Procyanidin dimer B1 and trimer C1 impair inflammatory response signalling in human monocytes.

The way specific procyanidins exert their anti-inflammatory effects is not fully understood. This study has investigated the capacity of different procyanidins to modulate lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production in THP1 human monocytes and their effects on the redox regulated protein kinases activity: IkB kinase beta (IKKb) and the extracellular signal-regulated kinase (ERK). LPS-triggered increase of ROS was prevented by cell pre-incubation with procyanidins. LPS induced ERK1/2 activation through phosphorylation, which was inhibited by all the compounds tested, the most active being epigallocatechin (EG), followed by epigallocatechin gallate (EGCG) and C1. Procyanidins inhibited IKKb activity in vitro. C1 and procyanidin extract (PE) exerted the maximal IKKb inhibition, followed by EGCG and dimer B1. Catechin exerted a slight but significant IKKb inhibition, in contrast to epicatechin, which was ineffective. In conclusion, procyanidins reduce the LPS-induced production of ROS and they exert their anti-inflammatory effects by inhibiting ERK1/2 and IKKb activity.

Grape seed proanthocyanidins correct dyslipidemia associated with a high-fat diet in rats and repress genes controlling lipogenesis and VLDL assembling in liver.

OBJECTIVE: To determine whether proanthocyanidins can protect against dyslipidemia induced by a high-fat diet (HFD) and to address the mechanisms that underlie this hypolipidemic effect. DESIGN AND MEASUREMENTS: Female Wistar rats were fed on a HFD for 13 weeks. They were divided into two groups, one of which was treated with a grape seed proanthocyanidin extract (25 mg kg(-1) of body weight) for 10 days. Plasma and liver lipids were measured by colorimetric and gravimetric analysis. Liver, muscle and adipose tissue were used to study the expression of genes involved in the synthesis and oxidation of fatty acids and lipoprotein homeostasis by real-time RT-PCR. RESULTS: The administration of proanthocyanidins normalized plasma triglyceride and LDL-cholesterol (both parameters significantly increased with the HFD) but tended to decrease hypercholesterolemia and fatty liver. Gene expression analyses revealed that proanthocyanidins repressed both the expression of hepatic key regulators of lipogenesis and very low density lipoprotein (VLDL) assembling such as SREBP1, MTP and DGAT2, all of which were overexpressed by the HFD. CONCLUSION: These findings indicate that natural proanthocyanidins improve dyslipidemia associated with HFDs, mainly by repressing lipogenesis and VLDL assembly in the liver, and support the idea that they are powerful agents for preventing and treating lipid altered metabolic states.

The different (sur)faces of Rap1p.

The DNA-binding protein Rap1p fulfills many different functions in the yeast cell. It targets 5% of the promoters, acting both as a transcriptional activator and as a repressor, depending on the DNA sequence context. In addition, Rap1p is an essential structural component of yeast telomeres, where it contributes to telomeric silencing. Here we review the evidence indicating that Rap1p function is modulated by the precise architecture of the its binding site and its surroundings: long tracts of telomeric repeats for telomeric functions, specific sequences and orientation for maximal transcriptional activation, and specific DNA recognition sequences for complementary factors in other cases. Many of these functions are probably related to chromatin organization around Rap1p DNA binding sites, resulting from the very tight binding of Rap1p to DNA. We propose that Rap1p alters its structure to bind to different versions of its DNA binding sequence. These structural changes may modulate the function of Rap1p domains, providing different interacting surfaces for binding to specific co-operating factors, and thus contributing to the diversity of Rap1p function.

Alternative mechanisms of transcriptional activation by Rap1p.

