Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Design of Cyanovinylene‐Containing Polymer Acceptors with Large Dipole Moment Change for Efficient Charge Generation in High‐Performance All‐Polymer Solar Cells

Designing polymers that facilitate exciton dissociation and charge transport is critical for the production of highly efficient all‐polymer solar cells (all‐PSCs). Here, the development of a new class of high‐performance naphthalenediimide (NDI)‐based polymers with large dipole moment change (Δµ_ge) and delocalized lowest unoccupied molecular orbital (LUMO) as electron acceptors for all‐PSCs is reported. A series of NDI‐based copolymers incorporating electron‐withdrawing cyanovinylene groups into the backbone (PNDITCVT‐R) is designed and synthesized with 2‐hexyldecyl (R = HD) and 2‐octyldodecyl (R = OD) side chains. Density functional theory calculations reveal an enhancement in Δµ_ge and delocalization of the LUMO upon the incorporation of cyanovinylene groups. All‐PSCs fabricated from these new NDI‐based polymer acceptors exhibit outstanding power conversion efficiencies (7.4%) and high fill factors (65%), which is attributed to efficient exciton dissociation, well‐balanced charge transport, and suppressed monomolecular recombination. Morphological studies by grazing X‐ray scattering and resonant soft X‐ray scattering measurements show the blend films containing polymer donor and PNDITCVT‐R acceptors to exhibit favorable face‐on orientation and well‐mixed morphology with small domain spacing (30–40 nm).     

Detached and attached Arabidopsis leaf assays reveal distinctive defense responses against hemibiotrophic Colletotrichum spp

The agriculturally important genus Colletotrichum is an emerging model pathogen for studying defense in Arabidopsis. During the process of screening for novel pathogenic Colletotrichum isolates on Arabidopsis, we found significant differences in defense responses between detached and attached leaf assays. A near-adapted isolate Colletotrichum linicola A1 could launch a typical infection only on detached, but not attached, Arabidopsis leaves. Remarkably, resistance gene-like locus RCH1-mediated resistance in intact plants also was compromised in detached leaves during the attacks with the virulent reference isolate C. higginsianum. The differences in symptom development between the detached leaf and intact plant assays were further confirmed on defense-defective mutants following inoculation with C. higginsianum, where the greatest inconsistency occurred on ethylene-insensitive mutants. In intact Arabidopsis plants, both the salicylic acid- and ethylene-dependent pathways were required for resistance to C. higginsianum and were associated with induced expression of pathogenesis-related genes PR1 and PDF1.2. In contrast, disease symptom development in detached leaves appeared to be uncoupled from these defense pathways and more closely associated with senescence: an observation substantiated by coordinated gene expression analysis and disease symptom development, and chemically and genetically mimicking senescence.     

Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis

Ribonuclease P (RNase P), a ribonucleoprotein (RNP) complex required for tRNA maturation, comprises one essential RNA (RPR) and protein subunits (RPPs) numbering one in bacteria, and at least four in archaea and nine in eukarya. While the bacterial RPR is catalytically active in vitro, only select euryarchaeal and eukaryal RPRs are weakly active despite secondary structure similarity and conservation of nucleotide identity in their putative catalytic core. Such a decreased archaeal/eukaryal RPR function might imply that their cognate RPPs provide the functional groups that make up the active site. However, substrate-binding defects might mask the ability of some of these RPRs, such as that from the archaeon Methanocaldococcus jannaschii (Mja), to catalyze precursor tRNA (ptRNA) processing. To test this hypothesis, we constructed a ptRNA-Mja RPR conjugate and found that indeed it self-cleaves efficiently (k(obs), 0.15 min(-1) at pH 5.5 and 55 degrees C). Moreover, one pair of Mja RPPs (POP5-RPP30) enhanced k(obs) for the RPR-catalyzed self-processing by approximately 100-fold while the other pair (RPP21-RPP29) had no effect; both binary RPP complexes significantly reduced the monovalent and divalent ionic requirement. Our results suggest a common RNA-mediated catalytic mechanism in all RNase P and help uncover parallels in RNase P catalysis hidden by plurality in its subunit make-up.     

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.     

