Study on the maltooligosaccharide composition of mucilage samples collected along the northern Adriatic coast

The mucilage phenomenon, a sporadic but massive accumulation of gelatinous material, can cause serious damage to the tourism and fishing industries along the Adriatic coast. Mucilage is presently thought to be the result of the aggregation of dissolved organic matter (DOM) into particulate organic matter (POM). Three principal classes of compounds have been identified in organic matter by spectrometric determination: carbohydrates, proteins and lipids. Carbohydrates are suspected to play a role in the first steps of DOM aggregation. Despite its importance in understanding the processes leading to mucilage formation, our present knowledge of the composition of the mucilage carbohydrate fraction is incomplete. Due to its high sensitivity and specificity, liquid chromatography coupled with electrospray-ionization tandem mass spectrometry (LC-ESIMS/MS) is gaining an increasing importance as a powerful technique for carbohydrate purification and characterization in complex samples. In this work, LC-ESIMS/MS is proposed as a useful method for the investigation of the oligosaccharide content in mucilage samples. The approach was applied using 3-7 unit maltooligosaccharides as reference compounds. The composition of the investigated mucilage sample was further investigated combining LC-ESIMS/MS with classic approaches, such as spectroscopic techniques and liquid chromatography coupled with the refractory index LC-RI.     

Identification of a Terminal Rhamnopyranosyltransferase (RptA) Involved in Corynebacterium glutamicum Cell Wall Biosynthesis

A bioinformatics approach identified a putative integral membrane protein, NCgl0543, in Corynebacterium glutamicum, with 13 predicted transmembrane domains and a glycosyltransferase motif (RXXDE), features that are common to the glycosyltransferase C superfamily of glycosyltransferases. The deletion of C. glutamicum NCgl0543 resulted in a viable mutant. Further glycosyl linkage analyses of the mycolyl-arabinogalactan-peptidoglycan complex revealed a reduction of terminal rhamnopyranosyl-linked residues and, as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues, which was fully restored upon the complementation of the deletion mutant by NCgl0543. As a result, we have now termed this previously uncharacterized open reading frame, rhamnopyranosyltransferase A (rptA). Furthermore, an analysis of base-stable extractable lipids from C. glutamicum revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase.A common feature of members of the Corynebacterineae is that they possess an unusual cell wall dominated by a heteropolysaccharide termed an arabinogalactan (AG), which is linked to both mycolic acids and peptidoglycan, forming the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex (5, 10, 12, 15, 24, 25, 34). The formation of the arabinan domain in the mAGP complex, consisting mainly of α1→5, α1→3, and β1→2 glycosyl linkages, results from the subsequent addition of arabinofuranose (Araf) from the lipid-linked sugar donor β-d-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) by a set of unique membrane-bound arabinofuranosyltransferases (5, 7, 12, 18, 34).The deletion of Corynebacterium glutamicum emb (emb_Cg) (4) and a chemical analysis of the cell wall revealed a novel truncated AG structure possessing only terminal Araf residues with a corresponding loss of cell wall-bound mycolic acids (4). The presence of a novel enzyme responsible for “priming” the galactan domain for further elaboration by Emb_Cg proteins led to the identification of AftA, which belongs to the glycosyltransferase C (GT-C) superfamily (5). Recently, additional GT-C enzymes have been identified, termed AftB, which is responsible for the attachment of terminal β(1→2) Araf residues (34), and AftC, which is involved in AG branching (12) before decoration with mycolic acids, both of which are conserved within the Corynebacterineae (12, 34). It is clear that additional glycosyltransferases involved in both AG and lipoarabinomannan biosynthesis still remain to be identified. Indeed, Liu and Mushegian (22) identified 15 members of the GT-C superfamily residing in the Corynebacterineae, representing candidates involved in the biosynthesis of cell wall-related glycans and lipoglycans (22). We have continued our earlier studies (5, 12, 34) to identify genes required for the biosynthesis of the core structural elements of the mAGP complex by studying mutants of C. glutamicum and the orthologous genes and enzymes of Mycobacterium tuberculosis.A particularly interesting feature of C. glutamicum is the presence of terminal rhamnopyranose (t-Rhap) residues attached to the C2 position of α(1→5)-linked Araf residues in the arabinan domain of AG (4). The biological function of these residues remains to be clarified; nevertheless, they are a feature of the corynebacterial cell wall, and the biosynthesis of which needs to be addressed. The current paradigm of AG biosynthesis follows a linear pathway which is built upon a decaprenyl pyrophosphate lipid carrier. The unique disaccharide linker and galactan domain is synthesized by a variety of GT-A and GT-B family glycosyltransferases, all of which utilizing a nucleotide diphosphate-activated sugar substrate for transferase activity. It has been hypothesized by us (3, 5) and others (8) that a major shift in the biosynthetic machinery takes place upon the initiation of arabinan polymerization. AftA, Emb, AftC, and AftB all belong to the GT-C family of glycosyltransferases, all of which utilize DPA as the sole lipid-activated phosphosugar donor for arabinose transfer into the cell wall. Since t-Rhap residues are present in the arabinan component of the cell wall, the enzyme(s) responsible for its addition is likely to belong to the GT-C family of glycosyltransferases and, as determined through deduction, is one which utilizes a lipid-phosphate-derived rhamnose substrate similar to DPA. Herein, we present the putative protein NCgl0543 as a distinct t-Rhap of the GT-C superfamily, which is responsible for the transfer of t-Rhap residues to the arabinan domain to form the branched 2,5-linked Araf motifs of C. glutamicum. In addition, we have identified a novel decaprenyl-monophosphorylrhamnose and discuss its role in substrate presentation for AG biosynthesis in C. glutamicum.     

