Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.     

Stock identification of the sciaenid fish Micropogonias undulatus in the western North Atlantic Ocean using parasites as biological tags

Proper fisheries management of the Atlantic croaker Micropogonias undulatus is necessary in the United States due to the commercial and recreational importance of this fish species. Croaker stock structure in the western North Atlantic has been investigated in the past by various authors, with inconclusive results. In this study, macroparasites were used as biological tags to identify putative croaker stocks in the area between New Jersey and Florida, which encompasses the Mid Atlantic Bight and the South Atlantic Bight separated at Cape Hatteras, North Carolina. The macroparasite community of the fish was identified, showing the presence of 30 species in four phyla, of which several were new host records, and one species, a monogenean, was new to science. A canonical correspondence analysis was applied to determine the variables responsible for parasite species composition, to resolve the question of croaker stock structure in the western North Atlantic Ocean. This analysis showed that latitude was the deciding variable delineating the parasite community composition of the Atlantic croaker. Among the 30 parasites, 15 were identified as putative tags according to qualitative criteria, and then 10 out of those 15 were selected as being appropriate tags using quantitative criteria. These parasite tags support the presence of two stocks roughly separated at the known biogeographical barrier at Cape Hatteras.     

Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.     

Philometrids of the southern flounder Paralichthys lethostigma: a multidimensional approach to determine their diversity

Two species of philometrid nematode, Philometra overstreeti and Philometroides paralichthydis, infect the southern flounder, Paralichthys lethostigma. Individuals of P. overstreeti are located between the teeth and inside the bony part of the branchial arches of the fish. Individuals of P. paralichthydis are associated with the bones of the buccal cavity and among muscles that control the dorsal and anal fins. Sequencing of part of the cytochrome oxidase I gene revealed 4 distinct genetic clades, each corresponding exactly to the 4 respective locations of the parasites in the host, suggesting the need for taxonomic revision. We hypothesized that each clade represented a separate species and, because the worms are morphologically indistinguishable, compared population level parameters of the clades comprising each currently recognized species. For each currently recognized species, the presence of worms from 1 clade was negatively correlated with the presence of worms from the other. Results also indicated significant differences between the clades in prevalences relative to both biotic and abiotic factors. Results clearly indicated major differences in the ecology of the philometrids constituting each clade. Taken as a whole, molecular and ecological data support the contention that the 4 genetic clades are likely 4 distinct species.     

The respective roles of polar/nonpolar binary patterns and amino acid composition in protein regular secondary structures explored exhaustively using hydrophobic cluster analysis

Several studies have highlighted the leading role of the sequence periodicity of polar and nonpolar amino acids (binary patterns) in the formation of regular secondary structures (RSS). However, these were based on the analysis of only a few simple cases, with no direct mean to correlate binary patterns with the limits of RSS. Here, HCA‐derived hydrophobic clusters (HC) which are conditioned binary patterns whose positions fit well those of RSS, were considered. All the HC types, defined by unique binary patterns, which were commonly observed in three‐dimensional (3D) structures of globular domains, were analyzed. The 180 HC types with preferences for either α‐helices or β‐strands distinctly contain basic binary units typical of these RSS. Therefore a general trend supporting the “binary pattern preference” assumption was observed. HC for which observed RSS are in disagreement with their expected behavior (discordant HC) were also examined. They were separated in HC types with moderate preferences for RSS, having “weak” binary patterns and versatile RSS and HC types with high preferences for RSS, having “strong” binary patterns and then displaying nonpolar amino acids at the protein surface. It was shown that in both cases, discordant HC could be distinguished from concordant ones by well‐differentiated amino acid compositions. The obtained results could, thus, help to complement the currently available methods for the accurate prediction of secondary structures in proteins from the only information of a single amino acid sequence. This can be especially useful for characterizing orphan sequences and for assisting protein engineering and design. Proteins 2016; 84:624–638. © 2016 Wiley Periodicals, Inc.     