Duration of alpha 1-antichymotrypsin gene activation by interleukin-1 is determined by efficiency of inhibitor of nuclear factor kappa B alpha resynthesis in primary human astrocytes

Expression of alpha1antichymotrypsin (ACT) is significantly activated by interleukin-1 (IL-1) in human astrocytes; however, it is barely affected by IL-1 in hepatocytes. This tissue-specific regulation depends upon an enhancer that contains both nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) elements, and is also observed for an NF-kappaB reporter but not for an AP-1 reporter. We found efficient activation of NF-kappaB binding in both cell types; however, this binding was persistent in glial cells and only transient in hepatocytes. IL-1-activated NF-kappaB complexes consisted of p65 and p50, with p65 transiently phosphorylated on serine 536 in glial cells whereas more persistently in hepatic cells. Overexpression of p65 or constitutively active IKKbeta (inhibitor of NF-kappaB kinase beta) resulted in an efficient activation of the ACT reporter in hepatic cells, indicating that a specific mechanism exists in these cells terminating IL-1 signaling. IL-1 effectively induced the degradation of inhibitor of NF-kappaBalpha (IkBalpha) and IkBepsilon in both cell types but IkBbeta was not affected. However, IkBalpha was resynthesized much more rapidly in hepatic cells in comparison to glial cells. In addition, the initial levels of IkBalpha were much lower in glial cells. We propose that the tissue-specific regulation of the ACT gene expression by IL-1 is determined by different efficiencies of IkBalpha resynthesis in glial and hepatic cells.     

Wound healing activity of NOE-aspirin: a pre-clinical study.

Aspirin, one of the oldest non-steroidal anti-inflammatory drugs, impedes tissue repair by virtue of retarding inflammation. The present study was undertaken to find out if linking of nitrooxyethyl ester to aspirin reverses its healing-depressant propensity. Nitrooxyethyl ester of aspirin (NOE-Asp) was synthesized in our laboratory through well-established synthetic pathway, starting from aspirin through esterification with ethylene glycol and nitration with a mixture of nitric and sulfuric acids at 0 degrees C. The effect of NOE-Asp on phases of healing such as collagenation, wound contraction and epithelialization and on scar size of healed wound was evaluated in three wound models-incision, dead space, and excision wounds. To assess its influence on the oxidative stress, the levels of glutathione (GSH) and thiobarbiturate reactive species (TBARS) were also determined in 10-day-old granulation tissue. NOE-Asp was further screened for its anti-inflammatory activity in rat paw edema model. NOE-Asp promoted collagenation (increase in breaking strength, granulation weight, and collagen content), wound contraction, and epithelialization phases of healing. NOE-Asp also showed a significant antioxidant effect in 10-day-old granulation tissue as compared to aspirin. The results vindicate our assumption that the esterification of aspirin with nirooxyethyl group reverses the healing-suppressant effect of aspirin. The compound also showed equipotent anti-inflammatory activity as aspirin.     

Crab shell chitin whisker reinforced natural rubber nanocomposites. 1. Processing and swelling behavior

Nanocomposite materials were obtained from a colloidal suspension of chitin whiskers as the reinforcing phase and latex of both unvulcanized and prevulcanized natural rubber as the matrix. The chitin whiskers, prepared by acid hydrolysis of chitin from crab shell, consisted of slender parallelepiped rods with an aspect ratio close to 16. After the two aqueous suspensions were mixed and strirred, solid composite films were obtained either by freeze-drying and hot-pressing or by casting and evaporating the preparations. The processing and swelling behavior of composite films were evaluated. It was concluded that the whiskers form a rigid network assumed to be governed by a percolation mechanism in the evaporated samples only. Comparatively, better resistance of evaporated samples than hot-pressed ones against swelling in an organic solvent medium is good evidence for the existence of a rigid chitin network. The values of diffusion coefficient, bound rubber content, and relative weight loss also supported the presence of a three-dimensional chitin network within the evaporated samples. The mechanical behavior of the composites gives additional insight and evidence for this fact (part 2).     

Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe

The protein subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2- aminoethyl 2-pyridyl disulfide (EPD-Fe). This reaction converts C5 protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA subunit of E. coli RNase P, in the presence of ascorbate and hydrogen peroxide. Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 protein) and the ribose units in M1 RNA. When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme. C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot. However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-protein interface in the RNase P holoenzyme. Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P.