Changes in the condition factor have an impact on metabolic rate and swimming performance relationships in Atlantic cod (Gadus morhua L.)

In the field, Atlantic cod face seasonal changes in food availability that in turn lead to changes in condition. To examine the physiological consequences of these changes in condition, we measured routine metabolic rate (RMR) to estimate standard metabolic rate (SMR), active metabolic rate (AMR), aerobic scope, critical swimming speed (Ucrit), cost of transport, sprint performance, time to exhaustion, and postexhaustion metabolic rate (EMR) for 24 Atlantic cod from the Gulf of St. Lawrence. Cod were measured at their initial condition (condition factor of 0.676+/-0.076) and after 9 wk of feeding (condition factor of 0.923+/-0.096). These levels of condition are representative of wild cod in the Gulf of St. Lawrence during the spring and during the fall-early winter, respectively. The improved condition did not change mass-specific SMR. However, mass-specific AMR, aerobic scope, and EMR decreased with the improvement in condition. The various measures of swimming performance were affected differently. Ucrit increased and cost of transport at 1.3 and 1.5 body lengths s(-1) decreased with improved condition, but the cost of transport at 0.3, 0.9, 1.1, 1.7, and 1.9 body lengths s(-1), sprint performance, and time to exhaustion did not change. Hierarchies for the speed at first burst-coast, the proportion of Ucrit supported by burst-coasts, and time to exhaustion were maintained with the improvement in condition. The relationships between metabolic rates and swimming performance differed with condition level, with stronger correlations apparent in the cod at their initial condition. Given the low condition of wild cod stocks, these responses indicate that reduced performance, due to both maintenance of metabolic expenditures and modified swimming capacities, may impair survival under conditions of reduced food availability.     

