Resveratrol decreases Rad51 expression and sensitizes cisplatin­resistant MCF­7 breast cancer cells.

Resveratrol (RES), a polyphenol compound with anti­proliferative properties, has been previously evaluated for its beneficial effects against a variety of tumour cells. The current study elucidated the means by which RES enhances the anti­proliferative effects of cisplatin (CIS) on MCF­7 cells, focusing on the inhibitory effects on DNA repair of double­strand breaks (DSBs). Chemoresistant MCF­7 cells (MCF­7R) were generated by continuous exposure to low concentrations of CIS (10 µM CIS­IC40) during 5 passages, with the IC50 value increasing ~3­fold. Using an MTT assay, we estimated the changes in IC50 for CIS in MCF­7, T47­D, MDA­MB­231 and MCF­7R cells in the presence of RES. The relative transcript level of Nbs­1, Mre­11 and Rad­50 genes was assessed using RT­qPCR analysis. Rad51 and H2AX [pSer139] protein expression was determined by western blot analysis. RES at 50 and 100 µM significantly enhanced the anti­proliferative effects of CIS in both MCF­7 and MCF­7R cells, decreasing the IC50 values for CIS to one­tenth and one­sixth, respectively. A total of 100 µM RES decreased the relative transcript levels of homologous recombination (HR) initiation complex components and the Rad51 protein level in MCF­7 and MCF­7R cells. After 48 h of CIS DNA damage, the levels of Rad51 protein increased, but this effect was inhibited by 100 µM RES. RES also maintained serine 139 phosphorylation of histone H2AX, suggesting that RES prevents the repair of DSBs. It was observed that RES exerts an antagonistic effect over CIS on the activation of Rad51 and sustained phosphorylation of H2AX. The results suggest that RES in combination with DNA damage­based therapy has potential as a strategy to overcome resistance and provide much safer and more effective treatment for breast cancer.

Induction of protection in murine experimental models against Trichinella spiralis: an up-to-date review.

The parasitic nematode Trichinella spiralis, an aetiological agent of the disease known as trichinellosis, infects wild and domestic animals through contaminated pig meat, which is the major source for Trichinella transmission. Prevention of this disease by interrupting parasite transmission includes vaccine development for livestock; however, major challenges to this strategy are the complexity of the T. spiralis life cycle, diversity of stage-specific antigens, immune-evasion strategies and the modulatory effect of host responses. Different approaches have been taken to induce protective immune responses by T. spiralis immunogens. These include the use of whole extracts or excretory-secretory antigens, as well as recombinant proteins or synthesized epitopes, using murine experimental models for trichinellosis. Here these schemes are reviewed and discussed, and new proposals envisioned to block the zoonotic transmission of this parasite.

MR (Mre11-Rad50) complex in Giardia duodenalis: In vitro characterization and its response upon DNA damage.

Giardia duodenalis is a well-known protozoan parasite of humans and other mammals. The repair of DNA double strand breaks (DSBs) is crucial for genomic stability and homologous recombination is one of the primary mechanisms used by cells to repair DNA. The Mre11 complex is comprised by Mre11, an endonuclease and 3'-5' exonuclease known to resect ends during homologous recombination, and Rad50, a member of the structural maintenance of chromosomes (SMC) family of ATPases. In this work we cloned, expressed and characterized the catalytic activities of the giardial Mre11 (GdMre11) and Rad50 (GdRad50) proteins. Our results show that while purified recombinant GdMre11 and GdRad50 proteins bind DNA, GdMre11 contains a 3'-5' exonuclease and purified recombinant GdRad50 has ATPase activity. The predicted structure for GdMre11 revealed a conserved Mn(2+) dependent binding pocket. We also explored the expression of giardial mre11 and rad50 genes after ionizing radiation, and our results indicate that both specific transcripts were increased after 1-2 h while their protein levels were found to be significantly increased 4 h after gamma radiation treatment. These proteins were localized in the nuclei before and after irradiation. The implication of these observations is discussed.

PCBs stimulate laccase production and activity in Pleurotus ostreatus thus promoting their removal.

Pleurotus ostreatus degrades polychlorinated biphenyls (PCBs) with an increase of laccase activity. Laccases are well known for their detoxifying activity. We show, using reverse transcription polymerase chain reaction and a biochemical assay, that reduction in PCBs (di, tri, tetra, and penta) levels are correlated with an increase in laccase activity. P. ostreatus cultures were obtained from 0 to 30 days in the presence or absence of 7,100 mg/L PCBs (from transformer oil) and a surfactant. After each selected time cultures were withdrawn and remaining PCBs were determined, a maximal removal percentage of PCBs was obtained at 20 (63.5 ± 2.0) and 30 days (63.8 ± 4.6) post-induction. Also, the activity of the enzyme was analyzed and it was found to increase at 10 (6.9-fold) and 20 (6.77-fold) days post-induction in the presence of PCBs, as determined by its activity. Taken together, these data suggest that PCBs induce laccase expression and that laccase catalyzes PCBs removal.

