The last exon of SNAP-23 regulates granule exocytosis from mast cells

SNAP-25 and its ubiquitous homolog SNAP-23 are members of the SNARE family of proteins that regulate membrane fusion during exocytosis. Although SNAP-23 has been shown to participate in a variety of intracellular transport processes, the structural domains of SNAP-23 that are required for its interaction with other SNAREs have not been determined. By employing deletion mutagenesis we found that deletion of the amino-terminal 18 amino acids of SNAP-23 (encoded in the first exon) dramatically inhibited binding of SNAP-23 to both the target SNARE syntaxin and the vesicle SNARE vesicle-associated membrane protein(VAMP). By contrast, deletion of the carboxyl-terminal 23 amino acids (encoded in the last exon) of SNAP-23 does not affect SNAP-23 binding to syntaxin but profoundly inhibits its binding to VAMP. To determine the functional relevance of the modular structure of SNAP-23, we overexpressed SNAP-23 in cells possessing the capacity to undergo regulated exocytosis. Expression of human SNAP-23 in a rat mast cell line significantly enhanced exocytosis, and this effect was not observed in transfectants expressing the carboxyl-terminal VAMP-binding mutant of SNAP-23. Despite considerable amino acid identity, we found that human SNAP-23 bound to SNAREs more efficiently than did rat SNAP-23. These data demonstrate that the introduction of a "better" SNARE binder into secretory cells augments exocytosis and defines the carboxyl terminus of SNAP-23 as an essential regulator of exocytosis in mast cells.     

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.     

The mitochondrial SDHD gene is required for early embryogenesis, and its partial deficiency results in persistent carotid body glomus cell activation with full responsiveness to hypoxia

The SDHD gene encodes one of the two membrane-anchoring proteins of the succinate dehydrogenase (complex II) of the mitochondrial electron transport chain. This gene has recently been proposed to be involved in oxygen sensing because mutations that cause loss of its function produce hereditary familiar paraganglioma, a tumor of the carotid body (CB), the main arterial chemoreceptor that senses oxygen levels in the blood. Here, we report the generation of a SDHD knockout mouse, which to our knowledge is the first mammalian model lacking a protein of the electron transport chain. Homozygous SDHD(-/-) animals die at early embryonic stages. Heterozygous SDHD(+/-) mice show a general, noncompensated deficiency of succinate dehydrogenase activity without alterations in body weight or major physiological dysfunction. The responsiveness to hypoxia of CBs from SDHD(+/-) mice remains intact, although the loss of an SDHD allele results in abnormal enhancement of resting CB activity due to a decrease of K(+) conductance and persistent Ca(2+) influx into glomus cells. This CB overactivity is linked to a subtle glomus cell hypertrophy and hyperplasia. These observations indicate that constitutive activation of SDHD(+/-) glomus cells precedes CB tumor transformation. They also suggest that, contrary to previous beliefs, mitochondrial complex II is not directly involved in CB oxygen sensing.     

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.     

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.     

In vitro effects of wastewater treatment plant effluent on sea bass red blood cells

Red blood cells of marine fish have been used in suitable biological assays to study the (eco)toxicity of wastewater treatment plant effluents. The aim of the present work was to draw upon their more relevant effects on cell hemolysis, ATP content, osmotic resistance and cell volume regulation. Following physico-chemical treatment, treatment plant effluents showed a residual toxicity resulting from multiple impairment of cell metabolism and structures. The earliest and most sensitive effects were related to the regulation of intracellular osmotic pressure leading to decreased cell water volume. Such effects were also observed following short-term incubation in 10-fold diluted effluent. Other damages were found following incubation in non-diluted effluent. Membrane structure was affected leading to increased osmotic resistance. Later, a decrease of the intracellular ATP level was found, followed by hemolysis. The presence of glucose in the incubation medium lessened the fall in ATP content and hemolysis in the treated cells but also in control cells.     

Genetic architecture of susceptibility to herbivores in hybrid willows

We performed a common garden experiment using parental, F1, F2, and backcross willow hybrids to test the hypothesis that hybrid willows experience breakdown of resistance to herbivores. After exposing plants to herbivores in the field, we measured the densities/damage caused by 13 insect herbivores and one herbivorous mite. Using joint-scaling tests, we determined the contribution of additive, dominance, and epistasis to variation in susceptibility to herbivores (measured either as density or damage level) among the six genetic classes. We found the genetic architecture of susceptibility/resistance in the parental species to be complex, involving additive, dominance, and epistasis for each herbivore species. Although genic interactions altered plant susceptibility for each of the 14 herbivores, three distinct patterns of response of herbivores to hybrids were expressed. One pattern, observed in four herbivore species, supported the hypothesis of breakdown of resistance genes in recombinant hybrids. A second pattern, shown by six other herbivore species, supported the hypothesis of hybrid breakdown of host recognition genes. In other words, epistatic interactions for host recognition traits (probably oviposition/feeding stimulants or attractants) appeared to be important in determining herbivore abundance for those six species. The final patterns supported a structure of dominance, either for host recognition traits (in the case of three herbivore species) or for host resistance traits (for one herbivore species). The combination of differing responses of herbivore species, including members of the same genus and tribe, and the ubiquitous importance of epistasis suggests that many genes affect herbivore resistance in this hybrid willow system.     

The effect of estradiol benzoate or a synthetic gonadotropin-releasing hormone used at the start of a progesterone treatment on estrous response in cattle

The aim was to compare the estrous response in heifers given either gonadotropin-releasing hormone (GnRH) or estradiol benzoate (EDB) at the start of a progesterone treatment initiated at emergence or dominance of the first or second follicular wave of the estrous cycle. Cross-bred beef heifers (n=134) were assigned to 1 of 3 treatments; 0.75 mg EDB given at insertion of a progesterone-releasing intravaginal device (PRID) treatment of 10 days duration (10dE2), 0.75 mg EDB at insertion of a PRID treatment of 8 days duration with 15 mg luprostiol (PGF) a luteolytic agent, given 1 day before PRID removal (8dE2) or 250 microg GnRH at insertion of a PRID treatment of 8 days duration with 15 mg PGF given 1 day before PRID removal (8dGnRH). Treatments were initiated on Days 2, 5, 10 or 13 of the estrous cycle. Estrous detection was conducted six times daily. Twice daily blood samples were taken, from 2 days before PRID insertion until detection of estrus. The proportion of heifers detected in estrus was higher (P < 0.05) for heifers in the 8dE2 treatment group (40/40) compared with those in the 8dGnRH group (38/42) and tended to be higher (P = 0.08) than heifers in the 10dE2 group (38/41). The onset of estrus was earlier (P < 0.05) for heifers in the 10dE2 treatment group (median 41 h, range 92 h) compared with either the 8dE2 (median 49 h, range 64 h) or 8dGnRH groups (median 49 h, range 92 h). Submission rate at 72 h was higher (P < 0.01) in the 8dE2 (95%) group than for those in the 10dE2 (74%) and 8dGnRH (69%) groups. In conclusion, EDB given at PRID insertion, with PGF given 1 day before PRID removal, was more effective at synchronizing estrus than was GnRH at PRID insertion. Decreasing the length of treatment and the use of PGF 1 day before the end of an EDB and progesterone treatment improved estrous synchrony.     

Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.