Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

A new oocyte-holding pipette for intracytoplasmic sperm injection without cytoplasmic aspiration: An experimental study in mouse oocytes

We assessed the feasibility of using a new oocyte-holding pipette (pipette without aspiration, PiWA) for intracytoplasmic sperm injection (ICSI), which prevents cytoplasmic aspiration during microinjection. A pilot experimental study in eight mature mouse oocytes to assess the feasibility of the oocyte-holding PiWA for ICSI procedure. The absence of oocyte degeneration after microinjection and the viability of correct embryo development were also evaluated. The pipette comprises a suction conduit inside an elongated cylindrical body and a funnel-shaped working end, which is dimensioned to hold the oocyte in a tight-fitting manner. Upon aspirating via the suction conduit, the oocyte remains partially trapped inside the funnel and becomes deformed changing the spherical shape of its resting state to an oval shape that tensions the surface and increases the turgor. In all ICSI procedures using the new PiWA, the oocyte membrane presented some resistance but was easily broken when exerting some pressure or small aspiration. The eight oocytes developed, six of which reached the blastocyte stage. The results obtained in this study indicate that the increase in oocyte membrane turgidity caused by PiWA prevents vigorous aspiration of the cytoplasm during spermatozoa microinjection.     

The Hfr status of Pseudomonas aeruginosa is stabilized by integrative suppression

Summary A temperature-sensitive mutant (dna-11) with the phenotype of a mutant defective in the initiation of DNA replication, was isolated from an Hfr-like FP2 donor of Pseudomonas aeruginosa. Reversion of its temperature-sensitive character was achieved by integrative suppression rather than by backmutation or an additional suppressor mutation. The dna-11 mutant proved to be helpful in stabilizing the Hfr status of the original host.     

Gene expression modifications in the liver caused by binge drinking and S-adenosylmethionine feeding. The role of epigenetic changes

Chronic ethanol ingestion, achieved by feeding ethanol at a constant rate using intragastric tube feeding, alters the expression of genes in the liver. This is done by epigenetic mechanisms, which depend on the blood alcohol levels at the time of killing. However, acute bolus feeding of ethanol changes gene expression without lasting epigenetic changes. This occurs with histone 3 methylation and acetylation modifications. The gene expression response to an acute bolus of ethanol might be modified by feeding S-adenosylmethionine (SAMe), a methyl donor. In the present study, rats were given a bolus of ethanol (6 g/kg body weight (bw), SAMe (1 g/kg bw), ethanol + SAMe, or isocaloric glucose. The group of rats (n = 3) were killed at 3 and 12 h post bolus, and gene microarray analysis was performed on their liver cells. SAMe reduced the 3 h blood ethanol levels and increased the ALT levels at 3 h. Venn diagrams showed that alcohol changed the expression of 646 genes at 3 h post bolus and 586 genes at 12 h. SAMe changed the expression of 1,012 genes when fed with ethanol 3 h post ethanol bolus and 554 genes at 12 h post ethanol bolus. SAMe alone changed the expression of 1,751 genes at 3 h and 1,398 at 12 h. There were more changes in gene expression at 3 h than at 12 h post ethanol when ethanol alone was compared to the dextrose control. The same was true when SAMe was compared to SAMe + ethanol. Ethanol up regulated gene expression in most functional pathways at 3 h. However, when SAMe was fed with ethanol at 3 h, most pathways were down regulated. At 12 h, however, when ethanol was fed, the pathways were half up regulated and half down regulated. The same was true when SAMe + ethanol was fed. The expression of epigenetically important genes, such as BHMT and Foxn3, was up regulated 3 h post alcohol bolus. At 3 h, SAMe down regulated the expression of genes, such as BHMT, Mat2a, Jun, Tnfrs9, Ahcy 1, Tgfbr1 and 2, and Pcaf. At 12 h, the insulin signaling pathways were half down regulated by ethanol, which was partly prevented by SAMe. The MAPK pathway was up regulated by ethanol, but SAMe did not prevent this. In conclusion, profound changes in gene expression evolved between 3 h and 12 post ethanol bolus. SAMe down regulated these changes in gene expression at 3 h, and less so at 12 h.     

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20–24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.     

A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2-2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100-1800 μmol photons m⁻² s⁻¹). Simultaneous measurements of FI and 820-nm transmission kinetics (I₈₂₀) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2-0.4 s) is accompanied by a decrease of I₈₂₀, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4-2 s) of the dip is not paralleled by the change in I₈₂₀. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I-P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I₈₂₀ kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin-NADP⁺-oxidoreductase or Mehler-peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.     

Molecular docking of novel donor–π–acceptor dicyanomethylenedihydrofuran-based fluorescent chromophores

A series of donor–π–acceptor dicyanomethylenedihydrofuran (DCDHF)-based chromophores comprising different π-aryl bridges and different terminal groups was synthesized and characterized. The chromophores were synthesized via Knoevenagel condensation of the active methyl-bearing DCDHF (electron acceptor) with a tertiary amine-containing arylaldehyde (electron donor) in dry pyridine at room temperature in the presence of a few drops of acetic acid. The synthesis approach involved the development of phenyl(thienyl)vinyl-bridged dicyanomethylenedihydrofuran dyes with a tertiary amine terminal group. Both absorption and emission spectra were explored. The strong emission properties detected using the synthesized chromophores could be attributed to intramolecular charge transfer. The chemical structures of the synthesized chromophores were verified using ¹H/¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopy. Both tertiary amine-containing and arylaldehyde groups were found to influence the biological properties of the synthesized chromophores. The synthesized (DCDHF)-based hybrids were tested to examine antibacterial effectiveness. Derivatives 1 and 2 demonstrated activity towards Gram-positive bacteria rather than Gram-negative bacteria when compared with an amoxicillin antibiotic reference. Finally, molecular docking inspiration was undertaken to determine their binding relationships (PDB code: 1LNZ).     

Photoreduction of menaquinone in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

Photochemically active reaction centers were isolated from the facultatively aerobic gliding green bacterium Chloroflexus aurantiacus. The absorption difference spectrum, obtained after a flash, reflected the oxidation of P-865, the primary donor, and agreed with that observed in a purified membrane preparation from the same organism (Bruce, B.D., Fuller, R.C. and Blankenship, R.E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6532–6536). By analysis of the kinetics in the presence of reduced N-methylphenazonium methosulfate to prevent accumulation of oxidized P-865, the absorption difference spectrum of an electron acceptor was obtained. The electron acceptor was identified as menaquinone (vitamin K-2), which is reduced to the semiquinone anion in a stoichiometry of approximately one molecule per reaction center. Reduction of menaquinone was accompanied by changes in pigment absorption in the infrared region. Our results indicate that the electron-acceptor chain of C. aurantiacus is very similar to that of purple bacteria.