Destabilization of tetraplex structures of the fragile X repeat sequence (CGG)n is mediated by homolog-conserved domains in three members of the hnRNP family

Hairpin or tetrahelical structures formed by a d(CGG)_n sequence in the FMR1 gene are thought to promote expansion of the repeat tract. Subsequent to this expansion FMR1 is silenced and fragile X syndrome ensues. The injurious effects of d(CGG)_n secondary structures may potentially be countered by agents that act to decrease their stability. We showed previously that the hnRNP-related protein CBF-A destabilized G′2 bimolecular tetraplex structures of d(CGG)_n. Analysis of mutant proteins revealed that the CBF-A-conserved domains RNP1₁ and ATP/GTP binding box were sufficient and necessary for G′2 d(CGG)_n disruption while the RNP2₁ motif inhibited the destabilization activity. Here, we report that a C-terminal fragment of CBF-A whose only remaining conserved domain was the ATP/GTP binding motif, disrupted G′2 d(CGG)_n more selectively than wild-type CBF-A. Further, two additional members of the hnRNP family, hnRNP A2 and mutant hnRNP A1 effectively destabilized G′2 d(CGG)_n. Examination of mutant hnRNP A2 proteins revealed that, similar to CBF-A, their RNP1₁ element and ATP/GTP binding motif mediated G′2 d(CGG)_n disruption, while the RNP2₁ element blocked their action. Similarly, the RNP1₁ and RNP2₁ domains of hnRNP A1 were, respectively, positive and negative mediators of G′2 d(CGG)_n destabilization. Last, employing the same conserved motifs that mediated disruption of the DNA tetraplex G′2 d(CGG)_n, hnRNP A2 destabilized r(CGG)_n RNA tetraplex.     

Increased arachidonic acid concentration in the brain of Flinders Sensitive Line rats, an animal model of depression

Depression may be associated with impaired membrane PUFA composition, especially decreased n-3 PUFA. This assumption has not been tested at the level of brain tissue. Moreover, most studies were confounded by dietary variability. We examined the FA composition of selected brain areas in an animal model of depression, the Flinders Sensitive Line (FSL) rat, and compared the findings with those in controls fed identical diets. In all brain regions studied, the concentration of arachidonic acid (AA) was significantly higher in the FSL rats: in the hypothalamus by 21%, in the nucleus accumbens by 24%, in the prefrontal cortex by 31%, and in the striatum by 23%. No significant differences were observed for n-3 PUFA or for the saturated and monounsaturated FAs. Our results confirm the existence of altered brain PUFA composition in an animal model of depression. The finding of increased AA, an n-6 PUFA, rather than decreased n-3 PUFA, emphasizes the importance of both PUFA families in the pathophysiological processes underlying depression. The FSL rat is a useful tool for further elucidation of the FA disturbances in depression.     

A Substrate Trapping Approach Identifies Proteins Regulated by Reversible S-nitrosylation

