Leucobacter denitrificans sp. nov., isolated from cow dung

The bacterial strain M1T8B10^T was isolated from cow dung in Suwon, Republic of Korea. The strain was a Gram stain-positive rod, nonmotile, and non-spore-forming. According to 16S rRNA gene sequence analysis, the strain fell within the clade of the genus Leucobacter, showing the highest sequence similarities with Leucobacter aridicollis L-9^T (98.7%), Leucobacter iarius 40^T (98.4%), and Leucobacter komagatae JCM 9414^T (98.2%). Cell-wall peptidoglycan contained the diagnostic diamino acid 2,4-diaminobutyric acid of the genus Leucobacter, showing B-type cross-linked peptidoglycans. The major fatty acids were anteiso-C_15:0, iso-C_16:0, and anteiso-C_17:0. The quinone system consisted of the menaquinones MK-11 (78%) and MK-10 (22%). The polar lipid profiles contained diphosphatidylglycerol, phosphatidylglycerol, and an unidentified glycolipid. Differences in several physiological features including nitrate reduction enabled the isolate to be differentiated from all recognized Leucobacter species. Based on these phylogenetic, chemotaxonomic, and phenotypic results, the isolate represents a novel species, for which the name Leucobacter denitrificans sp. nov. is proposed. The type strain is M1T8B10^T (=KACC 14055^T =NBRC 106309^T).     

Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5^S163R/wt), and homozygous knock‐in (Ctrp5^S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5^S163R/S163R and Ctrp5^S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.     

Compartmentation of ATP:citrate lyase in plants

Extracts prepared from young leaves of Pea (Pisum sativum), tobacco (Nicotiana tabacum), rape (Brassica napus), and spinach (Spinacia oleracea) all contained ATP:citrate lyase (ACL) activity, which was most active in rape leaflets (130 nmol min(-1) g fresh weight). In rape and spinach, ACL activity was predominantly localized in the plastids (between about 78% and 90% of the total activity), whereas in pea and tobacco, distribution was mainly cytosolic (about 85% and 78%, respectively, of the total). These distributions were calculated from the relative distributions of plastid and cytosol marker enzymes. Cross-reactivity between plant and rat ACL antibody was carried out by immunoblot analysis and, in rape and spinach, showed that a 120-kD protein, presumably indicating homomeric ACL proteins, was present in both cytosolic and plastidic fractions. In pea, two cross-reacting proteins were detected, the major material being in the cytosol fraction. Therefore, ACL occurs both in the cytosol and plastids of higher plants, but the distribution of activity changes according to the species. The plastidic ACL is proposed to function for the supply of acetyl-coenzyme A for lipid biosynthesis de novo, whereas the cytosolic ACL may provide acetyl-coenzyme A for the mevalonate pathway or fatty acid elongation.     

A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris

Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.     

Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase

This study aimed to examine the induction of defense responses in tomato elicited by Methylobacterium oryzae CBMB20 as a consequence of reduced stress ethylene level possibly through its ACC deaminase activity. Significantly increased activities of pathogenesis-related (PR) proteins and defense enzymes such as β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were noted in M. oryzae CBMB20 pretreated and challenged with Pseudomonas syringae pv. tomato (Pst) compared to either control or M. oryzae-treated tomato plants in both growth chamber and greenhouse conditions. Increased PR proteins and defense enzyme activities were correlated with the reduction of stress ethylene level. M. oryzae CBMB20 reduced the stress ethylene level about 27% and 55% when challenged with Pst, in growth chamber and greenhouse on day 7 respectively and the effect was comparable to that of the chemical ethylene biosynthesis inhibitor AVG, L-α-(2-aminoethoxyvinyl)-glycine hydrochloride. As a consequence of reduced stress ethylene level and its effect on defense response in crop plants, the disease severity was reduced 26% in M. oryzae CBMB20-treated plants challenged with pathogen. Therefore, inoculation of M. oryzae CBMB20 would induce the defense enzymes and contribute to the enhanced resistance of tomato plants against the pathogen Pst.     

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.     

Identification of Pak1 inhibitors using water thermodynamic analysis

Abstract

p21-activated kinases (Paks) play an integral component in various cellular diverse processes. The full activation of Pak is dependent upon several serine residues present in the N-terminal region, a threonine present at the activation loop, and finally the phosphorylation of these residues ensure the complete activation of Pak1. The present study deals with the identification of novel potent candidates of Pak1 using computational methods as anti-cancer compounds. A diverse energy based pharmacophore (e-pharmacophore) was developed using four co-crystal inhibitors of Pak1 having pharmacophore features of 5 (DRDRR), 6 (DRHADR), and 7 (RRARDRP and DRRDADH) hypotheses. These models were used for rigorous screening against e-molecule database. The obtained hits were filtered using ADME/T and molecular docking to identify the high affinity binders. These hits were subjected to hierarchical clustering using dendritic fingerprint inorder to identify structurally diverse molecules. The diverse hits were scored against generated water maps to obtain WM/MM ΔG binding energy. Furthermore, molecular dynamics simulation and density functional theory calculations were performed on the final hits to understand the stability of the complexes. Five structurally diverse novel Pak1 inhibitors (4835785, 32198676, 32407813, 76038049, and 32945545) were obtained from virtual screening, water thermodynamics and WM/MM ΔG binding energy. All hits revealed similar mode of binding pattern with the hinge region residues replacing the unstable water molecules in the binding site. The obtained novel hits could be used as a platform to design potent drugs that could be experimentally tested against cancer patients having increased Pak1 expression.     

Extrachromosomal and chromosomal gene conversion in mammalian cells.

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.