Expression of the Bacillus thuringiensis Mosquitocidal Toxin Cry11Aa in the Aquatic Bacterium Asticcacaulis excentricus

A mosquitocidal aquatic bacterium has been developed by introducing an operon containing the cry11Aa, and p20 genes from Bacillus thuringiensis subsp. israelensis (Bti) into the gram-negative aquatic bacterium Asticcacaulis excentricus. After transformation, the cry11Aa gene was successfully expressed in recombinant A. excentricus under the tac promoter, at the level of 0.04 pg/cell. The recombinant bacteria were toxic to Aedes aegypti larvae with an LC₅₀ of 6.83 × 10⁵ cells/mL. We believe that these bacteria may have potential as genetically engineered microorganisms for the control of mosquito larvae.     

An ionic model for rhythmic activity in small clusters of embryonic chick ventricular cells

We recorded transmembrane potential in whole cell recording mode from small clusters (2-4 cells) of spontaneously beating 7-day embryonic chick ventricular cells after 1-3 days in culture and investigated effects of the blockers D-600, diltiazem, almokalant, and Ba2+. Electrical activity in small clusters is very different from that in reaggregates of several hundred embryonic chick ventricular cells, e.g., TTX-sensitive fast upstrokes in reaggregates vs. TTX-insensitive slow upstrokes in small clusters (maximum upstroke velocity approximately 100 V/s vs. approximately 10 V/s). On the basis of our voltage- and current-clamp results and data from the literature, we formulated a Hodgkin-Huxley-type ionic model for the electrical activity in these small clusters. The model contains a Ca2+ current (ICa), three K+ currents (IKs, IKr, and IK1), a background current, and a seal-leak current. ICa generates the slow upstroke, whereas IKs, IKr, and IK1 contribute to repolarization. All the currents contribute to spontaneous diastolic depolarization, e.g., removal of the seal-leak current increases the interbeat interval from 392 to 535 ms. The model replicates the spontaneous activity in the clusters as well as the experimental results of application of blockers. Bifurcation analysis and simulations with the model predict that annihilation and single-pulse triggering should occur with partial block of ICa. Embryonic chick ventricular cells have been used as an experimental model to investigate various aspects of spontaneous beating of cardiac cells, e.g., mutual synchronization, regularity of beating, and spontaneous initiation and termination of reentrant rhythms; our model allows investigation of these topics through numerical simulation.     

Two L1-peptides are excellent tools for serological detection of HPV-associated cervical carcinoma lesions

A persistent high risk human papillomavirus (HR-HPV) infection causes cervical intraepithelial lesions and cervical carcinoma. There is evidence that detecting anti-L1 antibodies could be successfully used for discriminating between cervical lesion patients and women having normal cytology. It was found that peptides 18283 (55PNNNKILVPKVSGLQYRVFR74) and 18294 (284LYIKGSGSTANLASSNYFPT300) from the L1-surface exposed regions were specifically recognised by antibodies from the cervical lesion patient sera. These peptides were tested against 165 womens' normal cytology sera and 148 cervical lesion or cervical cancer patients' sera. Less than 3.6% of women's normal cytology sera recognised peptides 18283 or 18294; on the contrary, 91% to 96% of the cervical lesion (CIN I to CIN III) or cervical cancer patient sera recognised peptides 18283 and 18294. These data show that anti-peptide 18283 and 18294 antibodies in the patients' sera are strongly associated with the presence of HR-HPV associated cervical lesions, showing 92-97% sensitivity and 89-95% specificity in recognising precancerous and cervical cancer patients. These two peptides could be excellent tools for use in large-scale serological screening of women populations at risk of developing cervical carcinoma.     

