The splicing-factor related protein SFPQ/PSF interacts with RAD51D and is necessary for homology-directed repair and sister chromatid cohesion

DNA double-stranded breaks (DSBs) are among the most severe forms of DNA damage and responsible for chromosomal translocations that may lead to gene fusions. The RAD51 family plays an integral role in preserving genome stability by homology directed repair of DSBs. From a proteomics screen, we recently identified SFPQ/PSF as an interacting partner with the RAD51 paralogs, RAD51D, RAD51C and XRCC2. Initially discovered as a potential RNA splicing factor, SFPQ was later shown to have homologous recombination and non-homologous end joining related activities and also to bind and modulate the function of RAD51. Here, we demonstrate that SFPQ interacts directly with RAD51D and that deficiency of both proteins confers a severe loss of cell viability, indicating a synthetic lethal relationship. Surprisingly, deficiency of SFPQ alone also leads to sister chromatid cohesion defects and chromosome instability. In addition, SFPQ was demonstrated to mediate homology directed DNA repair and DNA damage response resulting from DNA crosslinking agents, alkylating agents and camptothecin. Taken together, these data indicate that SFPQ association with the RAD51 protein complex is essential for homologous recombination repair of DNA damage and maintaining genome integrity.     

Glucose Transporter of the Blood-Brain Barrier and Brain in Chronic Hyperglycemia

The effect of chronic hyperglycemia on the glucose transporter moiety of the blood-brain barrier and cerebral cortex was studied in rats 3 weeks after the administration of a single intravenous dose of streptozotocin (60 mg/kg), using specific [3H]cytochalasin B binding methods. Streptozotocin-treated rats developed hyperglycemia, as well as polydipsia and polyuria, and failed to gain weight. The density of D-glucose-displaceable cytochalasin B binding sites in the brain microvessels of streptozotocin-treated hyperglycemic rats was increased by about 30% compared with those of control rats, without change in the affinity of binding. Chronic hyperglycemia had no effect on the density or affinity of specific binding of cytochalasin B to cerebral cortical membranes. These findings do not support the hypothesis that glucose transporters in brain microvessels comprising the blood-brain barrier are "down-regulated" in chronic hyperglycemia.     

A Variant Macaque-Tropic Human Immunodeficiency Virus Type 1 Is Resistant to Alpha Interferon-Induced Restriction in Pig-Tailed Macaque CD4+ T Cells

Human immunodeficiency virus type 1 (HIV-1) antagonizes innate restriction factors in order to infect and persistently replicate in a host. In a previous study, we demonstrated that HIV-1 NL4-3 with a simian immunodeficiency virus mne (SIVmne) vif gene substitution (HSIV-vif-NL4-3) could infect and replicate in pig-tailed macaques (PTM), indicating that APOBEC3 proteins are primary barriers to transmission. Because viral replication was persistent but low, we hypothesized that HSIV-vif-NL4-3 may be suppressed by type I interferons (IFN-I), which are known to upregulate the expression of innate restriction factors. Here, we demonstrate that IFN-α more potently suppresses HSIV-vif-NL4-3 in PTM CD4⁺ T cells than it does pathogenic SIVmne027. Importantly, we identify a variant (HSIV-vif-Yu2) that is resistant to IFN-α, indicating that the IFN-α-induced barrier can be overcome by HSIV-vif chimeras in PTM CD4⁺ T cells. Interestingly, HSIV-vif-Yu2 and HSIV-vif-NL4-3 are similarly restricted by PTM BST2/Tetherin, and neither virus downregulates it from the surface of infected PTM CD4⁺ T cells. Resistance to IFN-α-induced restriction appears to be conferred by a determinant in HSIV-vif-Yu2 that includes env su. Finally, we show that the Yu-2 env su allele may overcome an IFN-α-induced barrier to entry. Together, our data demonstrate that the prototype macaque-tropic HIV-1 clones based on NL4-3 may not sufficiently antagonize innate restriction in PTM cells. However, variants with resistance to IFN-α-induced restriction factors in PTM CD4⁺ T cells may enhance viral replication by overcoming a barrier early in the viral replication cycle.     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

Photon flux density and light quality induce changes in growth,stomatal development,photosynthesis and transpiration of <Emphasis Type="Italic">Withania Somnifera</Emphasis> (L.) Dunal. plantlets

