HERG1 Currents in Native K562 Leukemic Cells

The human ether-a-go-go related gene (HERG1) K⁺ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to −40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC₅₀) of the blocker was 4.69 nm. The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at −120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K⁺ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H₂O₂.     

Contribution of endoplasmic reticulum stress,MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells

We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways.

     

Cytogenetic mapping of the Muller F element genes in Drosophila willistoni group

Comparative genomics in Drosophila began in 1940, when Muller stated that the ancestral haploid karyotype of this genus is constituted by five acrocentric chromosomes and one dot chromosome, named A to F elements. In some species of the willistoni group such as Drosophila willistoni and D. insularis, the F element, instead of a dot chromosome, has been incorporated into the E element, forming chromosome III (E + F fusion). The aim of this study was to investigate the scope of the E + F fusion in the willistoni group, evaluating six other species. Fluorescent in situ hybridization was used to locate two genes of the F element previously studied—cubitus interruptus (ci) and eyeless (ey)—in species of the willistoni and bocainensis subgroups. Moreover, polytene chromosome photomaps corresponding to the F element (basal portion of chromosome III) were constructed for each species studied. In D. willistoni, D. paulistorum and D. equinoxialis, the ci gene was located in subSectction 78B and the ey gene in 78C. In D. tropicalis, ci was located in subSection 76B and ey in 76C. In species of the bocainensis subgroup, ci and ey were localized, respectively, at subsections 76B and 76C in D. nebulosa and D. capricorni, and 76A and 76C in D. fumipennis. Despite the differences in the subsection numbers, all species showed the same position for ci and ey. The results confirm the synteny of E + F fusion in willistoni and bocainensis subgroups, and allow estimating the occurrence of this event at 15 Mya, at least.     

Karyotypic evolution of ribosomal sites in buffalo subspecies and their crossbreed

Domestic buffaloes are divided into two group based on cytogenetic characteristics and habitats: the “river buffaloes” with 2n = 50 and the “swamp buffaloes”, 2n = 48. Nevertheless, their hybrids are viable, fertile and identified by a 2n = 49. In order to have a better characterization of these different cytotypes of buffaloes, and considering that NOR-bearing chromosomes are involved in the rearrangements responsible for the karyotypic differences, we applied silver staining (Ag-NOR) and performed fluorescent in situ hybridization (FISH) experiments using 18S rDNA as probe. Metaphases were obtained through blood lymphocyte culture of 21 individuals, including river, swamp and hybrid cytotypes. Ag-NOR staining revealed active NORs on six chromosome pairs (3p, 4p, 6, 21, 23, 24) in the river buffaloes, whereas the swamp buffaloes presented only five NOR-bearing pairs (4p, 6, 20, 22, 23). The F1 cross-breed had 11 chromosomes with active NORs, indicating expression of both parental chromosomes. FISH analysis confirmed the numerical divergence identified with Ag-NOR. This result is explained by the loss of the NOR located on chromosome 4p in the river buffalo, which is involved in the tandem fusion with chromosome 9 in this subspecies. A comparison with the ancestral cattle karyotype suggests that the NOR found on the 3p of the river buffalo may have originated from a duplication of ribosomal genes, resulting in the formation of new NOR sites in this subspecies.     

Variation in fine‐scale genetic structure and local dispersal patterns between peripheral populations of a South American passerine bird

The distribution of suitable habitat influences natal and breeding dispersal at small spatial scales, resulting in strong microgeographic genetic structure. Although environmental variation can promote interpopulation differences in dispersal behavior and local spatial patterns, the effects of distinct ecological conditions on within‐species variation in dispersal strategies and in fine‐scale genetic structure remain poorly understood. We studied local dispersal and fine‐scale genetic structure in the thorn‐tailed rayadito (Aphrastura spinicauda), a South American bird that breeds along a wide latitudinal gradient. We combine capture‐mark‐recapture data from eight breeding seasons and molecular genetics to compare two peripheral populations with contrasting environments in Chile: Navarino Island, a continuous and low density habitat, and Fray Jorge National Park, a fragmented, densely populated and more stressful environment. Natal dispersal showed no sex bias in Navarino but was female‐biased in the more dense population in Fray Jorge. In the latter, male movements were restricted, and some birds seemed to skip breeding in their first year, suggesting habitat saturation. Breeding dispersal was limited in both populations, with males being more philopatric than females. Spatial genetic autocorrelation analyzes using 13 polymorphic microsatellite loci confirmed the observed dispersal patterns: a fine‐scale genetic structure was only detectable for males in Fray Jorge for distances up to 450 m. Furthermore, two‐dimensional autocorrelation analyzes and estimates of genetic relatedness indicated that related males tended to be spatially clustered in this population. Our study shows evidence for context‐dependent variation in natal dispersal and corresponding local genetic structure in peripheral populations of this bird. It seems likely that the costs of dispersal are higher in the fragmented and higher density environment in Fray Jorge, particularly for males. The observed differences in microgeographic genetic structure for rayaditos might reflect the genetic consequences of population‐specific responses to contrasting environmental pressures near the range limits of its distribution.     

The role of CMP-N-acetylneuraminic acid hydroxylase in determining the level of N-glycolylneuraminic acid in porcine tissues

The biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc) occurs by the action of cytidine monophosphate-N-acetylneuraminate (CMP-Neu5Ac) hydroxylase. Previous investigations on a limited number of tissues suggest that the activity of this enzyme governs the extent of glycoconjugate sialylation with Neu5Gc. Using improved analytical procedures and a panel of nine porcine tissues, each expressing different amounts of Neu5Gc, we have readdressed the issue of the regulation of Neu5Gc incorporation into glycoconjugates. The following parameters were measured for each tissue: the molar ratio Neu5Gc/Neu5Ac, the activity of the hydroxylase, and the relative amount of hydroxylase protein, as determined by enzyme-linked immunosorbent assay (ELISA). A positive correlation between the activity of the hydroxylase and the molar ratio Neu5Gc/Neu5Ac was observed for each tissue. In addition, the hydroxylase activity correlated with the amount of enzyme protein, though in heart and lung disproportionately large amounts of immunoreactive protein were detected. Taken together, the results suggest that the incorporation of Neu5Gc into glycoconjugates is generally controlled by the amount of hydroxylase protein expressed in a tissue.