Effects of CO<Subscript>2</Subscript> inhalation exposure on mice vomeronasal epithelium

Nasal epitheliums are the first sites of the respiratory tract in contact with the external environment and may therefore be susceptible to damage from exposure to many toxic volatile substances (i.e., volatile organic components, vapors, and gases). In the field of inhalation toxicology, a number of studies have considered the main olfactory epithelium, but few have dealt with the epithelium of the vomeronasal organ (VNO). However, in several species such as in rodents, the VNO (an organ of pheromone detection) plays an important role in social interactions, and alterations of this organ are known to induce adaptative behavioral disturbances. Among volatile toxicants, health effects of inhaled gases have been thoroughly investigated, especially during CO₂ inhalation because of its increasing atmospheric concentration. Therefore, this work was designed to examine the effects of 3% CO₂ inhalation on VNO in two different exposure conditions (5 h/day and 12 h/day) in mice. Behavioral sensitivity tests to urine of congener and histological measurements of VNO were conducted before, during (weeks 1–4), and after (weeks 5–8) CO₂ inhalation exposures. Results showed no significant modifications of behavioral responses to urine, but there were significant changes of both cell number and thickness of the VNO epithelium. Moreover, the findings indicated a selectively dose-dependent effect of CO₂, and further research could use other gases in the same manner for comparison.     

Biochemical and Immunocytological Characterizations of Arabidopsis Pollen Tube Cell Wall

