The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves

Shoot regeneration from Rubus leaves was obtained on a medium containing MS salts, vitamins and sugars, Staba vitamins, casein hydrolysate (100 mg l^–1) and 10 M thidiazuron. Shoot regeneration from Malus leaves was obtained on N⁶ rice anther medium with 5 M thidiazuron. In vitro pretreatment of source shoots with either colchicine or thidiazuron enhanced the organogenic potential of detached leaves of two Rubus hybrids. The response to colchicine was quadratic and occurred at non-mutagenic concentrations (75–250 M). The response to thidiazuron was exponential between 0 and 5 M. When applied as a pretreatment, the effectiveness of several different cytokinins (benzyladenine, thidiazuron, zeatin) at enhancing Malus and Rubus organogenesis was related to the shoot proliferation activity of the cytokinin and to treatment-induced variation in leaf and petiole size.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - MS Murashige & Skoog basal medium devoid of plant growth regulators - OI organogenesis-initiating subculture - PTI colchicine pretreatment subculture - PTII cytokinin pretreatment subculture - NAA naphthaleneacetic acid - TDZ thidiazuron - zeatin trans-zeatin     

Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.

BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.     

Effect of ethanol and fatty acids on malolactic activity ofLeuconostoc oenos

Medium-chain fatty acids (C₆ to C₁₂), produced by yeast metabolism during alcoholic fermentation, are known to be inhibitory to lactic acid bacteria. The purpose of this work was to clarify the effect of both ethanol and decanoic and dodecanoic acids on the growth and malolactic activity of aLeuconostoc oenos strain isolated from Portuguese red wine. Ethanol in concentrations up to 12% had no significant effect on malolactic activity but strongly inhibited cell growth. The fatty acids decanoic acid, in concentrations up to 12.5 mg l^–1, and, dodecanoic acid up to 2.5 mg l^–1 seemed to act as growth factors stimulating also malolactic activity; at higher concentrations they exerted an inhibitory effect. We found clear pH dependence between pH 3.0 and pH 6.0, between decanoic acid concentration and its effect on malolactic activity, indicating that the undissociated molecule is the active form. At pH 3.0 the results can be explained by considering that fatty acids enter the cell as protonated molecules and dissociate in the cytoplasm due to the higher internal pH, leading to increased intracellular hydrogenous concentration. This may be the basis of two different effects that contribute to the observed inhibition: decrease in the intracellular pH and dissipation of the transmembrane proton gradient, thus inhibiting intracellular enzymes and ApH-dependent transport systems.     

The direction of mothers' and daughters' preferences and the heritability of male ornaments in red jungle fowl (Gallus gallus)

We used a breeding design involving 18 sires and 108 dams tostudy the heritabilities of male ornaments in red jungle fowl(Gallus gallus). Ornaments used by females to choose mates showedlow heritabilities, with the exception of comb and wattle measures.The general absence of heritability suggests that a geneticcovariance did not exist at the time of this study between mostmale ornaments and female preferences for those ornaments. Thisresult is contrary to a key prediction of the arbitrary or Fisherianhypothesis of sexual selection. Comb size and color are condition-dependenttraits that reflect short-term changes in health, and comb sizeof males was positively correlated with offspring weight. Ourresults are consistent with the expectation of good-genes hypothesesthat male ornaments reflect the ability of males to withstandenvironmental stresses.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

Sarafotoxin expression in the bronchopulmonary tract: immunohistochemical occurrence and colocalization with endothelins

The immunohistochemical occurrence of sarafotoxin (SRTX), a snake venom peptide under strong evolutionary control, was investigated in the pulmonary diffuse neuroendocrine system (PDNES) of newborn cats and rats. By applying the avidin-biotin-peroxidase complex method on serial lung sections, we have demonstrated its distribution and colocalization with different endothelin (ET) isoforms. A light microscopic study revealed apparent immunostaining for SRTX in neuronal components and smooth muscle tissue and in neuroepithelial bodies (NEB), while isolated neuroendocrine cells (NEC) remain unlabelled. Comparison of the SRTX reactivity pattern with that of different ET peptides on adjacent lung sections showed colocalization of SRTX-b with ET-3 in NEB, intrapulmonary ganglion cells and nerve fibres, on the one hand, and with ET-1 in airway and vascular smooth muscle cells, on the other. These findings, in addition to the remarkable functional and structural similarities between SRTX and ET peptides, suggest a common evolutionary origin and biological significance of sarafotoxin and endothelins. Moreover, this is the first time that a toxic peptide has been demonstrated in the PDNES.     

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

Summary A simple procedure is described for removing Epon resin from semi-thin 1 m sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.     

Responses of benthic macroinvertebrates in small intermittent streams to silvicultural practices

We examined macroinvertebrate communities in small(0.1-1.0 m²) pools of intermittent streams (alwayscontainingsome water but without perennial flow) with small watersheds(2-6 ha) subjected to five types of forest harvest to assesspotential impacts of the different harvest methods. Bufferstrips10 m wide were left on each side of the streams. Each harvesttreatment was coupled with a similar unharvested referencestand.An incomplete block design included three 0.05 m² vacuumsamples from each treatment paired with three from theadjacentreferences. There was a high degree of similarity amongreferencesfor parameters other than taxonomic composition (e.g.macroinvertebrate density, number of species, Shannondiversity,functional groups, etc.). Statistically significantdifferenceswere found between references and treatments and among harvestmethods but the responses varied among response variables(density,Shannon-Weiner diversity, species composition), differentspeciesassemblages (all invertebrates, chironomids,Ephemeroptera-Plecoptera-Trichoptera [EPT], isopods), andfunctional group categories (shredders, collector-gatherers).Wecollected 56 taxa, 7–16 per site, with low communitysimilarity(mean Jaccards=0.18, mean Bray-Curtis percentdissimilarity=81). The most severe harvest treatmentsresultedin the highest diversities of total invertebrates in thesesmallspring pool communities.