Single Rap1p DNA-binding sites are poor activators of transcription of yeast minimal promoters, even when fully occupied in vivo. This low efficiency is due to two independent repression mechanisms as follows: one that requires the presence of histones, and one that requires Hrs1p, a component of the RNA polymerase II mediator complex. Both repression mechanisms were greatly reduced for constructs with tandemly arranged sites. In these constructs, UASrpg sequences (ACACCCATACATTT) activated better than telomere-like sequences (ACACCCACACACCC) in an orientation-dependent manner. Both mutations in the SWI/SNF complex and a deletion of amino acids 597--629 of Rap1p (Tox domain) decreased synergistic effects of contiguous telomeric sites. Conversely, deletion of amino acids 700--798 of Rap1p (Sil domain) made UASrpg and telomeric sites functionally indistinguishable. We propose that the Sil domain masks the main transactivation domain of Rap1p in Rap1p-telomere complexes, where the Tox domain behaves as a secondary activation domain, probably by interacting with chromatin-remodeling complexes. Rap1p DNA-binding sites in ribosomal protein gene promoters are mainly UASrpg-like; their replacement by telomeric sequences in one of these promoters (RPS17B) decreased transcription by two-thirds. The functional differences between UASrpgs and telomeric sequences may thus contribute to the differential expression of Rap1p-regulated promoters in vivo.

The carboxy-terminal cysteine of the tetraspanin L6 antigen is required for its interaction with SITAC, a novel PDZ protein.

PDZ domains are protein modules that mediate protein-protein interactions. Here, we present the identification and characterization of a protein similar to the recently identified PDZ-containing protein TACIP18, which we have named SITAC (similar to TACIP18). SITAC is preferentially expressed in cells of the digestive tract, associated with intracellular membranes. Despite the high degree of sequence identity between the PDZ domains of TACIP18 and those of SITAC, none of the known ligands of the former shows interaction with the latter, as judged by two-hybrid analysis. SITAC interacts with peptides containing bulky hydrophobic amino acids two positions upstream of the C-terminal residue. Surprisingly, SITAC also shows interaction with peptides ending in C, a previously unacknowledged ability of PDZ domains. The sequence -Y-X-C-COOH, bound in vitro by SITAC, is present in the member of the tetraspanin superfamily, the L6 antigen. Coimmunoprecipitation experiments show that SITAC interacts with L6A, but not with an L6A C-terminal mutant, confirming the capacity of SITAC to interact with proteins ending in C. Confocal analysis shows that the interaction between L6A and SITAC is necessary for the precise colocalization of both molecules in the same subcellular compartment. In summary, the characterization of the protein SITAC has unveiled novel sequences recognized by PDZ domains, and it suggests that L6A is a natural ligand of this PDZ protein.

A role for a PDZ protein in the early secretory pathway for the targeting of proTGF-alpha to the cell surface.

In general, plasma membrane integral proteins, such as the membrane-anchored growth factor proTGF-alpha, are assumed to be transported to the cell surface via a nonregulated, constitutive pathway. proTGF-alpha C-terminal mutants are retained in an early secretory compartment. Here, using a two-hybrid screen, we identify two TACIPs (proTGF-alpha cytoplasmic domain-interacting proteins) that contain PDZ domains and do not interact with proTGF-alpha C-terminal mutants. The binding specificity of one of them, TACIP18 (previously identified and named Syntenin or mda-9), coincides with that of the component that possibly mediates the normal trafficking of proTGF-alpha. TACIP18 colocalizes and interacts specifically with immature, intracellular forms of proTGF-alpha. Therefore, it appears that the interaction of TACIP18 with proTGF-alpha in the early secretory pathway is necessary for the targeting of the latter to the cell surface.

Analysis and dynamics of the chromosomal complements of wild sparkling-wine yeast strains.

We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations.

Structural and functional heterogeneity of Rap1p complexes with telomeric and UASrpg-like DNA sequences.

Rap1p binds to a variety of related DNA sequences. We studied complexes of Rap1p and its DNA-binding domain with two of these sequences, the UASrpg sequence (5'-ACACCCATACATTT-3') and the Saccharomyces cerevisiae telomeric consensus (5'-ACACCCACACACCC-3'). When cloned in front of a minimal CYC1 promoter, the two sequences differed in their transcriptional potential. Whereas UASrpg or telomeric single binding sites activated transcription with approximately the same strength, adjacent UASrpg sequences showed higher synergistic activity and orientation-dependence than telomeric sequences. We also found different sequence requirements for Rap1p binding in vitro to both sequences, since a single base-pair that severely reduced binding of Rap1p to UASrpg sequences had very little effect on the telomeric sequence. The Rap1p binding domain distorted DNA molecules encompassing the UASrpg sequence or the telomeric-like sequence, as revealed by both KMnO4 hypersensitivity and by hydroxyl radical foot-printing analysis. We propose that Rap1p is able to form structurally and functionally different complexes, depending on the type of DNA sequence the complex is assembled from. This functional and structural heterogeneity may be responsible for the multiple functions that Rap1p binding sites appear to have in vivo.

