Mutant barley (1-->3,1-->4)-beta-glucan endohydrolases with enhanced thermostability

The similar three-dimensional structures of barley (1-->3)-beta-glucan endohydrolases and (1-->3,1-->4)-beta-glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms. However, the (1-->3)-beta-glucanases hydrolyze polysaccharides of the type found in fungal cell walls and are members of the pathogenesis-related PR2 group of proteins, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall metabolism. The (1-->3)-beta-glucanases have evolved to be significantly more stable than the (1-->3,1-->4)-beta-glucanases, probably as a consequence of the hostile environments imposed upon the plant by invading microorganisms. In attempts to define the molecular basis for the differences in stability, eight amino acid substitutions were introduced into a barley (1-->3,1-->4)-beta-glucanase using site-directed mutagenesis of a cDNA that encodes the enzyme. The amino acid substitutions chosen were based on structural comparisons of the barley (1-->3)- and (1-->3,1-->4)-beta-glucanases and of other higher plant (1-->3)-beta-glucanases. Three of the resulting mutant enzymes showed increased thermostability compared with the wild-type (1-->3,1-->4)-beta-glucanase. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), a mutation that was likely to decrease the entropy of the unfolded state of the enzyme. Furthermore, the three amino acid substitutions which increased the thermostability of barley (1-->3,1-->4)-beta-glucanase isoenzyme EII were all located in the COOH-terminal loop of the enzyme. Thus, this loop represents a particularly unstable region of the enzyme and could be involved in the initiation of unfolding of the (1-->3,1-->4)-beta-glucanase at elevated temperatures.     

Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells

Anti-dsDNA B cells are actively tolerized in nonautoimmune BALB/c mice, as manifested by their developmental arrest, follicular exclusion, and rapid turnover rate. Previously, we have documented changes in the maturation status and follicular localization of anti-dsDNA B cells in autoimmune-prone MRL (+/+ and lpr/lpr) mice. To determine whether these differences in developmental status and follicular localization affect the functional capacity of anti-dsDNA B cells, we have now compared their in vivo life spans and their responses to in vitro stimuli. Our study shows that although anti-dsDNA B cells from both BALB/c and MRL-+/+ mice are localized to the T/B interface, only those in BALB/c mice have a rapid turnover rate. Therefore, the immature status and not the exclusion from the B cell follicle correlates with a shortened life span. Interestingly, apoptotic anti-dsDNA B cells were not detected at the T/B interface in BALB/c mice, suggesting that they are not dying there. This study also demonstrates that anti-dsDNA B cells, regardless of maturation status or follicular localization, are able to proliferate and up-regulate the costimulatory molecule B7-2 in response to CD40 ligand and IL-4. Therefore, one of the critical in vivo differences between anti-dsDNA B cells in BALB/c and MRL-+/+ mice compared with MRL-lpr/lpr mice may be the availability of T cell help.     

Significance of plasmalemma disruption in bovine and equine spermatozoa

We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their incidence in both fresh and frozen-thawed material from both animals. The vesicles were formed by the separation and expansion of the plasmalemma away from the underlying structure but were not caused by the freeze-thaw process. We suggest that these structures form part of the normal ultrastructure of spermatozoa and are damaged during preparation of the samples for transmission electron microscopy, resulting in a structure previously and incorrectly identified as damaged by the freezing and thawing process.     

Matrix metalloproteinases collagenase-2, macrophage elastase, collagenase-3, and membrane type 1-matrix metalloproteinase impair clotting by degradation of fibrinogen and factor XII

The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity.     

TNF-alpha, leptin, and lymphocyte function in human aging

Aging is associated with increased inflammatory activity and concomitant decreased T cell mediated immune responses. Leptin may provide a link between inflammation and T cell function in aging. The aim of the study was to investigate if plasma levels of tumor necrosis factor (TNF)-alpha were associated with leptin, circulating interleukin-2 receptors (sIL-2R), and phytohaemagglutinin (PHA) induced IL-2 production in whole blood in elderly humans. Circulating levels of TNF-alpha and sIL-2R were higher in elderly humans (N=42) compared to a young control group (N=37) whereas there was no difference with regard to IL-2 production. Furthermore, there were no age-related differences in serum levels of leptin. However, women had higher levels than men. In the elderly people, serum levels of leptin were correlated with TNF-alpha in univariate regression analysis and in a multiple linear regression analysis adjusting for the effect of gender and body mass index. Furthermore, TNF-alpha, but not leptin, was positively correlated to sIL-2R and negatively correlated to IL-2 production. In conclusion, increased plasma levels of TNF-alpha in aging is associated with poor IL-2 production ex vivo and lymphocyte activation in vivo. These associations do not seem to involve leptin.     

休眠期间低温累积对杏枝芽生理生化的影响

实验以３个不同低温需求量的杏品种为材料,在休眠时期进行１１种生理生化指标的测定,结果表明,随着低温累积量的增加,枝条与花芽总含水量、自由水含量,呼吸强度逐渐下降,萌芽前回升,束缚水、束／自比值、可溶性糖、可溶性蛋白质含量逐渐增加,到萌芽前减少,游离氨基酸与脯氨酸含量以及ＳＯＤ活性在完成低温需求量时最高,然后逐渐下降,游离氨基酸、脯氨酸含量与低温需求量存在着相关关系,相关系数为ｒ低需,游离＝０．９８     

Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia

Background

White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown.

Methodology/Principal Findings

We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval.

Conclusions/Significance

G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.