Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.     

Abnormal Pelage Color in an Isolated Population of <Emphasis Type="Italic">Alouatta guariba clamitans</Emphasis> Cabrera, 1940 in South Brazil

We located 4 brown howlers (1 adult male, 2 adult females, and 1 juvenile male) showing abnormally lighter pelage in 3 social groups comprising 5, 6, and 9 individuals in a 20 ha-forest fragment in the State of Rio Grande do Sul, Brazil. Two additional groups composed only of normally colored individuals also live in the fragment, which is isolated from nearby fragments by 267–1009 m. They were the only brown howlers with abnormal pelage color out of a total of 386 individuals belonging to 67 groups in 21 fragments in the 5876-ha study area. The isolation of the forest fragment, its high howler density (2.2 individuals⁄ha), and large group size (8.8 ± 2.4 individuals) may decrease the likelihood of successful immigration into the population, leading to an increased probability of inbreeding that may facilitate the expression of rare alleles.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mitotracker Green Is a P-Glycoprotein Substrate

P-glycoprotein has a widespread expression on normal tissues. The protein has also been strongly associated with the multidrug resistance phenotype (MDR) on tumor cells. The employment of flow cytometry and confocal microscopy has contributed to the discovery and application of new particular fluorescent dyes. Nevertheless, several studies are being performed in different cellular types neglecting the expression/activity of MDR proteins. Because many fluorochromes have been reported as P-glycoprotein substrates, an especial attention must be given to the properties of new dyes in the presence of MDR proteins. Flow cytometric analyzes of Mitotracker Green (MTG) fluorescence profile were performed in a human erythroleukemic cell line and its resistant counterpart. In this report we demonstrated that MTG, a probe used to evaluate the mitochondrial mass, is a P-glycoprotein substrate and its staining profile is dependent on the activity of this protein. In vitro studies on a human erythroleukemic cell line and its resistant counterpart revealed that MDR modulators (Cyclosporin A, Verapamil, and Trifluoperazine) alter the MTG fluorescence pattern on a resistant cell line. The findings suggest that attention should be given to the expression of P-glycoprotein when performing an evaluation of mitochondria properties with MTG.     

Effect of the removal of different endocrine glands upon insulin secretion and B-cell ultrastructure

Glucose-induced insulin secretion and B-cell ultrastructure were studied in islets obtained from normal, adrenalectomized, radiothyroidectomized, ovariectomized and orchidectomized rats. Both parameters were also studied in the same experimental groups submitted to specific substitutive therapy. Insulin secretion in response to high glucose was significantly diminished in adrenalectomized, hypothyroid and male castrated rats. Conversely, this secretion was enhanced in ovariectomized rats. These abnormal insulin responses were restored to normal range by specific substitutive therapy. B-cell ultrastructure was markedly altered in hypothyroid and in female and male castrated rats. No significant changes were observed in the adrenalectomized rats. No conspicuous alterations were depicted in the other islet cell populations. The features of the morphological alterations were mainly related to changes in the B-granules and the rough endoplasmic reticulum. Modifications of the other B-cell organelles were less frequent. In the castrated rats, a distinctive feature was the appearance of a finely granulated colloid material. These B-cell alterations, consecutive to changes in the circulating levels of a given hormone, seemed to depend on the chemical structure of the hormone itself rather than on the changes induced in the B-cell secretory function. The ultrastructural changes described were reversed, as in the case of insulin release, by specific substitutive therapy. It is concluded that changes in the circulating levels of the hormones studied are followed by specific alterations in both B-cell secretion and ultrastructure.     

Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Crithidia fasciculata. Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase

The non-invasive technique of 13C nuclear magnetic resonance was applied to study glucose metabolism in vivo in the insect parasite Crithidia fasciculata. It was found that under anaerobic conditions [1-13C]glucose underwent a glycolytic pathway whose main metabolic products were identified as [2-13C]ethanol, [2-13C]succinate and [1,3-13C2]glycerol. These metabolites were excreted by C. fasciculata into the incubation medium, while in the cells [3-13C]phosphoenolpyruvate was also detected in addition to the aforementioned compounds. The C3 acid is apparently the acceptor of the primary CO2 fixation reaction, which leads in Trypanosomatids to the synthesis of succinate. By addition of sodium bicarbonate to the incubation mixture L-[3-13C]malate was detected among the excretion products, while the ethanol:succinate ratio of 2.0 in the absence of bicarbonate changed to a ratio of 0.6 in the presence of the latter. This was due to a shift of the balance between carboxylation of phosphoenolpyruvate, leading to succinate, and pyruvate decarboxylation leading to ethanol. The addition of 25% 2H2O to the incubation mixture led to the formation of [2-13C, 2-2H]ethanol derived from the prior incorporation of 2H+ into pyruvate in the reactions mediated by either pyruvate kinase or malic enzyme. However, no 2H+ incorporation into L-malate was detected, excluding the possibility that the latter was formed by carboxylation of pyruvate, and lending support to the idea that L-malate results from the carboxylation of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxykinase. The formation of [2-13C, 2-2H]-succinate under the same conditions reflected the uptake of 2H+ during the reduction of fumarate. When the incubations were carried out in the presence of 100% 2H2O, several [1-13C, 1-2H]ethanol species were detected, as well as [2-13C, 2-2H]malate and [1,3-13C2, 1,3-2H2]glycerol. The former deuterated compounds reflect the existence of NAD2H species when the incubations were carried out in 100% 2H2O, while the incorporation of 2H+ into [1,3-13C2]glycerol must be attributed to the phosphoglucose-isomerase-mediated reaction during glycolysis.     

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.     

Species diversity of the genus Osmundea (Ceramiales,Rhodophyta) in the Macaronesian region

Species diversity within the genus Osmundea in the Macaronesian region was explored by conducting a comprehensive sampling in the Azores, the Canary, and the Madeira archipelagos. Toward identification, all specimens were first observed alive to verify the absence of corps en cerise, a diagnostic character for the genus and morphometric data were measured (thallus length and width, first‐order branches length and width, branchlets length and width, cortical cell length and width in surface view, cortical cell length and width in transverse section). Specimens were sequenced for COI‐5P (39 specimens) and three species delimitation methods (Generalized Mixed Yule Coalescent, Automatic Barcode Gap Discovery method, and Poisson Tree Processes) were used to assess the threshold between infra‐ and interspecific relationships. Subsequently, one or several sequences of plastid‐encoded large subunit of RuBisCO (21 specimens) per delimited species were generated to assess the phylogenetic relationships among Macaronesian Osmundea. Moreover, for each delineated species, vegetative and reproductive anatomy was thoroughly documented and, when possible, specimens were either assigned to existing taxa or described as novel species. This integrative approach has provided data for (i) the presence of O. oederi, O. pinnatifida, and O. truncata in Macaronesia; (ii) the proposal of two novel species, O. prudhommevanreinei sp. nov. and O. silvae sp. nov.; and (iii) evidence of an additional species referred as “Osmundea sp.1,” which is a sister taxon of O. hybrida.