Pro-tumor necrosis factor-alpha processing activity is tightly controlled by a component that does not affect notch processing.

The extracellular domain of a heterogeneous group of transmembrane proteins can be proteolytically released from the cell surface, a process known as protein ectodomain shedding. Despite the biomedical importance of several substrates of the shedding system, such as the beta-amyloid precursor protein (betaAPP), little is known about the regulation of protein ectodomain shedding, and the only protease known to be involved is the metalloprotease disintegrin, tumor necrosis factor-alpha converting enzyme (TACE). Here, we show that previously described pro-transforming growth factor-alpha shedding-defective cell mutants (M2 cells), known to be defective in ectodomain shedding of several molecules, that include betaAPP, fail to shed the ectodomain of pro-TNF-alpha. The target of the mutation is a component required for TACE activity, since transfection of TACE into M2 cells has no effect on the shedding of pro-TNF-alpha and somatic cell fusions between M2 cells and TACE null cells recover the ability to shed pro-TNF-alpha, pro-transforming growth factor-alpha, and betaAPP. Furthermore, we show that TACE is also necessary for the shedding of betaAPP since TACE null cells show defective betaAPP shedding. Biochemical evidence shows that the component that controls TACE is different from protein kinase C, the only known activator of protein ectodomain shedding, and that this component does not affect biosynthesis or processing of TACE or other metalloprotease disintegrins. The component mutated in M2 cells is likely to control only a subset of metalloprotease disintegrins involved in regulated ectodomain shedding, since Notch processing, a process known to be dependent on the activity of another metalloprotease disintegrin, Kuzbanian, is normal in M2 cells.

Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197.

A new lignin-degrading basidiomycete, strain I-62 (CECT 20197), isolated from decayed wood exhibited both a high dephenolization activity and decolorization capacity when tested on effluents from the sugar cane by-product fermentation industry. It has been classified as a member of the Polyporaceae family. The major ligninolytic activity detected in culture supernatants of basidiomycete I-62 was a phenoloxidase (laccase), in conjunction with small amounts of manganese peroxidase. No lignin peroxidase was detected. Laccase activity was produced in either defined or complete media. Addition of veratryl alcohol as the inducer, in defined medium, enhanced laccase production 10-fold. The use of fructose instead of glucose as a carbon source resulted in a 100-fold increase in laccase specific activity. Native isoelectrofocusing gels stained with guaiacol revealed the presence of at least seven laccase isozymes, with the most intense band being detected at pI 3. Southern hybridization analysis indicated the presence of a laccase gene family in strain I-62. Three different genes coding for phenoloxidases, lcc1, lcc2, and lcc3, were cloned and characterized. The high degree of homology between laccases from strain I-62 and laccases from Trametes species suggests a phylogenetic proximity between this new isolated fungus and the genus Trametes.

Isolation and characterization of a laccase gene from Podospora anserina.

The genome of the filamentous ascomycete Podospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene of Neurospora crassa. One of these regions contains a gene (lac2) encoding a protein that displays 62% identity with the N. crassa laccase. In shaken cultures, lac2 mRNA is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. Addition of aromatic xenobiotics (guaiacol, hydroquinone, benzoquinone) to the medium during the growth phase results in a rapid, drastic and temporary increase in the abundance of lac2 mRNA. The promoter region of lac2 contains two sequences which display complete homology with the eukaryotic Xenobiotic Responsive Element and two sequences homologous to the eukaryotic Antioxidant Responsive Element. The identity and function of the laccase encoded by lac2 are discussed.

Reversion of a long-living, undifferentiated mutant of Podospora anserina by copper.

The Podospora anserina nuclear mutant grisea displays an undifferentiated growth phenotype (diminished production of aerial hyphae), is female sterile (lack of perithecia), has a prolonged life-span compared to the wild-type strain, and lacks detectable phenoloxidase (laccase and tyrosinase) activity. Reversion of all of these characteristics to those of the wild-type phenotype was accomplished by supplementing the growth medium with extra amounts of copper salts. These results indicate that the primary defect of the grisea strain is in its copper uptake and/or distribution in the cells.