Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.     

Elastolytic activity of Pseudomonas aeruginosa elastase

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.     

Utilization of IncP-1 plasmids as vectors for transposon mutagenesis in myxobacteria

No free plasmid has ever been found in the myxobacterium Myxococcus xanthus, but IncP-1 plasmids are able to integrate into the chromosome of this bacterium. The frequency of integration depends greatly upon the structure of the IncP-1 plasmid used. This property has been used to devise new delivery systems for transposon mutagenesis in this species. Plasmids with low integration efficiencies have proved to be efficient donors of Tn5, while plasmids with very high frequencies of integration could be used directly to generate mutations. These vectors have also proved efficient for Tn5 transfer into other species of myxobacteria, which have not so far been susceptible to genetic analysis.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

Carbonic anhydrase,carbondioxide levels and growth of Nitrosomonas

The ammonia oxidizing bacterium Nitrosomonas europaea was grown either (a) with added bicarbonate in the absence of added CO₂ (bubbled through the culture), (b) with added bicarbonate plus low added CO₂ (0.03% v/v), or (c) without added bicarbonate with high added CO₂ (1% v/v). Cell doubling times of 12 h were observed in 1% cultures; doubling times of 2 to 3-fold longer wre found with 0.03% CO₂ and/or bicarbonate grown cultures. The specific activity of carbonic anhydrase was 40–80% lower in cultures grown on 1% CO₂. These results are compared with those in heterotrophic and photosynthetic microorganisms.Scientific Contribution Number 1241 from the New Hampshire Agricultural Experiment Station     

FURIN and placental syncytialisation: a cautionary tale

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.Subject terms: Proteases, Differentiation     

Hotspots of biotic compositional change in lakes along vast latitudinal transects in northern Canada

Ecotones mark zones of rapid change in ecological structure at various spatial scales. They are believed to be particularly susceptible to shifts caused by environmental transformation, making them key regions for studying the effects of global change. Here, we explored the variation in assemblage structure of aquatic primary producer and consumer communities across latitudinal transects in northeastern North America (Québec‐Labrador) to identify spatial patterns in biodiversity that indicated the location of transition zones across the landscape. We analyzed species richness and the cumulative rate of compositional change (expressed as beta‐diversity) of diatoms and chironomids to detect any abrupt shifts in the rate of spatial taxonomic turnover. We used principal coordinates analysis to estimate community turnover with latitude, then applied piecewise linear regression to assess the position of ecotones. Statistically significant changes in assemblage composition occurred at 52 and 55°N, corresponding to the transition between closed‐ and open‐crown forest, and to the southern onset of the forest tundra (i.e., the forest limit), respectively. The spatial distribution of ecotones was most strongly related to air temperature for chironomids and to vegetation‐ and soil‐related chemical attributes of lake water for diatoms, including dissolved organic carbon content and water color. Lakes at mid‐ to high‐latitudes currently face pressures from rapidly rising temperatures, accompanied by large increases in organic carbon inputs from their catchments, often leading to browning and its associated effects. The biota at the base of food webs in lakes located in transition zones are disproportionately affected by the cascading effects of these multi‐factorial changes, concurrent with pronounced terrestrial greening observed in these regions. Similar patterns of biotic shifts have been observed along alpine aquatic transects, indicating the potential for widespread restructuring of cold, high‐altitude and high‐latitude freshwater communities due to global change.     

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.