Nus A is involved in transcriptional termination on lambda tI.

The transcriptional terminator tI generates the 3'end of the integrase (int) gene transcript that is read from the lambda PI promoter in lambda phage. We have studied the factors that affect transcription termination in vitro and in vivo at the lambda tI terminator. In vitro transcriptional studies showed that tI is about 80% efficient in the presence of purified NusA protein, whereas it is only about 50% efficient in its absence. In vivo studies, where the readthrough transcript of lambda tI was measured by quantitative dot blot analysis, gave about 80% efficiency in wild-type strains, but only 60% in the nusA1 mutant strain at non-permissive temperatures. These results support the idea that termination at lambda tI in vivo involves interaction with the NusA factor.

Characterization of a bovine herpesvirus 4(BHV-4) 1.1-kb RNA and its transactivation by BHV-4 immediate-early 2 gene product.

We determined the structure of a 1.1-kb cytoplasmic polyadenylated RNA transcribed from a region of the bovine herpesvirus 4 (BHV-4) genome not conserved among gammaherpesviruses by sequencing its cDNA, by S1 nuclease analysis, and by primer extension analysis. We found that the RNA consists of a short, approximately 193-nucleotide (nt), 5' exon spliced to a 799-nt 3' exon and contains two short (53 and 57 codons) overlapping open reading frames (ORFs). The 53-codon ORF was previously designated BORFE1. Neither ORF exhibits detectable amino acid sequence homology with ORFs of other gammaherpesviruses. The 1.1-kb RNA promoter-regulatory region was specifically transactivated by the BHV-4 IE2 gene product, a homolog of the Epstein-Barr virus R transactivator, in cotransfection assays. In gel retardation experiments, IE2 protein formed a complex with DNA in a 129-bp fragment between -23 and -151 relative to the transcription start site of the 1.1-kb RNA, and less efficiently with a 57-bp subfragment between -78 and -22. A sequence similar to sequences of IE2-binding fragments of other BHV-4 IE2 responsive promoters was found partly in the 57-bp subfragment, extending into the portion of the 129-bp fragment not found in the 57-bp fragment. The 129-bp fragment, but not the 57-bp fragment, was sufficient for transactivation of a promoterless chloramphenicol acetyl transferase (CAT) reporter gene by IE2. However, the 129-bp fragment did not function efficiently as an IE2-responsive enhancer when inserted approximately 140 base pairs (bp) 5' to the transcription start site of a CAT reporter gene driven by an enhancerless simian virus 40 early promoter. Based on this and other observations, we propose that IE2 functions as a promoter factor rather than an enhancer factor.

Characterization of an abundant, unique 1.7-kilobase bovine herpesvirus 4 (BHV-4) late RNA and mapping of a BHV-4 IE2 transactivator-binding site in its promoter-regulatory region.

We characterized an abundant late 1.7-kb cytoplasmic polyadenylated RNA (L1.7 RNA) transcribed from the bovine herpesvirus 4 (BHV-4) HindIII W fragment, in a region of the genome not conserved with Epstein-Barr virus and herpesvirus saimiri. L1.7 RNA contains only extremely short (<100 nucleotides) open reading frames followed by two repeat arrays. The first repeat array contains 11 copies of a 23-bp unit, TGGCACTA GTAGCATTTAACCCC. The second and third copies are each interrupted by 15- to 17-bp sequences that are identical to each other for the first 15 bp. In addition, the second and third copies of the repeat unit each contain two copies of nucleotides 5 to 9 (ACTAG) of the repeat unit, one at each end of the interruption. The second repeat array contains 12 copies of a 25-bp sequence, GCTGTGTATTATTGAGTATTTTTTA. The promoter-regulatory region of L1.7 was activated by the BHV-4 immediate-early gene 2 product (IE2), a homolog of the Epstein-Barr virus R transactivator, in cotransfection assays. We mapped an IE2 recognition site within a 167-bp fragment approximately 10 bp 5' to the start of L1.7 RNA transcription, using cotransfection assays and gel retardation assays. Using gel retardation assays, we mapped an IE2-binding site within this fragment to a 31-bp region from 56 to 86 bp 5' to the start of L1.7 RNA transcription. This IE2-binding site was able to transfer IE2 responsiveness to a heterologous promoter. However, IE2 responsiveness was affected by both position and orientation. Alignment of the L1.7 IE2-binding site sequence with sequences of two other BHV-4 IE2-binding sites resulted in a provisional IE2-binding site consensus sequence different from the Epstein-Barr virus R transactivator-binding site.

Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators.

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)