Elastolytic Mechanism of a Novel M23 Metalloprotease Pseudoalterin from Deep-sea Pseudoalteromonas sp. CF6-2: CLEAVING NOT ONLY GLYCYL BONDS IN THE HYDROPHOBIC REGIONS BUT ALSO PEPTIDE BONDS IN THE HYDROPHILIC REGIONS INVOLVED IN CROSS-LINKING*

Elastin is a common insoluble protein that is abundant in marine vertebrates, and for this reason its degradation is important for the recycling of marine nitrogen. It is still unclear how marine elastin is degraded because of the limited study of marine elastases. Here, a novel protease belonging to the M23A subfamily, secreted by Pseudoalteromonas sp. CF6-2 from deep-sea sediment, was purified and characterized, and its elastolytic mechanism was studied. This protease, named pseudoalterin, has low identities (<40%) to the known M23 proteases. Pseudoalterin has a narrow specificity but high activity toward elastin. Analysis of the cleavage sites of pseudoalterin on elastin showed that pseudoalterin cleaves the glycyl bonds in hydrophobic regions and the peptide bonds Ala–Ala, Ala–Lys, and Lys–Ala involved in cross-linking. Two peptic derivatives of desmosine, desmosine-Ala-Ala and desmosine-Ala-Ala-Ala, were detected in the elastin hydrolysate, indicating that pseudoalterin can dissociate cross-linked elastin. These results reveal a new elastolytic mechanism of the M23 protease pseudoalterin, which is different from the reported mechanism where the M23 proteases only cleave glycyl bonds in elastin. Genome analysis suggests that M23 proteases may be popular in deep-sea sediments, implying their important role in elastin degradation. An elastin degradation model of pseudoalterin was proposed, based on these results and scanning electron microscopic analysis of the degradation by pseudoalterin of bovine elastin and cross-linked recombinant tropoelastin. Our results shed light on the mechanism of elastin degradation in deep-sea sediment.     

Sodium currents during differentiation in a human neuroblastoma cell line

The electrophysiological properties of a human neuroblastoma cell line, LA-N-5, were studied with the whole-cell configuration of the patch clamp technique before and after the induction of differentiation by retinoic acid, a vitamin A metabolite. Action potentials could be elicited from current clamped cells before the induction of differentiation, suggesting that some neuroblasts of the developing sympathetic nervous system are excitable. The action potential upstroke was carried by a sodium conductance, which was composed of two types of sodium currents, described by their sensitivity to tetrodotoxin (TTX) as TTX sensitive and TTX resistant. TTX-sensitive and TTX-resistant sodium currents were blocked by nanomolar and micromolar concentrations of TTX, respectively. The voltage sensitivity of activation and inactivation of TTX-resistant sodium current is shifted -10 to -30 mV relative to TTX-sensitive sodium current, suggesting that TTX-resistant sodium current could play a role in the initiation of action potentials. TTX-sensitive current comprised greater than 80% of the total sodium current in undifferentiated LA-N-5 cells. The surface density of total sodium current increased from 24.9 to 57.8 microA/microF after cells were induced to differentiate. The increase in total sodium current density was significant (P less than 0.05). The surface density of TTX-resistant sodium current did not change significantly during differentiation, from which we conclude that an increase in TTX-sensitive sodium current underlies the increase in total current.     

Zur Gedächtnisleistung von Wespen

Zusammenfassung Wespen beachten in Labyrinthen Farbsignale, vor denen sie sich auf eine bestimmte Seite wenden müssen. Sie richten sich auch dann nach den Farbzeichen, wenn sie erst hinter diesen, also zeitlich nach der Farbwahrnehmung, die vom Merkmal angezeigte Seite aufsuchen können. Das Farbsignal wurde während der Dressur von der Entscheidungsstelle weg bis an die für das Gelingen der Orientierungsaufgabe gerade noch erträgliche Grenze vorgerückt.Gedächtnisinhalt scheint nicht eine durch das Farbmerkmal ausgelöste starre Bewegungsfolge zu sein, sondern eine aufgeschobene Orientierungsreaktion.Herrn Prof. Dr. K. v. Frisch zum 70. Geburtstag in Verehrung gewidmet.     

