Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5′s helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.     

Farnesol kinase is involved in farnesol metabolism, ABA signaling and flower development in Arabidopsis

Farnesol, which is toxic to plant cells at high concentrations, is sequentially phosphorylated to farnesyl phosphate and farnesyl diphosphate. However, the genes responsible for the sequential phosphorylation of farnesol have not been identified and the physiological role of farnesol phosphorylation has not been fully elucidated. To address these questions, we confirmed the presence of farnesol kinase activity in Arabidopsis (Arabidopsis thaliana) membranes and identified the corresponding gene (At5g58560, FOLK). Heterologous expression in recombinant yeast cells established farnesol as the preferred substrate of the FOLK-encoded kinase. Moreover, loss-of-function mutations in the FOLK gene abolished farnesol kinase activity, caused an abscisic acid-hypersensitive phenotype and promoted the development of supernumerary carpels under water-stress conditions. In wild-type plants, exogenous abscisic acid repressed FOLK gene expression. These observations demonstrate a role for farnesol kinase in negative regulation of abscisic acid signaling, and provide molecular evidence for a link between farnesol metabolism, abiotic stress signaling and flower development.     

A novel binding assay identifies high affinity ligands to the rosiglitazone binding site of mitoNEET

A novel outer mitochondrial membrane protein containing [2Fe-2S] clusters, mitoNEET was first identified through its binding to the anti-diabetic drug pioglitazone. Pioglitazone belongs to a family of drugs that are peroxisome proliferator-activated receptor (PPAR) gamma agonists, collectively known as glitazones. With the lack of pharmacological tools available to fully elucidate mitoNEET's function, we developed a binding assay to probe the glitazone binding site with the aim of developing selective and high affinity compounds. We used multiple thiazolidine-2,4-dione (TZD), 2-thioxothiazolidin-4-one (TTD), and 2-iminothiazolidin-4-one (ITD) compounds to establish several trends to enhance ligand development for the purpose of elucidating mitoNEET function.     

Design and synthesis of caffeoyl-anilides as portmanteau inhibitors of HIV-1 integrase and CCR5

Designing multi-functional ligands is a recent strategy by which multiple targets can be inhibited by a single entity. A series of caffeoyl-anilide compounds based on structures of various integrase and CCR-5 inhibitors have been designed and synthesized as anti-HIV agents in the present study. Most of the compounds exhibited potent anti-HIV activity at micromolar concentration in CEM-GFP CD4+ T cells infected with HIV-1NL4.3 virus. Compound 14 showed a lower EC(50) and better TI as compared to AZT. Mechanism based studies suggest that these compounds inhibit either one or in some cases, both the targets. The experimental data and the docking results showed that these compounds are potential inhibitors for both HIV-1 IN and CCR5.     

Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs

Background

Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.

Methodology/Principal Findings

We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.

Conclusions/Significance

The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.     

Two nor-triterpene lactones from Caltha palustris

The chemical constituents present in the chloroform-soluble fraction of the ethanolic extract of Caltha palustris have been investigated. Two of these, caltholide and epicaltholide have been characterized as 24-nor-3β-hydroxylupan-13β,28-lactone and its 3α-isomer, respectively.     

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor genes in the brain of masu salmon from immaturity to spawning

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, -R2, -R3, -R4, and -R5, was examined in the brain of masu salmon (Oncorhynchus masou). In addition, responses of these genes to GnRH were examined in a GnRH analog (GnRHa) implantation experiment. Brain samples were collected one week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single forebrains was determined by real-time PCR assays. Among the five genes, R4 and R5 were dominantly expressed in both sexes. R1, R4, and R5 mRNAs showed similar changes throughout the experimental period in both sexes. Levels tended to be high in winter and low in the pre-spawning season, followed by elevations in the spawning period. The mRNA levels had weak to moderate negative correlations with the plasma level of estradiol-17beta (E2) in females. The effects of GnRHa on msGnRH-R mRNAs were not apparent for all the subtypes. These results indicate that the msGnRH-R1, -R4, and -R5 genes are synchronously expressed during sexual maturation. There was a trend toward decreased levels of their expression prior to the spawning period and then increased levels at spawning, possibly causing GnRH target neurons to sensitize to a GnRH stimulus. Furthermore, E2 may be involved in msGnRH-R gene expression in the brain of female masu salmon during sexual maturation.     

Evidence that estrogen regulates the sex change of honeycomb grouper (Epinephelus merra), a protogynous hermaphrodite fish

Circulating estradiol-17beta (E2) levels decrease precipitously during female to male (protogynous) sex change in fish. Whether this drop in E2 levels is a cause or consequence of sex change is still largely unknown. The present study treated adult female honeycomb groupers (Epinephelus merra) with aromatase inhibitor (AI, Fadrozole), either alone or in combination with E2, to investigate the role of estrogen in protogynous sex change. Control fish had ovaries undergoing active vitellogenesis; the gonads of AI-treated fish had already developed into testes, which produced sperm capable of fertilization. In contrast, co-treatment of fish with E2 completely blocked AI-induced sex reversal. AI treatment significantly reduced circulating levels of E2, whereas the addition of E2 to AI prevented the loss. The plasma androgen (testosterone and 11-ketotestosterone) levels were increased in the AI-treated fish, while the levels in the E2-supplemented fish were low compared to controls. Present results show that E2 plays an important role in maintaining female sex of hermaphrodite fishes, and that the inhibition of E2 synthesis causes oocyte degeneration leading to testicular differentiation in the ovary.     

Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.     

Mango ripening--chemical and structural characterization of pectic and hemicellulosic polysaccharides

Ripening of mango is characterized by a gradual, but natural softening of the fruit, which is due to progressive depolymerization of pectic and hemicellulosic polysaccharides with significant loss of galactose, arabinose and mannose residues at the ripe stage. Structural characterization employing permethylation followed by GC-MS analysis, IR and ¹³C NMR measurements revealed the major CWS fractions of both unripe and ripe mangoes to be of variable molecular weights and having a 1,4-linked galactan/galacturonan backbone, which is occasionally involved in side chain branches consisting of single residues of galactose and arabinose or oligomeric 1,5-linked arabinofuranose residues linked through 1,3-linkages; whereas the major hemicellulosic fractions of unripe mango to be of xyloglucan-type having 1,4-linked glucan backbone with branching by non-reducing terminal arabinose and xylose residues.