Protein S-nitrosylation, the nitric oxide-mediated posttranslational modification of cysteine residues, has emerged as an important regulatory mechanism in diverse cellular processes. Yet, knowledge about the S-nitrosoproteome in different cell types and cellular contexts is still limited and many questions remain regarding the precise roles of protein S-nitrosylation and denitrosylation. Here we present a novel strategy to identify reversibly nitrosylated proteins. Our approach is based on nitrosothiol capture and enrichment using a thioredoxin trap mutant, followed by protein identification by mass spectrometry. Employing this approach, we identified more than 400 putative nitroso-proteins in S-nitrosocysteine-treated human monocytes and about 200 nitrosylation substrates in endotoxin and cytokine-stimulated mouse macrophages. The large majority of these represent novel nitrosylation targets and they include many proteins with key functions in cellular homeostasis and signaling. Biochemical and functional experiments in vitro and in cells validated the proteomic results and further suggested a role for thioredoxin in the denitrosylation and activation of inducible nitric oxide synthase and the protein kinase MEK1. Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the role of thioredoxin-mediated denitrosylation in nitric oxide signaling. The approach described here may prove generally useful for the identification and exploration of nitroso-proteomes under various physiological and pathophysiological conditions.Protein S-nitrosylation, the covalent addition of a nitric oxide (NO)¹ group to a cysteine thiol, constitutes a widespread regulatory mechanism involved in various biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis (1–4). S-nitrosylation is known to modulate the functional properties of a large number of proteins and thereby influence normal cell function and emerging evidence implicates aberrant protein S-nitrosylation in multiple pathological conditions, including cardiovascular disease, neurodegeneration, and cancer (5, 6). Although significant advances have been made in the field of S-nitrosylation, there is still limited knowledge regarding the constituents of the proteome that become nitrosylated (that is, the nitrosoproteome) across different cell types and conditions. Therefore, much remains unknown about the specific roles and functional significance of S-nitrosylation in cellular function and disease. In addition, there is a need to better understand the mechanisms and consequences of denitrosylation, both for individual proteins and on a systems level.Protein denitrosylation is substantially mediated by two cellular denitrosylating systems, namely the glutathione and S-nitrosoglutathione reductase (GSH/GSNOR) and the thioredoxin and thioredoxin reductase (Trx/TrxR) systems, with the latter representing a direct mechanism of protein denitrosylation (7–11). Trx/TrxR-mediated denitrosylation has been specifically linked to several cellular processes, including apoptosis (7), cell adhesion (12), exocytosis (13) and heme protein maturation (14). Despite recent progress in characterizing protein nitrosylation and denitrosylation, the dynamic cellular nitrosoproteome remains relatively unexplored, particularly under physiologically relevant conditions. This may be in part because of methodological challenges inherent to the proteomic analysis of S-nitrosylation and denitrosylation (See Discussion and (15–17)).The disulfide and nitrosothiol (SNO) reductase activities of Trx depend on a highly conserved Cys-Gly-Pro-Cys active site (8, 18). Recent evidence suggests that, similar to reduction of disulfides, SNO reduction occurs in a two-step mechanism (7, 8). First, the more N-terminal cysteine (Cys32 in human Trx1) attacks the sulfur atom on the SNO moiety of the substrate protein, thereby displacing NO (formally, NO⁻) and generating an intermolecular disulfide between Trx and the substrate. The second step entails an intramolecular attack by the second active site cysteine (Cys35, known as the “resolving cysteine”) on the mixed disulfide intermediate, thus releasing the reduced target protein and the oxidized Trx. The normally transient disulfide intermediate formed in the first step is stabilized in a reaction that involves a Trx mutant that lacks the resolving cysteine. This so-called “trap mutant” has been employed in the identification of disulfide targets of Trx in several cell systems (19–22). However, the utility and value of such a trapping approach in the context of nitrosylation proteomics has not been evaluated.In this study we adapted the Trx trapping strategy for global profiling of cellular nitrosylation and denitrosylation processes. Using this approach, we report the identification of hundreds of potential new nitrosylated targets in monoctyes and macrophages, followed by validation using biochemical and functional assays. The findings presented herein greatly expand our knowledge of the monocyte and macrophage nitrosoproteome and suggest multiple roles for nitrosylation and denitrosylation in macrophage activation and function. The approach employed in this study may be applied to exploring nitrosoproteomes in different cells and under various physiological and pathological conditions.     

Transport of microorganisms to Israel during Saharan dust events

Dust storms are serious meteorological events that affect the East Mediterranean region, primarily during the spring season. The physical and chemical nature of dust storms, their origin, and the meteorological conditions leading to the generation of storms have been fully documented, but knowledge on their biological content is almost nonexistent. Four dust events that occurred in the period 2004–2005 were sampled in Haifa, Israel, an urban area on the East Mediterranean coast, for biological characterization. Samples were taken before or after (depending on the meteorological conditions) as well as during the dust events. Dust particles were collected as two size fractions using a dichotomous sampler, and their elemental content was determined using X-ray fluorescence analyses. Airborne bacteria and fungi were collected with the Six Stage Andersen Viable Impactor. Fungi were identified by optical microscopy. Compared to adjacent clear days, there was an increase in the concentration of both atmospheric particles and elements of geological and marine origin during the dust events. The concentration of airborne microorganisms during the dust events was also higher, and the fungal population content was affected. On a winter clear day the abundant airborne fungi were Paecilomyces variotii, Penicillium glabrum, and Alternaria alternata. On a spring clear day, the persisting airborne fungi were Alternaria alternata, Geotrichum candidum, Penicillium chrysogenum, and P. glabrum. However, during two dust events the fungal population was dominated by Alternaria alternata, Aspergillus fumigatus, A. niger, A. thomii, Cladosporium cladosporioides, Penicillium chrysogenum, and P. griseoroseum. This study suggests that Saharan and other desert dust events in the East Mediterranean have a significant effect on the airborne microbial populations, which might impact on health, agriculture, and ecology.     

