Systemic protection by antioxidants against UVL-induced erythema

An antioxidant supplemented diet provided marked systemic protection against ultraviolet light mediated erythema in hairless mice. Among the individual constituents of the diet, butylated hydroxytoluene was most effective whereas glutathione and vitamins C and E afforded negligible protection. The mixture of antioxidants, and butylated hydroxytoluene individually, demonstrated diminished, but significant, protection when applied topically. The safety of this systemic photoprotectant and its clinical relevance at present is unknown.     

Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions.

Quantitative analyses of cell walls from Streptococcus mutans Ingbritt grown under carbohydrate limitation in the chemostat showed that growth conditions had no statistically significant effect on the composition of polysaccharide, peptidoglycan, or the proportion of polysaccharide in the cell wall. Lysis of cell wall preparations with a muramidase supported this conclusion and further indicated that there was little difference in their overall structure. In contrast, there was a consistent difference between the rates of lysis by this enzyme of organisms grown in 0.2% glucose and 0.5% glucose. Extremes of pH or dilution rate essentially did not influence the immunogenicity of type c antigen in whole organisms irrespective of whether the carbohydrate source was glucose or sucrose. However, differences were found in the immunogenicity of lipoteichoic acid under similar circumstances. The results indicated there was an inherent phenotypic stability in the cell walls of S. mutans Ingbritt despite changes in pH, generation time, and carbohydrate source, and that any changes that did occur were probably due to associated cell-surface components.     

Alveolar macrophages from HIV-infected subjects are resistant to Mycobacterium tuberculosis in vitro

HIV-infected individuals frequently develop Mycobacterium tuberculosis (MTB) infection. Alveolar macrophages (AM) are the initial host defense against this organism. We measured MTB growth in AM from normal and HIV-infected subjects after in vitro exposure. Intracellular growth of MTB was reduced in AM from HIV-infected subjects compared with normal macrophages. This was confined to subjects with CD4 counts greater than 200/microl. Growth of avirulent mycobacteria in HIV macrophages was significantly less than virulent MTB. Because avirulent MTB is more sensitive to tumor necrosis factor-alpha (TNF-alpha), we examined the relationship between cytokine secretion and mycobacterial growth. Higher AM spontaneous TNF-alpha secretion was associated with reduced MTB growth in normal AM. This relationship was not seen in HIV-infected subjects, suggesting that other factors contributed to mycobacteria resistance. Mycobacteria-induced TNF-alpha secretion was inversely associated with growth in normal AM but not in HIV-infected subjects. Finally, binding and internalization of MTB was augmented in HIV macrophages compared with normal, demonstrating that reduced intracellular MTB growth was not due to impaired phagocytosis. In conclusion, the increased incidence of MTB infection in HIV-infected subjects does not appear to be due to a defect in macrophage innate immunity.     

Single prolonged stress decreases glutamate,glutamine, and creatine concentrations in the rat medial prefrontal cortex

Application of single prolonged stress (SPS) in rats induces changes in neuroendocrine function and arousal that are characteristic of post traumatic stress disorder (PTSD). PTSD, in humans, is associated with decreased neural activity in the prefrontal cortex, increased neural activity in the amygdala complex, and reduced neuronal integrity in the hippocampus. However, the extent to which SPS models these aspects of PTSD has not been established. In order to address this, we used high-resolution magic angle spinning proton magnetic resonance spectroscopy (HR-MAS ¹H MRS) ex vivo to assay levels of neurochemicals critical for energy metabolism (creatine and lactate), excitatory (glutamate and glutamine) and inhibitory (gamma amino butyric acid (GABA)) neurotransmission, and neuronal integrity (N-acetylaspartate (NAA)) in the medial prefrontal cortex (mPFC), amygdala complex, and hippocampus of SPS and control rats. Glutamate, glutamine, and creatine levels were decreased in the mPFC of SPS rats when compared to controls, which suggests decreased excitatory tone in this region. SPS did not alter the neurochemical profiles of either the hippocampus or amygdala. These data suggest that SPS selectively attenuates excitatory tone, without a disruption of neuronal integrity, in the mPFC.     

