Gliclazide may have an antiapoptotic effect related to its antioxidant properties in human normal and cancer cells

Experimental and clinical studies suggest that gliclazide may protect pancreatic β-cells from apoptosis induced by an oxidative stress. However, the precise mechanism(s) of this action are not fully understood and requires further clarification. Therefore, using human normal and cancer cells we examined whether the anti-apoptotic effects of this sulfonylurea is due to its free radical scavenger properties. Hydrogen peroxide (H₂O₂) as a model trigger of oxidative stress was used to induce cell death. Our experiments were performed on human normal cell line (human umbilical vein endothelial cell line, HUVEC-c) and human cancer cell lines (human mammary gland cell line, Hs578T; human pancreatic duct epithelioid carcinoma cell line, PANC-1). To assess the effect of gliclazide the cells were pre-treated with the drug. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was employed to measure the impact of gliclazide on cell viability. Generation of reactive oxygen species, mitochondrial membrane potential (∆Ψ_m), and intracellular Ca²⁺ concentration [Ca²⁺] were monitored. Furthermore, the morphological changes associated with apoptosis were determined using double staining with Hoechst 33258-propidium iodide (PI). Gliclazide protects the tested cells from H₂O₂-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨ_m and diminished intracellular [Ca²⁺] evoked by H₂O₂. Double staining with Hoechst 33258-PI revealed that pre-treatment with gliclazide diminished the number of apoptotic cells. Our findings indicate that gliclazide may protect both normal and cancer human cells against apoptosis induced by H₂O₂. It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress.     

Autosomal localization of interstitial telomeric sites (ITS) in brook trout, Salvelinus fontinalis (Pisces, Salmonidae)

Cytogenetic analysis of brook trout performed with molecular and conventional methods led to identification of interstitial telomeric sites on one or two subtelocentric chromosomes within the same pair. Morphology and specific patterns of these chromosomes using fluorochromes associated with A/T- or G/C-rich DNA proved that these chromosomes are not sex related. The chromomycin-positive region was located on the short arms of the ITS bearing chromosome pair and flanked by telomeric sequences, suggesting that this part of the chromosome had been translocated from another one. Our observations confirm that GC-rich regions are highly mobile genetic structures, and led to ITS formation on brook trout chromosomes.     

LC-MS/MS analysis of surface layer proteins as a useful method for the identification of lactobacilli from the Lactobacillus acidophilus group

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/ MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.     

Are seeds suitable for flow cytometric estimation of plant genome size?

BACKGROUND: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. METHODS: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. RESULTS: For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. CONCLUSIONS: The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.     

Dispersing of radioactive sodium and potassium preparations applied on the surface of plants,as a source of misinterpretations in biological experiments

Dispersing of splits of NaCl and KCl during the drying process has been found using labelled²⁴Na and⁴²K preparations. This bursting effect can cause some misinterpretations in biological experiments with plant material.