Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: A bona fide tracer

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF–Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group’s side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF–Cys2–heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF–Cys2–heparin complex might represent a biologically relevant complex in physiological media.     

Construction and Application of a luxABCDE Reporter System for Real-Time Monitoring of Enterococcus faecalis Gene Expression and Growth

The present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging of Enterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains of E. faecalis were constructed. pSL101 harbors the luxABCDE operon from pPL2lux and the pREG696 broad-host-range replicon and axe-txe toxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R(2) > 0.98) with the viable-cell count. We employed lux-tagged E. faecalis strains to monitor growth in real time in milk and urine in vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in the Galleria mellonella model system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI of E. faecalis and an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.     

Physical Mapping of Meiotic Crossover Events in a 200-Kb Region of Neurospora Crassa Linkage Group I

We propose a general restriction fragment length polymorphism-based strategy to analyze the distribution of meiotic crossover events throughout specific genetic intervals. We have isolated 64 recombinant chromosomes carrying independent meiotic crossover events in the genetic interval eth-1-un-2 on linkage group I of Neurospora crassa. Thirty-eight crossover events were physically mapped with reference to a 200-kb region cloned by chromosome walking, using N. crassa λ and cosmid libraries. Crossovers were homogeneously distributed at intervals of 5.0 +/- 2.3 kb along the entire cloned interval. The ratio of physical to genetic distance appears to be higher in the region than in the overall N. crassa genome, suggesting that recombinational activity is less in large chromosomes than in small ones. The present work provides a method for defining the centromeric-telomeric orientation of single cloned DNA fragments. Their physical distance can also be estimated with respect to linked loci, provided that crossover events are distributed homogeneously in the interval. This strategy overcomes typical difficulties in defining the position and direction of chromosome walking steps on conventional linkage maps.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses

Antimicrobial polypeptides (AMPPs), consisting of peptides and small proteins with antimicrobial activity, are an integral component of innate immunity. Their often potent properties and widespread prevalence in fish suggests that designing means of manipulating their levels has considerable potential for maintaining or improving fish health. There is evidence that a number of chronic stresses lead to significant downregulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak. Conversely, upregulation of AMPP expression could be used to enhance disease resistance in stressful environments, as well as improve the efficacy of traditional antimicrobial drugs. However, further work is required in linking levels of a number of AMPPs to physiological function since, while a number of studies have documented the down- or upregulation of AMPPs via gene expression, relatively few studies have quantitatively examined changes in protein expression. In addition, not all AMPPs appear to be expressed at microbicidal levels in vivo, suggesting that at least some may have functions other than being directly protective. Nonetheless, in fish, there is evidence that some constitutively expressed AMPPs, such as piscidins and histone-like proteins, are expressed at microbicidal levels and that they decline with stress. Furthermore, certain AMPPs derived from hemoglobin-β are upregulated to microbicidal levels after experimental challenge. The likely widespread distribution of these three AMPP groups in fish provides the opportunity to design strategies to greatly improve the health of cultured fish populations.     

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke

Purpose

Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.

Methods

Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.

Results

Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.

Conclusions

Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.     

TP53 and p16INK4A, but not H-KI-Ras, are involved in tumorigenesis and progression of pleomorphic adenomas

The putative role of TP53 and p16(INK4A) tumor suppressor genes and Ras oncogenes in the development and progression of salivary gland neoplasias was studied in 28 cases of pleomorphic adenomas (PA), 4 cases of cystic adenocarcinomas, and 1 case of carcinoma ex-PA. Genetic and epigenetic alterations in the above genes were analyzed by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP) and sequencing and by Methylation Specific-PCR (MS-PCR). Mutations in TP53 were found in 14% (4/28) of PAs and in 60% (3/5) of carcinomas. Mutations in H-Ras and K-Ras were identified in 4% (1/28) and 7% (2/28) of PAs, respectively. Only 20% (1/5) of carcinomas screened displayed mutations in K-Ras. p16(INK4A) promoter hypermethylation was found in 14% (4/28) of PAs and 100% (5/5) carcinomas. All genetic and epigenetic alterations were detected exclusively in the epithelial and transitional tumor components, and were absent in the mesenchymal parts. Our analysis suggests that TP53 mutations and p16(INK4A) promoter methylation, but not alterations in the H-Ras and K-Ras genes, might be involved in the malignant progression of PA into carcinoma.