Förster's resonance excitation transfer theory: not just a formula

After 65 years of increasing scrutiny and application, Theodor Fo?rster's treatment of resonance excitation transfer is widely quoted and has acquired the acronym FRET, in which "F" originally and rather curiously stood for "fluorescence." In this brief and mostly qualitative survey, we review some of its history, mention its important limitations, and relate some personal encounters with Fo?rster.     

Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects

Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.     

Prior information and oculomotor initiation: the effect of cues in gaps

Eye movements reflect not only an important output of various neural control systems, but also often reflect cognitive processing. For example, saccades are frequently used as a behavioural index of attentional processing. A second important eye movement type, smooth pursuit (SP), has received much less attention in this regard. These two types of eye movement were classically thought of as being separate, but recent results have suggested a closer linkage of their control mechanisms and perhaps their interactions with cognitive processes. Prior information, in the form of cues, alters saccade latency leading to characteristic cueing effects. When the period between the appearance of the cue and the appearance of the saccade target is sufficiently long, the latency of saccades to targets appearing at cued locations is increased. This "inhibition of return" is enhanced by a second type of stimulus manipulation, the early removal of the fixation target a few hundred milliseconds before the target appears (the gap paradigm). In the current experiments, the effect of cues, and interactions between cues and long gaps were investigated. In the main pursuit experiment, and in a separate saccade experiment, subjects were presented with interleaved runs of tasks with and without long gaps (gap duration = 1 s), and with and without cues. In tasks without cues, SP latency was reduced by long gaps (mean reduction 8 ms); unexpectedly, saccade latency for non-cue tasks was increased by long gaps (mean increase 41 ms). In a control experiment with only non-cue tasks, in which SP and saccade gap and non-gap tasks were run together, SP latency was again reduced in gap tasks, while saccade latency was increased, but by much less than in the first experiment. Analysis of individual subjects' data showed that while gaps increased saccade latency in two subjects who had participated in the main experiment (in which cues and gaps had been combined), in two naive subjects long gaps did not affect saccade latency. In the main pursuit experiment, cues had both spatially specific and non-spatially specific (warning) effects on pursuit latency. In non-gap conditions, latency was greater when contralateral cues were presented 250 ms prior to the appearance of the pursuit target, compared to ipsilateral cues, a pattern of effect consistent with inhibition of return. However, this was reversed when cues appeared during a gap--contralateral cues increased while ipsilateral cues decreased latency. For saccades, as expected, in both gap and non-gap conditions, cue effects were consistent with inhibition of return (latency was lower with contralateral cues), and the inhibition of return effect was larger in gap, compared to non-gap conditions. The results suggest that, in appropriate contexts (or as a result of appropriate training), there are distinct inhibitory mechanisms that operate on saccades but not pursuit. What appears to be an inhibition of return effect on pursuit latency when static cues are presented in pursuit tasks, may be better understood as the product of a modulation of mechanisms active in pursuit initiation, perhaps related to motion processing. In contrast to some recent evidence suggesting a close anatomical and functional linkage between pursuit and saccade initiation, the results are consistent with the involvement of a wider range of mechanisms, or a greater degree of flexibility, in programming the initiation of these two oculomotor behaviours.     

Lysophosphatidic Acid Induces αvβ6 Integrin-Mediated TGF-β Activation via the LPA2 Receptor and the Small G Protein Gαq

