Transformation of intact cells of Saccharomyces cerevisiae by square wave pulses using castellated microelectrodes

Genetic transformation of intact cells of Saccharomyces cerevisiae with an expression vector pYES 2, to efficiencies of 10⁵ to 10⁶ by high voltage electroporation is presented. Prototrophic transformants of yeast expressing resistance to ampicillin were obtained by subjecting the mixture of cells and DNA to a single square wave pulse at an amplitude of 2.5, 2.75 and 3.0 kV/cm in combination with a pulse width of 4, 5 and 3 msec for the three different strains Y915, Y742 and INVSC 1 respectively. The critical factors and electrical parameters which determine the transformation efficiency were examined. This communication describes the optimal conditions for reproducible and high efficiency transformation of yeast by the method of electroporation.     

Potency and pharmacokinetics of broad spectrum and isoform-specific p110γ and δ inhibitors in cancers

Emerging data on cancer suggesting that target-based therapy is promising strategy in cancer treatment. PI3K-AKT pathway is extensively studied in many cancers; several inhibitors target this pathway in different levels. Recent finding on this pathway uncovered the therapeutic applications of PI3K-specific inhibitors; PI3K, AKT, and mTORC broad spectrum inhibitors. Noticeably, class I PI3K isoforms, p110γ and p110δ catalytic subunits have rational therapeutic application than other isoforms. Therefore, three classes of inhibitors: isoform-specific, dual-specific and broad spectrum were selected for molecular docking and dynamics. First, p110δ structure was modelled; active site was analyzed. Then, molecular docking of each class of inhibitors were studied; the docked complexes were further used in 1.2?ns molecular dynamics simulation to report the potency of each class of inhibitor. Remarkably, both the studies retained the similar kind of protein ligand interactions. GDC-0941, XL-147 (broad spectrum); TG100-115 (dual-specific); and AS-252424, PIK-294 (isoform-specific) were found to be potential inhibitors of p110γ and p110δ, respectively. In addition to that pharmacokinetic properties are within recommended ranges. Finally, molecular phylogeny revealed that p110γ and p110δ are evolutionarily divergent; they probably need separate strategies for drug development.     

Modulation of Macrophage Polarization and HMGB1-TLR2/TLR4 Cascade Plays a Crucial Role for Cardiac Remodeling in Senescence-Accelerated Prone Mice

The aim of this study was to investigate the role of macrophage polarization in aging heart. Macrophage differentiation is pathogenically linked to many inflammatory and immune disorders. It is often preceded by myocardial inflammation, which is characterized by increased cardiac damage and pro-inflammatory cytokine levels. Therefore, we investigated the hypothesis that senescence accelerated-prone (SAMP8) mice cardiac tissue would develop macrophage polarization compared with senescence-resistant control (SAMR1) mice. Both SAMP8 and SAMR1 mice were sacrificed when they became six month old. We evaluated, histo-pathological changes and modifications in protein expression by Western blotting and immuno-histochemical staining for M1 and M2 macrophage markers, high mobility group protein (HMG)B1 and its cascade proteins, pro-inflammatory factors and inflammatory cytokines in cardiac tissue. We observed significant upregulation of HMGB1, toll-like receptor (TLR)2, TLR4, nuclear factor (NF)κB p65, tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, interferon (IFN)γ, interleukin (IL)-1β, IL-6 and M1 like macrophage specific marker cluster of differentiation (CD)68 expressions in SAMP8 heart. In contrast, M2 macrophage specific marker CD36, and IL-10 expressions were down-regulated in SAMP8 mice. The results from the study demonstrated that, HMGB1-TLR2/TLR4 signaling cascade and induction of phenotypic switching to M1 macrophage polarization in SAMP8 mice heart would be one of the possible reasons behind the cardiac dysfunction and thus it could become an important therapeutic target to improve the age related cardiac dysfunction.     

Multistrain probiotic production by co‐culture fermentation in a lab‐scale bioreactor

Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.     

