LlaFI, a Type III Restriction and Modification System in Lactococcus lactis

We describe a type III restriction and modification (R/M) system, LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactis LL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence. LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg²⁺ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.     

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.     

Nrf2 Negatively Regulates Melanogenesis by Modulating PI3K/Akt Signaling

Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway.     

1H, 13C and 15N resonance assignments for the full-length mammalian cytochrome b5 in a membrane environment

Microsomal cytochrome b₅ plays a key role in the oxidation of a variety of exogenous and endogenous compounds, including drugs, fatty acids, cholesterol and steroid hormones. To better understand its functional properties in a membrane mimic environment, we carried out high-resolution solution NMR studies. Here we report resonance assignments for full-length rabbit cytochrome b₅ embedded in dodecylphosphocholine micelles.     

Moths use multimodal sensory information to adopt adaptive resting orientations

Camouflage conceals animals from predators and depends on the interplay between the morphology and behaviour of animals. Behavioural elements of animals, such as the choice of a resting spot or posture, are important for effective camouflage, as well as the animals’ cryptic appearance. To date, the type of sensory input that mediates resting site choice remains poorly understood. Previously, we showed that bark‐like moths perceive and rely on bark structure to seek out cryptic resting positions and body orientations on tree trunks. In the present study, we investigated the sensory organs through which moths perceive the structure of bark when positioning their bodies in adaptive resting orientations. We amputated (or blocked) each one of the hypothetical sensory organs in moths (antennae, forelegs, wings, and eyes) and tested whether they were still able to perceive bark structure properly and adopt adaptive resting orientations. We found that visual information or stimulation is crucial for adaptively orienting their bodies when resting and tactile information from wings may play an additional role. The present study reveals multimodal information use by moths to achieve visual camouflage and highlights the sensory mechanism that is responsible for the adaptive behaviour of cryptic insects. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 900–904.     

Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study

Detailed residue-wise interactions involved in the binding of myosin to actin in the rigor conformation without nucleotides have been examined using molecular dynamics simulations of the chicken skeletal myosin head complexed with two actin monomers, based on the cryo-microscopic model of Holmes et al. (Nature 2003;425:423-427). The overall interaction is largely electrostatic in nature, because of the charged residues in the four loops surrounding the central primary binding site. The 50k/20k loop, disordered in crystal structures and in simulations of free myosin in solution, was found to be in a conformation stabilized with 1 - 2 internal salt bridges. The cardiomyopathy loop forms 2 - 3 interprotein salt bridges with actin monomers upon binding, whereas its Arg405 residue, the mutation site associated with the hypertrophic cardiomyopathy, forms a strong salt bridge with Glu605 in the neighboring helix away from actin in the actin-bound myosin. The myopathy loop of the R405Q mutant maintains a high degree of two-strand beta-sheet character when bound to actin with the corresponding salt bridges broken.     

Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC and MAPKs‐induced EGFR transactivation

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H₂O₂‐indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10^?4 M H₂O₂ for 1 h. In addition, H₂O₂ increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen‐activated protein kinase (MAPK), and JNK/SAPK. Moreover, H₂O₂ induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H₂O₂‐induced cytosolic phospholipase A₂ (cPLA₂) activation and AA release. H₂O₂ increased NF‐κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)‐2 proteins. Subsequently, H₂O₂ stimulated PGE₂ synthesis, which was reduced by the inhibition of NF‐κB activation. Moreover, each H₂O₂ or PGE₂ increased DNA synthesis and the number of cells. However, H₂O₂‐induced increase in DNA synthesis was inhibited by the suppression of cPLA₂ pathway. In conclusion, H₂O₂ increased AA release and PGE₂ production by the upregulation of cPLA₂ and COX‐2 via Ca²⁺/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009. © 2009 Wiley‐Liss, Inc.