Activation of latent transforming growth factor β (TGF-β) by αvβ6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes αvβ6 integrin-mediated TGF-β activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce αvβ6 integrin-mediated TGF-β activation is unknown; in addition, the αvβ6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces αvβ6-mediated TGF-β activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced αvβ6 integrin-mediated TGF-β activity is mediated via the LPA2 receptor, which signals via Gα_q. Finally, we show that the expression levels of both the LPA2 receptor and αvβ6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and αvβ6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces αvβ6 integrin-mediated TGF-β activation in epithelial cells via LPA2, Gα_q, RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.Transforming growth factor (TGF)-β includes a pleiotropic group of cytokines that exist in three mammalian isoforms (TGF-β1, -β2, and -β3) that are all secreted as latent complexes. This latent complex needs to be activated for TGF-β family members to exert their biological effect. The small latent complex contains the latency associated peptide (LAP), which, in TGF-β1 and TGF-β3, contains an arginine-glycine-aspartate (RGD) motif. This RGD motif can bind integrins, facilitating TGF-β activation. The LAP of TGF-β2 does not contain an RGD motif and no role for integrin mediated TGF-β2 activation has been described. TGF-β1 exerts profound effects on matrix deposition and is a central mediator of lung injury and fibrosis. There are several mechanisms by which TGF-β1 may be activated, including extremes of heat, oxidation, proteolytic cleavage, deglycosylation, and activation by thrombospondin-1.^{1,2,3,4,5,6,7,8} In vivo, activation by integrins appears to play a major role in activating TGF-β1 during development⁹ and in various disease models.^{10,11,12,13,14}Integrins are heterodimeric transmembrane proteins made up of α and β subunits. Six, of the 24 currently described integrins are able to bind the RGD motif in the LAP of TGF-β. Four of these integrins (αvβ3, αvβ5, αvβ6, and αvβ8) are thought to be able to activate TGF-β1.^13,14,15 The role of integrin-mediated TGF-β activation in vivo has only been confirmed for the αvβ6 and αvβ8 integrins.^13,14 Mice in which the aspartic acid in the RGD site of TGF-β1 is replaced by glutamic acid, preventing integrin-mediated TGF-β1 activation, completely phenocopy TGF-β1 null mice, highlighting the importance of TGF-β1 interactions with integrins.⁹ Furthermore, activation of TGF-β1 by the epithelially restricted αvβ6 integrin is central to the pathogenesis of acute lung injury and pulmonary fibrosis.^12,14Further regulation of TGF-β bioavailability is afforded by interaction of the small latent complex with the latent TGF-β binding proteins (LTBPs). There are four LTBPs (1, 2, 3, and 4) that belong to the LTBP/fibrillin family of extracellular glycoproteins. Of these, three, LTBP-1, -3, and -4, associate with the small latent complex through covalent attachment with the LAP, forming the large latent complex.¹⁶ The LTBPs are required to ensure correct post-translational modification of the small latent complex,¹⁷ and they target storage of TGF-β in the extracellular matrix by crosslinking the large latent complex to the matrix via the actions of tissue transglutaminase.^18,19 The LTBPs are also likely to determine, at least in part, the specificity of TGF-β activation. LTBPs-1 and -3 can bind all isoforms of TGF-β, whereas LTBP-4 can only bind TGF-β1.^16,20 There is further evidence of the importance of LTBP modulating TGF-β activation from in vivo studies using mice null for various LTBPs. LTBP-1 null mice have reduced TGF-β activity and are protected from hepatic fibrosis.²¹ LTBP-3 null mice have phenotypic features consistent with reduced TGF-β activity in the bones.²² Mice with a gene trap disruption of LTBP-4 show reduced epithelial Smad2 phosphorylation and abnormal cardiopulmonary development and develop colonic tumors similar to those seen in Smad3 null mice.²³Overexpression of the αvβ6 integrin is not sufficient to promote fibrosis and the αvβ6 integrin itself must be activated during injury to promote TGF-β1 activation.^12,24 αvβ6-dependent TGF-β activation also requires an intact actin cytoskeleton¹⁴ and is critically dependent on association of latent complexes with the specific LTBP family member, LTBP-1.²⁵ In cells lacking LTBP-1, αvβ6 cannot activate TGF-β, but this response can be rescued by expression of a short fusion protein composed of the region of LTBP-1 that forms a disulfide bond to TGF-β1 LAP and the region required to cross-link LTBP-1 to the extracellular matrix protein fibronectin.²⁵ These findings are consistent with a model by which the integrin-expressing cell activates latent TGF-β by exerting traction on the tethered latent complex. Such a mechanism received further support from a recent report that myofibroblasts can activate latent TGF-β using the αvβ5 integrin and cell contraction.²⁶ It is thought that cellular injury may induce contractile forces through the cytoskeleton and integrins to the latent TGF-β1 molecule, which is itself tethered by the LTBP-1 to either the cell surface or the extracellular matrix.^25,26 We identified that agonists of the seven-transmembrane domain, G protein-coupled receptor (GPCR), PAR1, can promote αvβ6 integrin-mediated TGF-β activation via RhoA, known to modulate cell contraction, and that this pathway is important in acute lung injury.¹² However, whether other GPCRs are able to contribute to αvβ6 integrin-mediated, TGF-β activation is not known. Furthermore, the G protein involved in mediating injury-induced αvβ6 integrin-mediated, TGF-β activation has not yet been identified.Lysophosphatidic acid (LPA), is a bioactive phospholipid known to mediate contraction in a number of cell types.²⁷ It is released from activated platelets at sites of injury,²⁸ contributing to wound repair. LPA is present in bronchoalveolar lavage fluid and increased during inflammation,²⁹ and can mediate pro-inflammatory effects on several cells types within the lung.^30,31,32 Furthermore, LPA is increased in patients with pulmonary fibrosis.³³ LPA mediates its cellular effects via the LPA class of GPCRs. Currently five subtypes have been identified (LPA1-5), and like PAR1, these receptors couple to the small G proteins Gα_i, Gα_q, and Gα_12/13.^34,35 It has recently been shown that LPA can induce fibroblast chemotaxis through an LPA1-dependent mechanism, and this could be important in the pathogenesis of pulmonary fibrosis.³³The purpose of the present study was to investigate whether LPA can activate TGF-β in epithelial cells via the αvβ6 integrin and to dissect the proximal signaling pathway from the relevant LPA receptor to RhoA. We demonstrate that the LPA2 receptor is the predominant receptor for transducing LPA-induced, αvβ6 integrin-mediated, TGF-β activation and that this pathway is coupled to the G protein, Gα_q. Finally we provide evidence that both the αvβ6 integrin and LPA2 receptor are induced in epithelial cells overlying areas of pulmonary fibrosis in the lungs of mice treated with intratracheal bleomycin and in samples from patients with idiopathic pulmonary fibrosis.     