A STUDY OF THE STRUCTURE OF SPLENIC SINUSES IN MAN AND IN THE ALBINO RAT WITH THE LIGHT MICROSCOPE AND THE ELECTRON MICROSCOPE

The splenic sinuses in the spleens of 5 human beings and 7 albino rats have been studied in the light microscope and electron microscope after fixation in Dalton's fluid and Palade's fluid and embedding in n-butyl methacrylate. Splenic sinuses are tortuous vascular channels of large but variable diameter which represent the first venous vessels in the spleen and make up almost the entire red pulp in man and in rats. These vessels are composed of reticulo-endothelial cells flattened to endothelial form and sheathed by a netted reticulum. The luminal surface of the endothelium is made highly irregular by delicate and variable cytoplasmic protrusions, slender corridors separating adjacent endothelial cells, anastomotic openings to other sinuses, bulgings of entire cells, and even thrusts of endothelium spanning the sinai lumen. The supporting reticulum presents a well developed latticed appearance in tangential sections of sinuses, but in most cuts is punctate or linear. The reticulum is composed of strands without limiting membranes, which, in substance, are amorphous and resemble basement membrane. Material identical in appearance to the substance of the reticulum may be present in the endothelium, suggesting that the reticulum is formed by endothelial cells. The endothelium also contains deposits of presumed ferritin and hemosiderin. The extreme luminal bulgings of endothelium suggest production of circulating monocytes or lymphocytes by detachment of endothelial cells. Sinuses are patent and collapsed to varying degrees. Patent sinuses are separated by collapsed sinuses and these collapsed sinuses appear to constitute splenic (Billroth) cords.     

Genomic analysis of the emergence of 20th century epidemic dysentery

Background

Shigella dysenteriae type 1 (Sd1) causes recurrent epidemics of dysentery associated with high mortality in many regions of the world. Sd1 infects humans at very low infectious doses (10 CFU), and treatment is complicated by the rapid emergence of antibiotic resistant Sd1 strains. Sd1 is only detected in the context of human infections, and the circumstances under which epidemics emerge and regress remain unknown.

Results

Phylogenomic analyses of 56 isolates collected worldwide over the past 60 years indicate that the Sd1 clone responsible for the recent pandemics emerged at the turn of the 20^th century, and that the two world wars likely played a pivotal role for its dissemination. Several lineages remain ubiquitous and their phylogeny indicates several recent intercontinental transfers. Our comparative genomics analysis reveals that isolates responsible for separate outbreaks, though closely related to one another, have independently accumulated antibiotic resistance genes, suggesting that there is little or no selection to retain these genes in-between outbreaks. The genomes appear to be subjected to genetic drift that affects a number of functions currently used by diagnostic tools to identify Sd1, which could lead to the potential failure of such tools.

Conclusions

Taken together, the Sd1 population structure and pattern of evolution suggest a recent emergence and a possible human carrier state that could play an important role in the epidemic pattern of infections of this human-specific pathogen. This analysis highlights the important role of whole-genome sequencing in studying pathogens for which epidemiological or laboratory investigations are particularly challenging.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-355) contains supplementary material, which is available to authorized users.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Influence of Interfacial Composition on in Vitro Digestibility of Emulsified Lipids: Potential Mechanism for Chitosan's Ability to Inhibit Fat Digestion

The objective of this study was to investigate the influence of interfacial composition and electrical charge on the in vitro digestion of emulsified fats by pancreatic lipase. An electrostatic layer-by-layer deposition technique was used to prepare corn oil-in-water emulsions (3 wt% oil) that contained droplets coated by (1) lecithin, (2) lecithin–chitosan, or (3) lecithin–chitosan–pectin. Pancreatic lipase (1.6 mg mL⁻¹) and/or bile extract (5.0 mg mL⁻¹) were added to each emulsion, and the particle charge, droplet aggregation, and free fatty acids released were measured. In the presence of bile extract, the amount of fatty acids released per unit amount of emulsion was much lower in the emulsions containing droplets coated by lecithin–chitosan (38 ± 16 μmol mL⁻¹) than those containing droplets coated by lecithin (250 ± 70 μmol mL⁻¹) or lecithin–chitosan–pectin (274 ± 80 μmol mL⁻¹). In addition, there was much more extensive droplet aggregation in the lecithin–chitosan emulsion than in the other two emulsions. We postulated that lipase activity was reduced in the lecithin–chitosan emulsion as a result of the formation of a relatively thick cationic layer around each droplet, as well as the formation of large flocs, which restricted the access of the pancreatic lipase to the lipids within the droplets. Our results also suggest that droplets initially coated by a lecithin–chitosan–pectin layer did not inhibit lipase activity, which may have been because the chitosan–pectin desorbed from the droplet surfaces thereby allowing the enzyme to reach the lipids; however, further work is needed to establish this. This information could be used to create food emulsions with low caloric level, or to optimize diets for individuals with lipid digestion problems.     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.