Resilience to Freezing in the Vegetative Cells of the Microalga Lobosphaera incisa (Trebouxiophyceae,Chlorophyta)

The chlorophyte microalga Lobosphaera incisa was isolated from the snowy slopes of Mt. Tateyama in Japan. This microalga stores exceptionally high amounts of the omega-6 LC-PUFA arachidonic acid in triacylglycerols, and therefore represents a potent photosynthetic source for this essential LC-PUFA. Assuming that freezing tolerance may play a role in adaptation of L. incisa to specific ecological niches, we examined the capability of L. incisa to tolerate extreme sub-zero temperatures. We report here, that the vegetative cells of L. incisa survived freezing at −20°C and −80°C (over 1 month), without cryoprotective agents or prior treatments. Cells successfully recovered upon thawing and proliferated under optimal growth conditions (25°C). However, cells frozen at −80°C showed better recovery and lower cellular ROS generation upon thawing, compared to those preserved at −20°C. Photosynthetic yield of PSII, estimated by F_v/F_m, temporarily decreased at day 1 post freezing and resumed to the original level at day 3. Interestingly, the thawed algal cultures produced a higher level of chlorophylls, exceeding the control culture. The polar metabolome of the vegetative cells comprised a range of compatible solutes, dominated by glutamate, sucrose, and proline. We posit that the presence of endogenous cryoprotectants, a rigid multilayer cell wall, the high LC-PUFA content in membrane lipids, and putative cold-responsive proteins may contribute to the retention of functionality upon recovery from the frozen state, and therefore for the survival under cryospheric conditions. From the applied perspective, this beneficial property holds promise for the cryopreservation of starter cultures for research and commercial purposes.     

Ascertaining the biochemical function of an essential pectin methylesterase in the gut microbe Bacteroides thetaiotaomicron

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II–derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella. Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.     

Diversity of the major capsid genes (g23) of T4-like bacteriophages in the eutrophic Lake Kotokel in East Siberia, Russia

Numerous studies revealed high diversity of T4-like bacteriophages in various environments, but so far, little is known about T4-like virus diversity in freshwater bodies, particularly in eutrophic lakes. The present study was aimed at elucidating molecular diversity of T4-like bacteriophages in eutrophic Lake Kotokel located near Lake Baikal by partial sequencing of the major capsid genes (g23) of T4-like bacteriophages. The majority of g23 fragments from Lake Kotokel were most similar to those from freshwater lakes and paddy fields. Despite the proximity and direct water connection between Lake Kotokel and Lake Baikal, g23 sequence assemblages from two lakes were different. UniFrac analysis showed that uncultured T4-like viruses from Lake Kotokel tended to cluster with those from the distant lake of the same trophic status. This fact suggested that the trophic conditions affected the formation of viral populations, particularly of T4-like viruses, in freshwater environments.     

Two newly identified exons in human GRM1 express a novel splice variant of metabotropic glutamate 1 receptor

To date, five human metabotropic glutamate (mGlu) 1 receptor splice variants (1a, 1b, 1d, 1f, and 1g) have been described, all of which involve alternative C-terminal splicing. mGlu1a receptor contains a long C-terminal domain (341 amino acids), which has been shown to scaffold with several proteins and contribute to the structure of the post-synaptic density. However, several shorter mGlu1 receptor splice variants lack the sequence required for these interactions, and no major functional differences between these short splice variants have been described. By using RT-PCR we have shown that two human melanoma cell lines express both mGlu1a and mGlu1b receptors. In addition, using 3′RACE, we identified three previously unknown mGlu1 receptor mRNAs. Two differ in the length of their 3′ untranslated region (UTR), and encode the same predicted protein as mGlu1g receptor—the shortest of all mGlu1 receptor splice variants. The third mRNA, named mGlu1h, encodes a predicted C-terminal splice variant of 10 additional amino acids. mGlu1h mRNA was observed in two different melanoma cell lines and is overexpressed, compared with melanoma precursor cells, melanocytes. Most importantly, this new splice variant, mGlu1h receptor, is encoded by two previously unidentified exons located within the human GRM1 gene. Additionally, these new exons are found exclusively within the GRM1 genes of higher primates and are highly conserved. Therefore, we hypothesize that mGlu1h receptors play a distinct role in primate glutamatergic signaling.     

Delivery and processing of exogenous double-stranded DNA in mouse CD34 + hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA

We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~ 45% of the cells correspond to CD34 + hematopoietic stem cells. Taking into account that CD34 + stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34 + cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34 + bone marrow cells.     

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.