ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Hepatitis C virus infection protein network.

A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFbeta pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

Modulation of P-glycoprotein activity by acridones and coumarins from Citrus sinensis.

Bioguided fractionation of the roots of Citrus sinensis (Rutaceae) led to the isolation and identification of five coumarins, namely, clausarin, suberosin, poncitrin, xanthyletin and thamnosmonin, seven acridones, namely, acrimarine B, 2-methoxycitpressine I, citpressine I, buntanine, acrimarine E, honyumine and acrimarine C, and one terpenoid, namely, limonin. Among these compounds, clausarin, 2-methoxycitpressine I and acrimarine E inhibited P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells over-expressing P-glycoprotein.

Modulation of cancer cell multidrug resistance by an extract of Ficus citrifolia.

Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Previous studies have shown that natural compounds such as prenyl flavonoids are able to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells. A fraction from the dichloromethane extract of a common Guadalupe Ficus, Ficus citrifolia was studied for its direct interaction with the purified C-terminal cytosolic domain of P-glycoprotein, and for its induced accumulation and cytotoxicity of vinblastine and daunomycin in two model cell lines overexpressing P-glycoprotein, namely K562/R7 and MESSA/Dx5. The fraction bound with high affinity to P-glycoprotein C-terminal cytosolic domain and was as efficient as cyclosporin A to increase intracellular accumulation of daunomycin in K562/R7 leukemic cells. Moreover, the fraction markedly enhanced the cytotoxic effect of vinblastine on the growth of MESSA/Dx5 cells. These results suggest that Ficus citrifolia possesses important therapeutic potential for improving the efficacy of cancer chemotherapy.

Natural and synthetic benzophenones: interaction with the cytosolic binding domain of P-glycoprotein.

A benzophenone glycoside has been isolated from Davallia solida. Its structure was elucidated by chemical and spectral means as 4-O-beta-D-glucopyranosyl-2,6,4'-trihydroxybenzophenone. It bound with moderate affinity to the purified C-terminal cytosolic domain of P-glycoprotein, but the binding affinity was 6- to 10-fold increased for its aglycone derivative and other related benzophenones.

Multiple S gene family members including natural antisense transcripts are differentially expressed during development of maize flowers.

Within the large Brassica S gene family, SLG (S locus glycoprotein) and SRK (S locus receptor kinase) participate to the control of pollen-stigma self-incompatibility. In the self-compatible species maize, S gene family members are predominantly expressed in vegetative organs but are also expressed to a lesser extent in the stigma (silk). To determine if the expression of any S gene family members correlates with female receptivity, we analyzed their expression in developing maize silks. We show that a large family of maize S transcripts is expressed in developing silks. Surprisingly, we isolated a cDNA complementary to a large portion of the antisense strand of the maize receptor kinase S domain. Rapid amplification of cDNA ends (RACE)-polymerase chain reaction, RNase protection, and Northern hybridization with single-stranded riboprobes confirmed that natural antisense S transcripts exist in leaves and seedling shoots and in all sexual tissues tested except mature pollen. These natural antisense S transcripts co-exist with several less abundant sense S transcripts. The accumulation of sense and antisense S transcripts is differentially regulated during pollen and silk development. Thus, these results support a role for S gene family members in sexual tissue development and/or compatible pollination and reveal a new level of complexity in the regulation and function of the S gene family in maize.

B chromosome behavior in maize pollen as determined by a molecular probe.

The B chromosomes of maize typically undergo nondisjunction during the second microspore division (generative cell division). When the microspore nucleus contains only one B chromosome, two kinds of sperm result, one with two B chromosomes and one with no B chromosomes. The sperm with the B chromosomes preferentially fertilizes the egg cell. Previous studies of these phenomena have been limited to genetic analysis and chromosome spreads. In this study we show that a B chromosome-specific probe can be used with fluorescence in situ hybridization (FISH) analysis to detect the presence, location, and frequency of B chromosomes in intact interphase nuclei within mature pollen of maize. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe.

Expression of heat shock factor and heat shock protein 70 genes during maize pollen development.

We have analysed the expression of heat shock protein 70 (HSP70) and heat shock factor (HSF) gene during maize pollen development, HSFs being the transcriptional activators of hsp genes. In order to eliminate the sporophytic tissues of anthers, we have isolated homogeneous cell populations corresponding to five stages of maize pollen development from microspores to mature pollen. We show that in the absence of heat stress, hsp70 genes are highly expressed late-bicellular pollen as compared to other stages. HSP70 transcripts are significantly accumulated in response to a heat shock at the late microspore stage but to a much lower extent than in vegetative tissues. The latest stages of pollen development, i.e. mid-tricellular and mature pollen, do not exhibit heat-induced accumulation of HSP70 transcripts. Therefore, we analysed the expression of hsf genes throughout pollen development. We demonstrate that at least three hsf genes are expressed in maize and that transcripts corresponding to one hsf gene, whose expression is independent of temperature in somatic as well as in microgametophytic tissues, are present at similar levels throughout pollen development. In addition, we show that the expression of the two other hsf genes is heat-inducible in maize vegetative tissues and is not significantly increased after heat shock at any stage of pollen development. These results indicate that the loss of hsp gene expression at late stages of pollen development is not due to a modification of hsf gene expression at the mRNA level and that hsf gene expression is differentially regulated in vegetative and microgametophytic tissues.

PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis.

One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 x 10(5) recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant embryos and represents a breakthrough to isolate genes involved in embryo differentiation.

Identification and Localization of Sugar Components of Rice (Oryza sativa L.) Root Cap Mucilage.

This study is devoted to the neutral sugar analysis of rice root exudate and root cap mucilage.The results indicate that most of the carbohydrate in the exudate is released in soluble dialysable form, mainly as glucose. Mucilage polymers represent only a minor fraction of the whole exudate containing glucose, galactose, xylose, arabinose, and minor amounts of fucose and mannose. In situ localization of some of these sugar residues is also reported. The strongest reaction detected is with fucose-binding lectin. The significance of such data on mucilage composition and its biological role within the rhizosphere - versus colonizing microorganisms - are suggested.