Vanadate-Induced Inhibition of BCR-Triggered Apoptosis is Coupled with Tyrosine Phosphorylation and Induction of G2M Growth Arrest in Ramos-BL B Cells

The regulation of the tyrosine phosphorylation of key signaling molecules by tyrosine kinases and phosphatases is essential for BCR-triggered signaling cascades during B cell selection process. We used the non-selective tyrosine phosphatase inhibitor vanadate to study the importance of the late regulation of the tyrosine phosphorylation for BCR-triggered G1 growth arrest and apoptosis in Ramos-BL B cells. Vanadate induces G2M growth arrest in a dose-dependent manner and prevents BCR-triggered apoptosis. Vanadate-induced upregulation of the tyrosine phosphorylation is concomitant with increased expression of cyclin B and inhibition of caspase-3 activation and PARP cleavage. The anti-apoptotic effect of vanadate was observed even when added up to 6 hours after the treatment of Ramos-BL B cells with anti-IgM. Vanadate increases BCR-triggered tyrosine phosphorylation of the cytosolic tyrosine phosphatases, SHP-1 and SHP-2 after 24 hours. Co-stimulation with anti-CD40 prevents anti-IgM-triggered tyrosine phosphorylation of these phosphatases and up-regulates the expression of SHP-1. We conclude that the regulation of the tyrosine phosphatase activity is indispensable for BCR-triggered execution of the apoptosis in Ramos-BL B cells.     

Human defined antigenic region on the nucleoprotein of Crimean-Congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. In this study, amino acid sequence data for the CCHFV nucleoprotein (NP) was used to identify potential linear epitopic regions which were subsequently included in the design of large and small truncated recombinant NP antigens and peptide libraries. Two truncated recombinant CCHFV NP antigens were prepared based on results of prediction studies to include epitopic regions and exclude hydrophobic regions that could influence protein expression and solubility. Serum samples were collected from acute and convalescent patients. An IgG antibody response was detected in 16/16 samples tested using the large recombinant NP-based ELISA and in 2/16 using the small recombinant NP-based ELISA. A total of 60 peptides covering predicted epitopic regions of the NP were synthesized and peptide NRGGDENPRGPVSR at amino acid position 182–195, reacted with 13/16 human serum samples. In summary, functional assays are required to determine the biological activity of predicted epitopes for development of peptide based assays for antibody detection. Bacterially expressed complete NP antigens have previously been shown to be useful tools for antibody detection. Truncation of the antigen to remove the hydrophobic C terminus had no impact on the ability of the antigen to detect IgG antibody in human sera. The results indicate that the region from amino acids 123 to 396 includes a highly antigenic region of the NP with application in development of antibody detection assays.