Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca²⁺ionophore, ionomycin, suggesting the involvement of extracellular Ca²⁺ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca²⁺ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca²⁺ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998. © 1998 Wiley-Liss, Inc.     

Regional Differences in Seasonal Timing of Rainfall Discriminate between Genetically Distinct East African Giraffe Taxa

Masai (Giraffa tippelskirchi), Reticulated (G. reticulata) and Rothschild''s (G. camelopardalis) giraffe lineages in East Africa are morphologically and genetically distinct, yet in Kenya their ranges abut. This raises the question of how divergence is maintained among populations of a large mammal capable of long-distance travel, and which readily hybridize in zoos. Here we test four hypotheses concerning the maintenance of the phylogeographic boundaries among the three taxa: 1) isolation-by-distance; 2) physical barriers to dispersal; 3) general habitat differences resulting in habitat segregation; or 4) regional differences in the seasonal timing of rainfall, and resultant timing of browse availability. We used satellite remotely sensed and climate data to characterize the environment at the locations of genotyped giraffes. Canonical variate analysis, random forest algorithms, and generalized dissimilarity modelling were employed in a landscape genetics framework to identify the predictor variables that best explained giraffes'' genetic divergence. We found that regional differences in the timing of precipitation, and resulting green-up associated with the abundance of browse, effectively discriminate between taxa. Local habitat conditions, topographic and human-induced barriers, and geographic distance did not aid in discriminating among lineages. Our results suggest that selection associated with regional timing of events in the annual climatic cycle may help maintain genetic and phenotypic divergence in giraffes. We discuss potential mechanisms of maintaining divergence, and suggest that synchronization of reproduction with seasonal rainfall cycles that are geographically distinct may contribute to reproductive isolation. Coordination of weaning with green-up cycles could minimize the costs of lactation and predation on the young. Our findings are consistent with theory and empirical results demonstrating the efficacy of seasonal or phenologically dictated selection pressures in contributing to the reproductive isolation of parapatric populations.     

Inversions of chromosome arms 4AL and 2BS in wheat invert the patterns of chiasma distribution

In many species, including wheat, crossing over is distal, and the proximal regions of chromosome arms contribute little to genetic maps. This was thought to be a consequence of terminal initiation of synapsis favoring distal crossing over. However, in an inverted rye chromosome arm, the pattern of metaphase I chiasmata was also inverted, suggesting that crossover frequencies were specific to chromosome segments. Here, wheat chromosome arms 2BS and 4AL, with essentially entire arms inverted in reverse tandem duplications (rtd), were studied in the MI of meiosis. Inversion–duplication placed the recombining segments in the middle of the arms. While the overall pairing frequencies of the inverted–duplicated arms were considerably reduced relative to normal arms, chiasmata, if present, were always located in the same regions as in structurally normal arms, and relative chiasma frequencies remained the same. The frequencies of fragment or fragment + bridge configurations in AI and AII indicated that of the two tandemly arranged copies of segments in rtds, the more distal inverted segments were more likely to cross over than the segments in their original orientations. These observations show that also in wheat, relative crossover frequencies along chromosome arms are predetermined and independent of the segment location. The segments normally not licensed to cross over do not do so even when placed in seemingly most favorable positions for it.     

Thermal and resonance neutrons generated by various electron and X-ray therapeutic beams from medical linacs installed in polish oncological centers

BackgroundHigh-energy photon and electron therapeutic beams generated in medical linear accelerators can cause the electronuclear and photonuclear reactions in which neutrons with a broad energy spectrum are produced. A low-energy component of this neutron radiation induces simple capture reactions from which various radioisotopes originate and in which the radioactivity of a linac head and various objects in the treatment room appear.AimThe aim of this paper is to present the results of the thermal/resonance neutron fluence measurements during therapeutic beam emission and exemplary spectra of gamma radiation emitted by medical linac components activated in neutron reactions for four X-ray beams and for four electron beams generated by various manufacturers’ accelerators installed in typical concrete bunkers in Polish oncological centers.Materials and methodsThe measurements of neutron fluence were performed with the use of the induced activity method, whereas the spectra of gamma radiation from decays of the resulting radioisotopes were measured by means of a portable high-purity germanium detector set for field spectroscopy.ResultsThe fluence of thermal neutrons as well as resonance neutrons connected with the emission of a 20 MV X-ray beam is ～10⁶ neutrons/cm² per 1 Gy of a dose in water at a reference depth. It is about one order of magnitude greater than that for the 15 MV X-ray beams and about two orders of magnitude greater than for the 18–22 MeV electron beams regardless of the type of an accelerator.ConclusionThe thermal as well as resonance neutron fluence depends strongly on the type and the nominal potential of a therapeutic beam. It is greater for X-ray beams than for electrons. The accelerator accessories and other large objects should not be stored in a treatment room during high-energy therapeutic beam emission to avoid their activation caused by thermal and resonance neutrons. Half-lives of the radioisotopes originating from the simple capture reaction (n,γ) (from minutes to hours) are long enough to accumulate radioactivity of components of the accelerator head. The radiation emitted by induced radioisotopes causes the additional doses to staff operating the accelerators.     

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients

Background

Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.

Methods

Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.

Results

The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38–76 %, depending on the gene. The highest MI value was found for RASSF1A (52 %) and the lowest for NPRL2/G21 (5 %). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71 % tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = −0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75–92 % NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.

Conclusions

The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn’t seem to be a critical determinate of its promoter hypermethylation.