The aim of the study was to establish whether the quantity and the quality of light affect growth and development of Withania somnifera plantlets. We have studied growth and histo-physiological parameters [stomatal characteristics, chloroplastic pigments concentrations, photosynthesis, and transpiration (E)] of W. somnifera plantlets regenerated under various light intensities, or monochromatic light or under a mixture of two colors of light in tissue culture conditions. Plantlets grown under a photon flux density (PFD) of 30 μmol m^-2 s^-1 showed greater growth and development than those raised under other PFDs. Chlorophylls and carotenoids, numbers of stomata, rate of photosynthesis (P_N) and transpiration (E), stomatal conductance (g_s), and water use efficiency (WUE) increased with increasing the PFD up to 60 μmol m^-2 s^-1. Light quality also affected plantlets growth and physiology. Highest growth was observed under fluorescent and in a mixture of blue and red light. Very few stomata were developed in any of the monochromatic light but under fluorescent or under a mixture of two colors stomatal numbers increased. Similarly, g_s, E, P_N, and WUE were also higher under fluorescent light and under a mixture of red and blue light. Regressional analysis showed a linear relationship between P_N (r ² = 70) and g_s and between E (r ² = 0.95) and g_s. In conclusion, both the quality and the quantity of light affect growth of plantlets, development of stomata and physiological responses differently depending on the intensity and the wavelength of light.     

Salicylic Acid-induced Nitric Oxide and ROS Generation Stimulate Ginsenoside Accumulation in Panax ginseng Roots

We evaluated the involvement of nitric oxide (NO) in salicylic acid (SA)-induced accumulation of ginsenoside in adventitious roots of Panax ginseng and its mediation by reactive oxygen species (ROS). Related effects of SA on components of the antioxidant system were also sought. Adventitious roots of P. ginseng were grown in suspension culture for 3 weeks in MS medium and treated over 5 days with SA (100 μM) alone, SA in combination with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), or PTIO alone. Nitric oxide, the superoxide anion (O₂^·−), H₂O₂, nitrite, nonprotein thiol, and ascorbate were monitored together with ginsenoside, NADPH oxidase activity, and several antioxidant enzymes. Salicylic acid did not inhibit root growth but induced accumulation of ginsenoside, lipid peroxidation, and generation of NO and O₂^·−. It also enhanced activities of NADPH oxidase, superoxide dismutase, catalase, and peroxidase, including ascorbate peroxidase. These effects were suppressed by PTIO. Salicylic acid also decreased glutathione reductase activity. Inclusion of PTIO with SA decreased the activity of glutathione reductase further. Treatment with SA plus PTIO also decreased nonprotein thiol and ascorbate contents but caused nitrite to overaccumulate. Salicylic acid applied to adventitious roots in culture induced accumulation of ginsenoside in an NO-dependent manner that was mediated by the associated increases in O₂^·−, which gave other antioxidant responses that were dependent on NO.     

Mutational analysis of conserved aspartic acid residues in the <Emphasis Type="Italic">Methanothermobacter thermautotrophicus</Emphasis> MCM helicase

Minichromosome maintenance (MCM) helicases are thought to function as the replicative helicases in archaea and eukarya, unwinding the duplex DNA in the front of the replication fork. The archaeal MCM helicase can be divided into three parts, the N-terminal, catalytic, and C-terminal regions. The N-terminal part of the protein is divided into three domains, A, B, and C, and was shown to be involved in protein multimerization and binding to single- and double-stranded DNA. Two Asp residues found in domain C are conserved among MCM proteins from different archaea. These residues are located in a loop at the interface with domain A. Mutations of these residues in the Methanothermobacter thermautotrophicus MCM protein, Asp202 and Asp203, to Asn result in a significant reduction in the ability of the enzyme to bind DNA and in lower thermal stability. However, the mutant proteins retained helicase and ATPase activities. Further investigation of the DNA binding revealed that the presence of ATP rescues the DNA binding deficiencies by these mutant proteins. Possible roles of these conserved residues in MCM function are discussed.     

Identification of flavonoids in the stems and leaves of Scutellaria baicalensis Georgi

Scutellaria baicalensis Georgi (S. baicalensis), a perennial herb of the Labiatae family, is a well-known traditional Chinese medicine. In the present study, a comprehensive qualitative analysis of flavonoids in the stems and leaves of S. baicalensis was performed. Under the optimized experimental conditions, 21 flavonoids were clearly detected. 17 of them were successfully identified based on the on-line UV and MS(n) data and were sequentially confirmed by the literature search. The rest 4 flavonones, which were not on-line identified, were successfully isolated and were identified by 1D and 2D NMR. One of them, 5,6,7,3',4'-pentahydroxy flavanone-7-O-glucuronide (2) is a new compound.     

Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.     

An artificial riboswitch for controlling pre-mRNA splicing

Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.