Microcin E492 Amyloid Formation Is Retarded by Posttranslational Modification

Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.     

Empty class II major histocompatibility complex created by peptide photolysis establishes the role of DM in peptide association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.     

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.     

Effects of temperature on the activity and kinetics of the granulovirus infecting the potato tuber moth Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae)

The granulovirus infecting the potato tuber moth (PoGV) is an important biocontrol agent, especially for managing the pest in rustic potato storerooms. For efficient propagation and use of baculoviruses in pest control strategies, information on the effects of temperature on virus multiplication and activity is crucial. The interaction between PoGV infection and incubation temperature on P. operculella was studied in laboratory bioassays by determining the survival, yield of virus-infected larvae, and the kinetics of virus in vivo increase. Bioassays for LC₅₀ determination by using the egg-dip method were repeated over a period of six years in controlled incubation chambers at six constant temperatures ranging from 16 to 28 °C. Additionally, at temperatures of 17 and 24 °C the kinetics of virus development and increase in larva were assessed in destructive time-series experiments. Three different virus concentrations were used for inoculation. Control mortality was significantly temperature-dependent and was well described by a second-order polynomial function, with lowest mortality at 25 °C (20%) and highest at 16 °C (>60%). LC₅₀ values and slopes of probit-mortality curves were not significantly different between temperatures. Numbers of virus-infected larvae increased exponentially with increasing log-concentration of virus inoculum; an effect of temperature was not evident. Virus granules per larva correlated highly with larval age and larval weight. Multiple regression revealed minor direct effects of temperature on virus numbers; however, with decreasing temperature, larval weight and hence virus numbers increased. As a result, temperature is an important factor to be considered in virus-production facilities. Rearing temperature in virus-production facilities should be maintained at temperatures around 24 °C.     

DNA barcoding of deep‐water notacanthiform fishes (Teleostei,Elopomorpha)

Notacanthiformes Goodrich, 1909, is an order of deep‐sea teleost fishes with a leptocephalus larval stage whose biology and systematics are not widely known. The aim of this work was to apply the DNA barcode standard, based on COI sequence variation, to the delimitation of the species of this order, which is composed by the families Halosauridae (halosaurus) and Notacanthidae (spiny eels). The sequence data used for the analyses were obtained from two sources: 71 samples collected during oceanographic surveys in the North Atlantic Ocean (including the rare species Lipogenys gillii Goode & Bean, 1895) and 95 sequences collected from the BOLD data set. The neighbor‐joining analysis of the barcodes was successful in identifying 96% of the specimens, representing 9 of 16 and 9 of 10 of the recognised species of halosaurus and spiny eels, respectively, including all the current genera. The comparison between the Atlantic and the BOLD data sets also flagged the possibility of occurrences of misidentification and cryptic species. A modern molecular tool like the DNA barcoding supports the previously morphological‐based systematics of the order Notacanthiformes and will provide better access to the taxonomic knowledge of these deep‐water fishes.     

Artificial endosperm of Cleopatra tangerine zygotic embryos: a model for somatic embryo encapsulation

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype-derived plants in a short time. In this work, a range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. Different complexing ions in the form of alginate capsules, zeolite as an ion exchanger and the relationship between capsule-nutrient gel on germination of zygotic embryos, were evaluated. Artificial endosperm assays showed that abscisic acid (1 μM) and mannitol (0.25 M) delayed germination and conversion of zygotic embryos, whereas amino acid supplements (proline, glutamic acid and arginine) accelerated the conversion process. An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

Background  

The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry.     

Lipid characterization of red alga Rhodymenia pseudopalmata (Rhodymeniales,Rhodophyta)

Rhodymenia pseudopalmata is a red alga that grows at the Caribbean coast of the Yucatan Peninsula and has been proven successful in cultivation. In this study we present the lipid composition of R. pseudopalmata collected from wild populations during three different seasons of 2013. Cultured material was also analyzed and compared in order to evaluate its value as feedstock for biotechnological uses. Thin layer chromatography, ¹ H and ¹³ C NMR spectroscopy and gas chromatography‐mass spectrometry were used to assess variations in their lipid composition. Our results showed that the dominant lipid classes were phospholipids both in wild and cultured materials. The phospholipids phosphatidylcholine and phosphatidylglycerol and the glycolipid monogalactosyldiacylglycerol were present in both wild and cultured R. pseudopalmata, whereas the phospholipid lysophosphatidylcholine was only found in wild material. Fatty acids (FAs) showed a high monounsaturated FAs (MUFAs) content with oleic acid (C18:1ω9) as the dominant compound (78 and 94% of the MUFAs for wild and culture materials, respectively). Saturated FAs (SFAs) represented approximately 90% of the total fatty acid content, with palmitic acid (C16:0) reaching approximately 83% of the SFA content. Rhodymenia pseudopalmata was low in polyunsaturated FAs when compared to other red algae. Other compounds such as 1‐heptadecene, 1‐hexadecene, 15‐heptadecenal, 3‐eicosene 6,10,14‐trimethyl‐2‐pentadecanone, phytol, and heptadecane were also found. Lipid composition differences between the wild and cultured algae suggest that light and nutrients can be manipulated to modify lipid composition. Based on its lipid composition and cultivation feasibility, R. pseudopalmata could be a potential source for nutraceuticals and biofuels production.