The PI3K-NF-kappaB signal transduction pathway is involved in mediating the anti-inflammatory effect of IB-MECA in adjuvant-induced arthritis

The anti-inflammatory effect of adenosine was previously found to be mediated via activation of the A3 adenosine receptor (A3AR). The aim of the present study was to decipher the molecular mechanism involved with the inhibitory effect of IB-MECA, an A3AR agonist, on adjuvant-induced arthritis. The adjuvant-induced arthritis rats responded to IB-MECA treatment with a decrease in the clinical score and the pathological score of the disease. The response to IB-MECA was neutralized by the antagonist MRS 1220, confirming that the efficacy of the synthetic agonist was A3AR mediated. The A3AR protein expression level was highly expressed in the synovia, in the peripheral blood mononuclear cells and in the drain lymph node (DLN) tissues of adjuvant-induced arthritis rats in comparison with na?ve animals. Downregulation of A3AR expression was noted upon treatment with IB-MECA. Analysis of synovia and DLN protein extracts revealed a decreased expression level of PI3K, PKB/Akt, IKK, NF-kappaB and tumor necrosis factor alpha, known to affect survival and apoptosis of inflammatory cells, whereas the caspase-3 level was upregulated.Taken together, high A3AR expression is found in the synovia, in the immune cells in the DLN and in peripheral blood mononuclear cells. IB-MECA, an orally bioavailable molecule, activates the A3AR, inducing receptor downregulation and the initiation of a molecular mechanism that involves de-regulation of the PI3K-NF-kappaB signaling pathway. As a result, a potent anti-inflammatory effect manifested in the improvement of the disease clinical score and pathological score occurs. The finding that the A3AR expression level in the peripheral blood mononuclear cells and in the DLN reflects the receptor status in the remote inflammatory site suggests use of the A3AR as a follow-up biomarker.     

The Efficacy of an Immunoisolating Membrane System for Islet Xenotransplantation in Minipigs

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.     

Dissociation between AKAP3 and PKARII Promotes AKAP3 Degradation in Sperm Capacitation

Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca²⁺-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca²⁺ in the medium or when intracellular Ca²⁺ was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH₄Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKA_RII anchoring during sperm capacitation.     

Different susceptibility of rat pancreatic alpha and beta cells to hypoxia

Insulin-producing beta cells are known to be highly susceptible to hypoxia, which is a major factor in their destruction after pancreatic islet transplantation. However, whether the glucagon-producing pancreatic islet alpha cells are sensitive to hypoxia is not known. Our objective was to compare the sensitivity of alpha and beta cells to hypoxia. Isolated rat pancreatic islets were exposed to hypoxia (1% oxygen, 94% N(2), 5% CO(2)) for 3 days. The viability of the alpha and beta cells, as well as the stimulus-specific secretion of glucagon and insulin, was evaluated. A quantitative analysis of the proportion of beta to alpha cells indicated that, under normoxic conditions, islet cells were composed mainly of beta cells (87 ± 3%) with only 13 ± 3% alpha cells. Instead, hypoxia treatment significantly increased the proportion of alpha cells (40 ± 13%) and decreased the proportion of beta cells to 60 ± 13%. Using the fluorescent TUNEL assay we found that only a few percent of beta cells and alpha cells were apoptotic in normoxia. In contrast, hypoxia induced an abundance of apoptotic beta cells (61 ± 22%) and had no effect on the level of apoptosis in alpha cells. In conclusion, this study demonstrates that hypoxia results in severe functional abnormality in both beta and alpha cells while alpha cells display significantly decreased rate of apoptosis compared to intensive apoptotic injury of beta cells. These findings have implications for the understanding of the possible role of hypoxia in the pathophysiology of diabetes.     

Activity of coliphage HK022 excisionase (Xis) in the absence of DNA binding

A mutated excisionase (Xis) protein of coliphage HK022 whose single Cys residue was replaced by Ser does not bind to its two tandem binding sites (X1, X2) on the P arm of attR. Despite its DNA-binding inability the protein showed 30% excision activity of the wild type Xis both in vitro and in vivo. This partial activity is attributed to the interaction of Xis with integrase that is retained in the mutant protein. This protein-protein interaction occurs in the absence of DNA binding.