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.Fertilization of flowering plants requires the delivery of the two sperm cells, carried by a fast growing tip-polarized pollen tube, to the egg cell. In plants with dry stigma and solid style such as Arabidopsis (Arabidopsis thaliana), this process begins with the deposition and specific adhesion of the pollen grains on the stigmatic tissue, subsequent hydration of the pollen grains, and germination of pollen tubes (Palanivelu and Preuss, 2000). Pollen tubes invade the papillae cell wall of the stigma, enter the short style, and grow through the apoplast of the specialized transmitting tract (TT) that is filled with a nutrient-rich extracellular matrix (Kandasamy et al., 1994; Lennon et al., 1998). During this invasive growth, pollen tubes are guided to the ovules via signals that need to pass through the cell wall to reach their membrane-associated or intracellular targets (Lord and Russell, 2002; Kim et al., 2003; Boavida et al., 2005; McCormick and Yang, 2005; Johnson and Lord, 2006). In plant species with wet stigma and hollow style such as lily (Lilium longiflorum), adhesion between the pollen tube wall and the TT epidermis extracellular matrix is important for the growth of the pollen tubes toward the ovules (Mollet et al., 2000, 2007; Park et al., 2000; Chae et al., 2007). In addition to being the interface between the tube cells and the surroundings (female sporophyte or culture medium), the pollen tube wall also controls the cell shape, protects the generative cells, and allows resistance against turgor pressure (Geitmann and Steer, 2006; Geitmann, 2010).Most of our knowledge on cell wall polymers of higher plants comes from investigations on vegetative organs in which cells have diffuse growth. The cell wall is mainly composed of polysaccharides (cellulose, hemicellulose, pectin, and occasionally callose, depending on the tissue) and proteoglycans (e.g. extensin and arabinogalactan proteins [AGPs]) forming a complex network with processing enzymes.Pectins are complex wall macromolecules with uncertain supramolecular organization (Vincken et al., 2003) consisting of homogalacturonan (HG) that can be methylesterified and acetylesterified, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (Carpita and McCann, 2000). HG is a polymer of repeated units of (1→4)-α-d-GalUA that can be cross-linked with calcium upon block-wise action of pectin methylesterases (PMEs) on methylesterified HG (Micheli, 2001). RG-II has the same homopolymer backbone as HG but is substituted with four different oligosaccharides composed of unusual sugars, such as apiose, aceric acid, and 3-deoxy-d-manno-2-octulosonic acid, of unknown function (for review, see Caffall and Mohnen, 2009). RG-I consists of the repeating disaccharide (1→4)-α-d-GalUA-(1→2)-α-l-Rha, with a wide variety of side chains attached to the rhamnosyl residues, ranging from monomers to large oligosaccharides such as (1→4)-β-d-galactan, (1→5)-α-l-arabinan, and/or type I arabinogalactan (Caffall and Mohnen, 2009).Xyloglucan (XyG) is the major hemicellulosic polysaccharide of the primary wall of flowering plants. Classic XyG consists of a (1→4)-β-d-glucan backbone substituted with Xyl, Gal-Xyl, or Fuc-Gal-Xyl motifs, which correspond, according to the one-letter code proposed by Fry et al. (1993), to X, L, and F, respectively, G being the unsubstituted glucosyl residue of the glucan backbone. The main XyG fragments released after endo-glucanase treatment of the cell wall from wild-type Arabidopsis vegetative organs are generally XXXG, XXLG/XLXG, XXFG, and XLFG (Zablackis et al., 1995; Lerouxel et al., 2002; Nguema-Ona et al., 2006; Obel et al., 2009). In addition, O-acetylation of XyG can occur, most generally on the galactosyl residues, but its biological function is unknown (Cavalier et al., 2008). In the primary wall, XyG interacts with cellulose microfibrils via hydrogen bonds and participates in the control of cell expansion (Cosgrove, 1999).AGPs and extensin belong to the Hyp-rich glycoproteins superfamily with very high levels of type II arabinogalactan glycosylation (Nothnagel, 1997; Showalter, 2001). These proteoglycans have been implicated in many aspects of plant development, including cell expansion, cell signaling and communication, embryogenesis, wound response, and pollen tube guidance (Wu et al., 1995; Nothnagel, 1997; Seifert and Roberts, 2007; Driouich and Baskin, 2008).Despite the importance of pollen tubes for the delivery of the sperm cells to the egg, little is known about the underlying molecular mechanisms that regulate the mechanical interaction of pollen tubes with female floral tissues. There are very scarce data concerning the different components of the pollen tube cell wall. Past approaches to characterize the pollen tube cell wall are limited to a few plant genera, including Camellia (Nakamura and Suzuki, 1981), Lilium (Jauh and Lord, 1996; Mollet et al., 2002), Nicotiana (Rae et al.,1985; Li et al., 1995; Ferguson et al., 1998; Qin et al., 2007), Pinus (Derksen et al., 1999), and Zea (Rubinstein et al., 1995), and are mostly based on immunocytochemistry. These studies revealed that, depending on the species, the pollen tube cell wall contains epitopes that are found in the polymers described above, including HGs with varying levels of methylesterification, AGPs, extensin-like proteins, and low amounts of cellulose. Unlike most other plant cells, callose, a (1→3)-β-glucan, is predominant and is deposited in the wall back from the tip. Moreover, it is deposited at regular intervals to form callose plugs that maintain the tube cell in the apical expanding region of the tube and separate the viable from the degenerating region of the tube (for review, see Geitmann and Steer, 2006). Only a few reports have investigated the pollen tube of the model plant Arabidopsis. They have focused either on in vivo-grown or on in vitro-grown pollen tubes using monoclonal antibodies (MAbs) directed against a subset of cell wall epitopes present in HG, XyG, and AGPs (Lennon and Lord, 2000; Freshour et al., 2003; Pereira et al., 2006), but quantitative chemical analyses are lacking. This lack of information is most likely due to the fact that substantial amounts of pollen tube material are needed for chemical analysis, and a reproducible and efficient method for liquid culture of Arabidopsis pollen tubes had not been established until recently (Boavida and McCormick, 2007; Bou Daher et al., 2009).Here, we report the composition and localization of different cell wall polymers of in vitro-grown wild-type Arabidopsis pollen tubes based on biochemical analyses coupled to immunocytochemical investigations both at light and transmission electron microscopy (TEM) levels using recently developed MAbs. Our results show distinct patterns of labeling (tip, whole tube, and shank of the tube) depending on the recognized epitope. The most striking observations are (1) the abundance of (1→5)-α-l-arabinan in the tube wall (greater than 40 mol % of Ara), mostly localized, with LM6 and LM13, in the outer wall layer of the tube and (2) an atypical XyG matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profile with over 68% of the oligosaccharide fragments being O-acetylated.     

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1–4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.