HPV18 Utilizes Two Alternative Branch Sites for E6~*I Splicing to Produce E7 Protein

Human papillomavirus 18(HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6 E7 pre-mRNA. The E6 ORF region in the bicistronic E6 E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6~*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences(BPS) upstream of its 30 splice site, with an identical heptamer AACUA■C, for E6~*I splicing. One heptamer has a branch site adenosine(underlined) at nt 384 and the other at nt 388. E6~*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6~*I splicing prefers the 30 ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6~*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment.     

Mre11 and Ku regulation of double-strand break repair by gene conversion and break-induced replication

The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.     

Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms

Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens.     

Morphological and agronomical diversity patterns in the Spanish barley core collection

Seven thousand years of barley cultivation under the environmental hardships typical of the Mediterranean climate have generated genetic singularity of the Spanish barleys, consistently reported in the literature. From the Spanish National Collection of 2289 accessions, a core subset with 159 landraces and 16 old varieties was constituted. Twenty-seven characters were evaluated for the core collection, to define the structure of the diversity. Several evaluation trials were carried out in 1999-2000, whereas yield trials were performed in earlier years. Phenotypic diversity was large for most of the characters studied. Comparisons of genetic diversity between the core and the original collections suggested that the core is a good representation of the existing diversity in the BNG. Comparisons with results of studies on Spanish materials from other collections seem to indicate that the Spanish diversity is not well represented in some world collections. Principal component analyses for quantitative and qualitative characters revealed a clear distinction between two- and six-row cultivars, and also between landraces and commercial varieties. Geographical origins of the landraces were correlated with grain yield, heading date, duration of grain filling period, and growth class. In relation to diseases, altitude played an important role on the resistance to powdery mildew and brown rust. For brown rust, all the resistant landraces came from low altitudes. These geographical gradients seemed consistent with prior knowledge about barley adaptation, and would confirm the agreement between passport data and true adaptive origin of these landraces from a geographical point of view.     

Imaging of carrageenan macrocycles and amylose using noncontact atomic force microscopy

Samples of kappa-carrageenan, iota-carrageenan, and synthetic amylose have been examined by atomic force microscopy (AFM). All samples were spray deposited from aqueous solutions onto freshly cleaved mica, air dried, and imaged in air using noncontact atomic force microscopy (NCAFM). Images of single stranded amylose and carrageenan are presented. At relatively low polymer concentrations in the presence of NaCl iota-carrageenan formed circles that appear to be predominantly head-to-tail associated unimeric duplex (double stranded) structures. At higher iota-carrageenan concentrations the polymer forms circles and aggregates that appear to involve dimeric duplex structure. Direct comparison of synthetic amylose molecular weights determined from NCAFM images with results from solution measurements showed that NCAFM provides an excellent way to measure amylose molecular weight and molecular weight distribution. It is shown that synthetic amylose is single stranded in aqueous solution and that the chain length distribution is broader than the Poisson distribution anticipated from polymerization theory.     

Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5′-UTR, a coding sequence of 1515 bp and a 104-bp 3′-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (55 kDa) and the tetrameric protein (230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.     

Thermal degradation of allicin in garlic extracts and its implication on the inhibition of the in-vitro growth of Helicobacter pylori

Allicin, the main active principle related to Allium sativum chemistry, is considered to be responsible for the bacteriostatic properties of garlic. The work described here has demonstrated the direct implication of the allicin present in solvent-free garlic extracts obtained with ethanol (ethanolic garlic extract, EGE) and acetone (acetonic garlic extract, AGE) in the inhibition of the in-vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. The evolution of allicin concentration as a function of time and temperature has been the subject of a kinetic study. The reaction order, activation energy, and preexponential factor (in accordance with Arrhenius theory) have been determined for the decomposition process of allicin in these organic media. First-order decomposition, an activation energy of 97.4 kJ/mol, and an Arrhenius preexponential factor of 8.9 x 10(10) s(-1) have been determined for allicin in EGE. For allicin in AGE the kinetic order determined was 1.5, the activation energy 184.5 kJ/mol, and the preexponential factor 3.1 x 10(24) s(-1) (mg/L)(-0.5). The presence or absence of allicin in these garlic products was found to be crucial for the inhibition of the in-vitro growth of Hp, as demonstrated by microbiological analysis for AGE. A relationship has been identified between the effectiveness and durability of the anti-Hp properties shown by AGE and the allicin content of these products. The bacteriostatic properties were active for up to 10 months if the samples were maintained at 6 degrees C.     

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.