Who ate whom? Adaptive Helicobacter genomic changes that accompanied a host jump from early humans to large felines

Helicobacter pylori infection of humans is so old that its population genetic structure reflects that of ancient human migrations. A closely related species, Helicobacter acinonychis, is specific for large felines, including cheetahs, lions, and tigers, whereas hosts more closely related to humans harbor more distantly related Helicobacter species. This observation suggests a jump between host species. But who ate whom and when did it happen? In order to resolve this question, we determined the genomic sequence of H. acinonychis strain Sheeba and compared it to genomes from H. pylori. The conserved core genes between the genomes are so similar that the host jump probably occurred within the last 200,000 (range 50,000-400,000) years. However, the Sheeba genome also possesses unique features that indicate the direction of the host jump, namely from early humans to cats. Sheeba possesses an unusually large number of highly fragmented genes, many encoding outer membrane proteins, which may have been destroyed in order to bypass deleterious responses from the feline host immune system. In addition, the few Sheeba-specific genes that were found include a cluster of genes encoding sialylation of the bacterial cell surface carbohydrates, which were imported by horizontal genetic exchange and might also help to evade host immune defenses. These results provide a genomic basis for elucidating molecular events that allow bacteria to adapt to novel animal hosts.     

"The stone which the builders rejected...": Delay of reinforcement and response rate on fixed-interval and related schedules

The article deals with response rates (mainly running and peak or terminal rates) on simple and on some mixed-FI schedules and explores the idea that these rates are determined by the average delay of reinforcement for responses occurring during the response periods that the schedules generate. The effects of reinforcement delay are assumed to be mediated by a hyperbolic delay of reinforcement gradient. The account predicts that (a) running rates on simple FI schedules should increase with increasing rate of reinforcement, in a manner close to that required by Herrnstein's equation, (b) improving temporal control during acquisition should be associated with increasing running rates, (c) two-valued mixed-FI schedules with equiprobable components should produce complex results, with peak rates sometimes being higher on the longer component schedule, and (d) that effects of reinforcement probability on mixed-FI should affect the response rate at the time of the shorter component only. All these predictions were confirmed by data, although effects in some experiments remain outside the scope of the model. In general, delay of reinforcement as a determinant of response rate on FI and related schedules (rather than temporal control on such schedules) seems a useful starting point for a more thorough analysis of some neglected questions about performance on FI and related schedules.     

Centriole movements in mammalian epithelial cells during cytokinesis

Background  

In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules.     

Enzyme activity in the aestivating Green-striped burrowing frog (Cyclorana alboguttata)

Green-striped burrowing frogs (Cyclorana alboguttata) can depress their resting metabolism by more than 80% during aestivation. Previous studies have shown that this species is able to withstand long periods of immobilisation during aestivation while apparently maintaining whole muscle mass and contractile performance. The aim of this study was to determine the effect of prolonged aestivation on the levels of metabolic enzymes (CCO, LDH and CS) in functionally distinct skeletal muscles (cruralis, gastrocnemius, sartorius, iliofibularis and rectus abdominus) and liver of C. alboguttata. CS activity was significantly reduced in all tissues except for the cruralis, gastrocnemius and the liver. LDH activity was significantly reduced in the sartorius and rectus abdominus, but remained at control (active) levels in the other tissues. CCO activity was significantly reduced in the gastrocnemius and rectus abdominus, and unchanged in the remaining tissues. Muscle protein was significantly reduced in the sartorius and iliofibularis during aestivation, and unchanged in the remaining muscles. The results suggest that the energy pathways involved in the production and consumption of ATP are remodelled during prolonged aestivation but selective. Remodelling and subsequent down-regulation of metabolic activity seem to target the smaller non-jumping muscles, while the jumping muscles retain enzyme activities at control levels during aestivation. These results suggest a mechanism by which aestivating C. alboguttata are able to maintain metabolic depression while ensuring that the functional capacity of critical muscles is not compromised upon emergence from aestivation.     

Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species

Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and ¹H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307^T, Halococcus saccharolyticus DSM 5350^T, Halococcus salifodinae DSM 8989^T, Halococcus dombrowskii DSM 14522^T, Halococcus hamelinensis JCM 12892^T, Halococcus qingdaonensis JCM 13587^T), Halorubrum sp. (Hrr. coriense DSM 10284^T, Halorubrum chaoviator DSM 19316^T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189^T, Natronococcus occultus DSM 3396^T) and Halobacterium noricense DSM 9758^T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757^T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.     

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.