Expression of distinct ERG proteins in rat, mouse, and human heart. Relation to functional I(Kr) channels

One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-go-go-related gene, which encodes a voltage-gated K(+) channel alpha subunit. Heterologous expression of HERG1 gives rise to K(+) currents that are similar (but not identical) to the rapid component of delayed rectification, I(Kr), in cardiac myocytes. In addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional I(Kr) channels. In the experiments here, antibodies generated against HERG1 were used to examine ERG1 protein expression in heart and in brain. In Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or RERG1 (rat ERG1) probed with antibodies targeted against the C terminus of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band is evident in blots of extracts from cells expressing MERG1b or HERG1b. In immunoblots of fractionated rat (and mouse) brain and heart membrane proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F, the 165- and 205-kDa proteins were replaced by two new bands at 175 and 130 kDa, suggesting that ERG1 is differentially glycosylated in rat/mouse brain and heart. In human heart, a single HERG1 protein with an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein (and I(Kr)) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue. Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play roles in the generation of functional cardiac I(Kr) channels.     

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.     

Binding of GDNF and neurturin to human GDNF family receptor alpha 1 and 2. Influence of cRET and cooperative interactions

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of (125)I-GDNF from GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.     

Tank spawning of first generation domestic green sturgeon

Green sturgeon Acipenser medirostris from hatchery spawned wild‐caught broodfish matured under aquaculture conditions. The first females (three out of four) matured at age 8 years, were successfully induced to ovulate by hormonal injections, and their eggs collected by caesarean surgery for fertilization. While the incision healed completely, unknown complications resulted in mortalities 5–12 months later. To minimize handling stress and to eliminate mortalities associated with surgery, a semi‐natural tank spawning technique was used in subsequent years. A female was placed into a 3.7 m diameter flat‐bottom circular fiberglass tank, depth 0.7 m, with two or three males, and the fish were injected with gonadotropin‐releasing hormone analog (GnRHa) and white sturgeon pituitary extracts to induce spawning. Fuller’s Earth was continually added to the spawning tank using a belt feeder to minimize egg adhesion to other eggs and to the tank surfaces. The tank was checked hourly for oviposition, and the eggs collected using a fine mesh dip net, every 1–3 h. Spawning lasted 15–21 h, and 53–80 000 eggs were collected from individual spawning females. Direct observation of spawning activity was not possible because of the high turbidity caused by Fuller’s Earth. However, the female and males occasionally breached at the water surface and appeared to be interacting. After oviposition was completed, viable milt was collected from all males, and fertilization success ranged from 7% to 83% in five spawning events. Over three consecutive years (2009–2011) all five females and 11 males used in semi‐natural tank spawning ovulated or spermiated, and were not subject to post‐spawning mortality. The relatively slow deposition of eggs and the continuous addition of Fuller’s Earth minimized the adhesion of eggs into large clumps. While the egg fertility and hatchability varied greatly among individual females, the tank spawning technique presented a feasible alternative to other more invasive methods for obtaining fertilized eggs of green sturgeon, and may be applicable for other sturgeons, particularly endangered species.     

No evidence for an association between the -871 T/C promoter polymorphism in the B-cell-activating factor gene and primary Sjögren's syndrome

Polyclonal B cell activation might be related to pathogenic over-expression of B-cell-activating factor (BAFF) in primary Sjögren's syndrome (pSS) and other autoimmune diseases. We therefore investigated whether BAFF over-expression in pSS could be a primary, genetically determined event that leads to the disease. The complete BAFF gene was sequenced in Caucasian pSS patients and control individuals. The only single nucleotide polymorphism frequently observed, namely -871 T/C in the promoter region, was then genotyped in 162 French patients with pSS and 90 French control individuals. No significant differences in allele (T allele frequency: 49.7% in patients with pSS versus 50% in controls; P = 0.94) and genotype frequencies of BAFF polymorphism were detected between pSS patients and control individuals. BAFF gene polymorphism was not associated with a specific pattern of antibody secretion either. T allele carriers had significantly increased BAFF protein serum levels (mean values of 8.6 and 5.7 ng/ml in patients with TT and TC genotypes, respectively, versus 3.3 ng/ml in patients with CC genotype; P = 0.01), although no correlation was observed between BAFF polymorphism and mRNA level. In conclusion, BAFF gene polymorphism is neither involved in genetic predisposition to pSS nor associated with a specific pattern of antibody production.     

Mechano-electrical Transduction: New Insights into Old Ideas

The gating-spring theory of hair cell mechanotransduction channel activation was first postulated over twenty years ago. The basic tenets of this hypothesis have been reaffirmed in hair cells from both auditory and vestibular systems and across species. In fact, the basic findings have been reproduced in every hair cell type tested. A great deal of information regarding the structural, mechanical, molecular and biophysical properties of the sensory hair bundle and the mechanotransducer channel has accumulated over the past twenty years. The goal of this review is to investigate new data, using the gating spring hypothesis as the framework for discussion. Mechanisms of channel gating are presented in reference to the need for a molecular gating spring or for tethering to the intra- or extracellular compartments. Dynamics of the sensory hair bundle and the presence of motor proteins are discussed in reference to passive contributions of the hair bundle to gating compliance. And finally, the molecular identity of the channel is discussed in reference to known intrinsic properties of the native transducer channel.     

Catalytic and DNA-binding properties of the human Ogg1 DNA N-glycosylase/AP lyase: biochemical exploration of H270, Q315 and F319, three amino acids of the 8-oxoguanine-binding pocket

The human Ogg1 protein (hOgg1) is an antimutator DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-7,8-dihydroguanine (8-oxoG) and the incision of apurinic and apyrimidinic (AP) sites in DNA. In this study, we have investigated the functional role of H270, Q315 and F319, three amino acids that are located in the 8-oxoG-binding pocket of hOgg1. Wild-type and mutant hOgg1 proteins (H270A, H270R, H270L, Q315A and F319A) were purified to apparent homogeneity. The catalytic activities and the DNA-binding properties of the various hOgg1 mutants were compared to those of the wild-type. The results show that hOgg1 mutated at H270 (H270A and H270L) or F319 (F319A) exhibits greatly reduced (50- to 1000-fold) DNA glycosylase activity, whereas the AP lyase activity is only moderately affected (<4-fold). The affinity of the hOgg1 mutants (H270A, H270L and F319A) for 8-oxoG.C-containing DNA is also greatly reduced (>30-fold), whereas their affinity for THF.C-containing DNA is only moderately reduced (<7-fold). The results also show that hOgg1 mutated at Q315 (Q315A) exhibits catalytic and DNA-binding properties similar to those of the wild-type. Therefore, H270 and F319 are essential to form the functional 8-oxoG-binding pocket, whereas Q315 is less crucial. In contrast, H270, Q315 and F319 are not required for efficient binding of THF.C and cleavage of AP sites. Finally, hOgg1 mutant proteins with a substitution of H270A or F319A are members of a new type of hOgg1 that is deficient in DNA glycosylase but proficient in AP lyase.     

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.