts were higher than those after 18 months. Furthermore, the LAB numbers in the cheese core was lower than those under the rind or in the intermediate section. The main LAB species isolated from milk (Lactococcus lactis, Pediococcus pentosaceus, Streptococcus uberis and Lactococcus garvieae) were not found in the corresponding cheeses. Some differences were observed in the species composition among the three cheese sections. Microbiota under the rind and in the intermediate section was similar and dominated by Lactobacillus paracasei and Lactobacillus rhamnosus. The core, after 18 months of ripening, was characterized by a total absence of LAB. In each sample, all LAB were genotypically grouped and the different biotypes were subjected to several technological tests indicating that some non-starter LAB (NSLAB) displayed technological features that are favorable for the production of Grana Trentino cheese.     

Molecular insight into substrate recognition by human cytosolic sialidase NEU2

Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide α(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide α(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid.     

The mouse: An “average” homeotherm

Mice, rats, and nearly all mammals and birds are classified as homeothermic, meaning that their core temperature is regulated at a constant level over a relatively wide range of ambient temperatures. In one sense, this homeothermic designation has been confirmed by the advent of radiotelemetry and other techniques that allow for the remote monitoring of awake, unrestrained animals in laboratory or natural settings. This technology confirmed that, when averaged over many hours, core temperature of mammals is regulated at a nearly constant level. On the other hand, telemetric sampling in relatively small mammals such as mice and rats also revealed that their core temperature often varies markedly from hour to hour. In other words, the mouse could be defined as a homeotherm only when core temperature is averaged over a relatively long period. Many researchers ascribe equal homeothermic capabilities to mice and other small rodents as they do to humans. Such an assumption could lead to errors in extrapolating physiological, pharmacological, and toxicological findings from experimental test species to humans.