A detailed study of the periodate oxidation of sialic acids in glycoproteins

Periodate oxidation of terminalN-acetyl- andN-glycoloylneuraminic acid residues in the mucins from edible bird nest substance and pig submandibular gland, respectively, can be carried out under conditions which exclusively give rise to the formation of the C-7 analogues of these sialic acids. In contrast, the C-8 compounds can be obtained in a maximum yield of about 40%. Under identical conditions,N-glycoloylneuraminic acid is oxidized about 1.5 times faster than theN-acetylated derivative. After release of the sialic acids by acid hydrolysis, the characterization of the oxidation products was carried out by TLC, by GLC and GLC-MS of the corresponding pertrimethylsilyl derivatives, and by 500-MHz¹H-NMR spectroscopy. In addition, molar response factors for GLC analysis and extinction coefficients in the orcinol/Fe³⁺/HCl assay were determined.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Seasonal variations in spatial usage by the estuarine dolphin, Sotalia guianensis (van Bénéden, 1864) (Cetacea; Delphinidae) at its southern limit of distribution.

The use of space by the resident population of estuarine dolphins (Sotalia guianensis) in Norte Bay, southern Brazil was studied by boat surveys between 2000 and 2003. Data such as the geographical position and group size was collected at 5-minute intervals using focal-group sampling. The distribution and seasonal and daily group home-ranges were estimated using a GIS (Geographic Information System) environment. The dolphins did not use the area homogeneously, restricting their activities to a well-defined area located in the western part of the bay, while the use of the eastern part was not observed. A discrepancy between the Environment Protection Area of Anhatomirim, created especially to protect this population and the distribution of the dolphins was observed. Seasonal group home-range varied both qualitatively and quantitatively among the four consecutive seasons analyzed. The dolphins tended to use a larger area in cold seasons, when apparently the abundance of major prey items is lower. Core areas of intense use were identified, and also varied seasonally. These core areas may be regarded as critical habitats and require special legislation and control. The mean daily group home-range did not differ statistically among seasons. The present study corroborates a spatially strict residency pattern of the estuarine dolphin in highly productive coastal ecosystems.     

Assessing spatial patterns and density of a dolphin population through signature whistles

Some dolphin species produce signature whistles, which may allow the identification of individual dolphins using passive acoustic monitoring (PAM). Identifying individuals by their sounds may enhance the opportunities for monitoring and addressing biological and ecological questions about these species. Here, we explored the potential of signature whistles to investigate ecological aspects of a resident bottlenose dolphin population. Using a limited data set, with few individuals recognized by signature whistles, combined with spatial capture-recapture (SCR) methods, we investigated how effective such approach is describing spatial use patterns and estimating density for this population. The data were collected using 4–6 stationary bottom-moored recorders. Since only eight signature whistles were identified, our density estimate may represent a subset of the entire population. However, even with only a few signature whistles identified, our results confirmed the center of the core area used by these dolphins as the area with the highest encounter probability. In addition, our results provided evidence that these dolphins have the same spatial use pattern at night as during the day. This study shows that SCR analysis of signature whistle data can improve our ecological knowledge and understanding of dolphin populations.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

Background

The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.

Methods

Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.

Results

As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.

Conclusions

Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.     

Lobomycosis-like disease in wild bottlenose dolphins Tursiops truncatus of Laguna, southern Brazil: monitoring of a progressive case

Lobomycosis is a chronic dermal infection affecting humans and small cetaceans. In 1993, a study identified the presence of the etiologic agent of lobomycosis in a resident population of Tursiops truncatus (bottlenose dolphin) in Laguna, Brazil. Until now, no additional information relating to the persistence or prevalence of this pathogen in this population has been available. Numbering less than 60 animals, the residency of these dolphins in an impacted lagoon system has raised concerns about the health and viability of this small population. Using photo-identification data collected between September 2007 and September 2009, this study evaluated the occurrence of lobomycosis-like disease (LLD) throughout this population. Of 47 adult dolphins and 10 calves identified, 7 (12%) presented some form of epidermal lesion and 5 (9%) had evidence of LLD. The lesions were stable in all but 2 cases, in which a progressive development was recorded in a presumed adult female and her calf (referred to here as the LLD pair). During the first few months of observation, the lesion grew slowly and at a constant rate on the adult. However, in the fourteenth month, the growth rate increased rapidly and the first lesions appeared on the calf. Compared to the rest of the population, the LLD pair also presented a different spatial ranging pattern, suggesting a possible social or geographic factor. Current and previous records of LLD or lobomycosis indicate that the disease is endemic in this population. These findings highlight the importance of monitoring both the health of these cetaceans and the quality of their habitat.     

The structure of a bottlenose dolphin society is coupled to a unique foraging cooperation with artisanal fishermen

Diverse and localized foraging behaviours have been reported in isolated populations of many animal species around the world. In Laguna, southern Brazil, a subset of resident bottlenose dolphins (Tursiops truncatus) uses a foraging tactic involving cooperative interactions with local, beach-casting fishermen. We used individual photo-identification data to assess whether cooperative and non-cooperative dolphins were socially segregated. The social structure of the population was found to be a fission-fusion system with few non-random associations, typical for this species. However, association values were greater among cooperative dolphins than among non-cooperative dolphins or between dolphins from different foraging classes. Furthermore, the dolphin social network was divided into three modules, clustering individuals that shared or lacked the cooperative foraging tactic. Space-use patterns were not sufficient to explain this partitioning, indicating a behavioural factor. The segregation of dolphins using different foraging tactics could result from foraging behaviour driving social structure, while the closer association between dolphins engaged in the cooperation could facilitate the transmission and learning of this behavioural trait from conspecifics. This unique case of a dolphin-human interaction represents a valuable opportunity to explore hypotheses on the role of social learning in wild cetaceans.     

Anatomy of flower and fruit of <i>Vassobia breviflora</i> (Solanaceae) in the south of the southern Yungas (Argentina)

Solanaceae is a family with nearly 2400 species of cosmopolitan distribution. Vassobia breviflora is the only species of the genus present in Argentina. The goal of this work was to review and characterize the anatomy of the flower and fruit of V. breviflora from samples collected in populations of Yungas in the argentine Northwest. Conventional anatomical techniques were applied. The results showed that most flower, fruit and seed structures did not differ from those previously reported regarding the structural organization described for other species of the Solanaceae family. However, for the first time, we described the androecium, fruit, seed, floral and fruit pedicels, five types of tricomes and five types of stomata in the perianth. We found some differences in the shape of the transmission tissue and in the type of ovule with respect to that previously reported. Also, we located the parenchyma and the epidermic secretory cells of the nectary. In the context of the family Solanaceae, we discussed the function and diagnostic value of the described structures.