Salt-induced alterations of the fluorescence yield and of emission spectra in Chlorella pyrenoidosa

1. The addition of salts to the suspending medium induces a decreasein the yield of chlorophyll a fluorescence in normal and DCMU-poisonedintact algal cells of Chlorella pyrenoidosa. Potassium and sodiumacetate cause a pronounced lowering of the fluorescence at relativelylow concentrations (0.01–0.1 M). MgCl₂ and KCl cause asimilar lowering of fluorescence but at much higher concentrations(0.1–0.4 M). In contrast to sodium acetate, ammonium acetatedoes not cause any significant change in the fluorescence transient.
2. Unlike the case in isolated chloroplasts, MgCl₂ decreasestheratio of short wavelength (mainly system 2) to long wavelength(mainly system 1) emission bands in both DCMU poisoned and normalcells. Since these salt-induced changes do not appear to berelated to the redox reactions of photosynthesis, the saltsmight have caused a decrease in the mutual distance betweenthe two photosystems by changing the microstructure of the chloroplastsin vivo thereby facilitating the spillover of excitation energyfrom strongly fluorescent system 2 to weakly fluorescent system1.
3. The light induced turbidity changes in intact algal cells,asmeasured by the increase in optical density at 540 nm, isreducedin the presence of these salts. However, MgCl₂ producesthegreatest reduction while Na acetate the least, even thoughbothof these salts (at the concentrations used) cause largesuppressionof the fluorescence transient. Moreover, the lightinduced turbiditychanges were, essentially irreversible.
4. Ashigh concentrations of salts increase the osmotic potentialof the bathing medium, it seems that the osmotic changes aswell as the ionic changes in the intact algal cells are responsiblefor the fluorescence quenching and changes in the mode of excitationtransfer observed in this study. In the case of low concentrationsof salts (e.g., 0.1 M Na or K acetate) the effects are predominantlyionic, and in the case of very high concentrations of MgCl₂(0.4 M), the osmotic effects play a much larger role.

(Received July 30, 1973; )     

Reactivation by chloride of hill activity in heat- and tris-treated thylakoid membranes from Beta vulgaris

Hill activity (photoreduction of 2,6,dichlorophenol indophenol) of heat inactivated (40°C, 3 min) and Tris-washed (0.8M, pH 8.3) thylakoids of Beta vulgaris (beet-spinach) was partially restored if they were incubated with 150 mM MgCl₂ prior to the assay. Mg(NO₃)₂ or MgSO₄ were unable to restore this activity. The extent of this reactivation was dependent upon the degree of inactivation by heating and upon the composition of the isolation and the resuspension buffer used during the heat treatment. Washing of heat-treated thylakoids with phosphate-EDTA buffer prior to incubation with MgCl₂ did not affect the extent of this reactivation. Chloride ions seem to be required for the reactivation of Hill activity damaged either by heat or by Tris.Most commonly used chloroplast isolation and resuspension media, except for Tris-HCl as resuspension medium, were suitable for restoration of Hill activity in heat-damaged thylakoids by preincubation with 150 mM MgCl₂ prior to the assay. Pretreatment with MgCl₂ stimulated Hill activity in Tris-treated and heat-damage thylakoids if phosphate buffer was used for their resuspension. However, the same pretreatment inhibited Hill activity in unheated thylakoids isolated in Tris medium and resuspended in the same medium. On the other hand, MgCl₂ pretreatment induced restoration of the Hill activity of the heated thylakoids when Tricine or Hepes was used as the resuspension medium. It appears that the presence of Tris somehow hampers the Cl^– induced reactivation. The stimulation of Hill activity by MgCl₂ treatment in unheated (control) thylakoids is possibly induced by Mg²⁺ ions and not by Cl^– ions.Abbreviations Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1. 1-dimethyl-urea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2 hydroxyethyl piperazine-N, 2 ethano-sulfonic acid - HT heat-treated - PS II photosystem II - Tricine N-tri (hydroxymethyl) methyl glycine - Tris Tris-(hydroxymethyl) amino-methane     

Temperature dependent changes in absorption and fluorescence properties of the cyanobacterium Anacystis nidulans

Temperature dependent changes in absorbance and fluorescence of chlorophyll a (Chl a) were analyzed in membrane fragments and in a Chl-protein complex reconstituted with lipids isolated from the cyanobacterium Anacystis nidulans. Absorbance versus temperature curves measured at 656 nm showed an inflection point at 23–24°C and at 14–16°C in the membrane fragments prepared from A. nidulans cells, grown at 39° and 25°C, respectively. Temperature-induced absorbance changes measured at 680 and 696 nm did not show clear break points. The presence of lipids was essential in order to see a clear maximum in the fluorescence versus temperature curve of Chl a in a Chl-protein complex. It is suggested that a specific form of Chl a may be associated with lipids in the thylakoid membranes and that this form of Chl a may be responsible for temperature-induced absorbance and fluorescence yield changes in this cyanobacterium.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - SDS sodium dodecyl sulphate DPB-CIW No. 802.