Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean

Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.     

Development of the deep‐sea viviparous quill worm Leptoecia vivipara (Hyalinoeciinae,Onuphidae, Annelida)

Development of Leptoecia vivipara, a brooding deep‐sea onuphid polychaete with a circum‐Antarctic distribution, was studied using light microscopy, scanning and transmission electron microscopy, and histology. All specimens examined were brooding viviparous females or juveniles; no male gametes were detected. Anterior segments of juvenile and adult worms bore paired compact ovaries with clusters of vitellogenic oocytes. In adults, the mid‐body region formed a chamber containing up to 12 offspring at different stages of development, from oocyte to 13 chaetigers. Mature oocytes freely floated in the coelomic fluid, while embryos and juveniles were enclosed in peritoneal envelopes. Chaetal replacement in juveniles and the morphology of the provisional maxillae are described. Leptoecia vivipara is argued to be a progenetic species with juvenile‐like external morphology and accelerated sexual maturation. These traits may have arisen as adaptations to epibenthic life in a high‐latitude deep‐sea environment affected by seasonal pulses of organic matter.     

Targeted Deletion of FGL2 Leads to Increased Early Viral Replication and Enhanced Adaptive Immunity in a Murine Model of Acute Viral Hepatitis Caused by LCMV WE

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4⁺CD25⁺ Foxp3⁺ regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2^−/−) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2^−/− had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2^+/+). Frequencies of CD8⁺ and CD4⁺ T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2 ^−/− mice. Increased frequencies of CD8⁺ T cells specific for LCMV tetramers GP₃₃ and NP₃₉₆ were detected within the liver of fgl2^−/− mice. Plasma from fgl2^−/− mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2^−/− mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.     

The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.