In vitro propagation of the neotropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation

Guadua angustifolia Kunth was successfully propagated in vitro from axillary buds. Culture initiation, bud sprouting, shoot and plant multiplication, rooting and acclimatization, were evaluated. Best results were obtained using explants from greenhouse-cultivated plants, following a disinfection procedure that comprised the sequential use of an alkaline detergent, a mixture of the fungicide Benomyl and the bactericide Agri-mycin, followed by immersion in sodium hypochlorite (1.5% w/v) for 10 min, and culturing on Murashige and Skoog medium containing 2 ml l^?1 of Plant Preservative Mixture^®. Highest bud sprouting in original explants was observed when 3 mg l^?1 N⁶-benzylaminopurine (BAP) was incorporated into the culture medium. Production of lateral shoots in in vitro growing plants increased with BAP concentration in culture medium, up to 5 mg l^?1, the highest concentration assessed. After six subcultures, clumps of 8–12 axes were obtained, and their division in groups of 3–5 axes allowed multiplication of the plants. Rooting occurred in vitro spontaneously in 100% of the explants that produced lateral shoots. Successful acclimatization of well-rooted clumps of 5–6 axes was achieved in the greenhouse under mist watering in a mixture of soil, sand and rice hulls (1:1:1).     

Productive replication and evolution of HIV-1 in ferret cells

A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1 animal model.     

Response of Endogenous Hormone Concentrations to Two Floral Inductive Treatments,viz. KNO<Subscript>3</Subscript> and PBZ,in Mango cv. ‘Tommy Atkins’ Growing Under Tropical Conditions

Floral induction (FI) has been intensively studied in mango, more under sub-tropical than under tropical environments. Decreases in temperature below 20 °C, which is common in sub-tropical regions but seldom occurs in many tropical ones, has been considered a critical factor for FI in this species. Trying to understand the way by which two FI treatments, potassium nitrate (KNO₃) and paclobutrazol (PBZ), can regulate flowering by modulating the endogenous concentrations of plant hormones, the following compounds were analyzed in terminal buds, wood and bark sections of lateral branches from treated and untreated ‘Tommy Atkins’ mango trees growing under tropical conditions: indole-acetic acid (IAA), gibberellins (GAs), zeatin/zeatin riboside (Z/ZR) and N ⁶ (Δ²-isopentenyl) adenine/N ⁶ (Δ²-isopentenyl) adenosine. Behavior in the contents of these endogenous hormones was often irregular but their course was in general similar for all three treatments. However, levels of GAs were consistently lower in most evaluations of wood and bark sections of PBZ-treated trees compared to KNO₃-treated and control plants. In contrast, the endogenous levels of the presumably FI promoting Z/ZR raised considerably at the time close to FI in buds of KNO₃-treated trees. These KNO₃-treated trees flowered earlier and more profusely than those from other treatments. Although PBZ could be related in this work to a reduction in GA contents, no direct influence of this compound over FI could be established. KNO₃ might partially exert its promoting effect on mango FI by increasing Z/ZR contents.     

Intrinsic caspase-8 activation mediates sensitization of erlotinib-resistant tumor cells to erlotinib/cell-cycle inhibitors combination treatment

Inhibitors of the tyrosine kinase activity of epidermal growth factor receptor, as erlotinib, have an established role in treating several cancer types. However, resistance to erlotinib, particularly in breast cancer cell lines, and erlotinib treatment-associated disorders have also been described. Also, methods and combination therapies that could reverse resistance and ameliorate non-desirable effects represent a clinical challenge. Here, we show that the ATP non-competitive CDK2/cyclin A inhibitor NBI1 sensitizes erlotinib-resistant tumor cells to the combination treatment (co-treatment) for apoptosis-mediated cell death. Furthermore, in erlotinib-sensitive cells, the effective dose of erlotinib was lower in the presence of NBI1. The analysis in the breast cancer MDA-MB-468 erlotinib-resistant and in lung cancer A549 cell lines of the molecular mechanism underlying the apoptosis induced by co-treatment highlighted that the accumulation of DNA defects and depletion of cIAP and XIAP activates the ripoptosome that ultimately activates caspases-8 and -10 and apoptosis. This finding could have significant implications for future treatment strategies in clinical settings.