Newly discovered neutral glycosphingolipids in aureobasidin A-resistant zygomycetes: Identification of a novel family of Gala-series glycolipids with core Gal alpha 1-6Gal beta 1-6Gal beta sequences

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.     

Single-nucleotide polymorphism g.1548G > A (E469K) in human ICAM-1 gene affects mRNA splicing pattern and TPA-induced apoptosis

The single-nucleotide polymorphism (SNP) g.1548G > A (E469K) in the human intercellular adhesion molecule-1 (ICAM-1) gene has been suggested to have an association with several types of inflammatory diseases. The polymorphism is located at the three-base position upstream of the splice donor site that produces an alternatively spliced short isoform (ICAM-1-S). To clarify its functional relevance, we studied RNA splicing patterns by comparing cells with different genotype (G/G cells and A/A cells). G/G cells expressed a lower amount of ICAM-1-S mRNA than A/A cells. Since ICAM-1-S has no transmembrane or intracellular domain, ICAM-1 signal transduction and cell-cell contact including Fas-FasL interaction may be influenced. In addition, we studied the effect of this change on FLIP-L mRNA and apoptosis. FLIP-L mRNA tended to decrease, while cell death induced by phorbol 12-myristate 13-acetate was increased. These results suggest that the g.1548 polymorphism modifies inflammatory immune responses by changing cell-cell interaction and then regulating apoptosis.     

alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion.     

VopB1 and VopD1 are essential for translocation of type III secretion system 1 effectors of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a pathogenic Vibrio species that causes food-borne acute gastroenteritis, often related to the consumption of raw or undercooked seafood. Vibrio parahaemolyticus has 2 type III secretion systems (T3SS1 and T3SS2). Here, we demonstrate that VP1657 (VopB1) and VP1656 (VopD1), which share sequence similarity with Pseudomonas genes popB (38%) and popD (36%), respectively, are essential for translocation of T3SS1 effectors into host cells. A VP1680CyaA fusion reporter system was constructed to observe effector translocation. Using this reporter assay we showed that the VopB1 and VopD1 deletion strains were unable to translocate VP1680 to host cell but that the secretion of VP1680 into the culture medium was not affected. VopB1 or VopD1 deletion strains did not enhance cytotoxicity and failed to activate mitogen-activated protein kinases and secretion of interleukin-8, which depend on VP1680. Thus, we conclude that VopB1 and VopD1 are essential components of the translocon. To target VopB1 and VopD1 may have therapeutic potential for the treatment or prevention in V.?parahaemolyticus infection.     

Stem cells in asexual reproduction of Enchytraeus japonensis (Oligochaeta, Annelid): proliferation and migration of neoblasts

Enchytraeus japonensis is a small oligochaete that reproduces mainly asexually by fragmentation (autotomy) and regeneration. As sexual reproduction can also be induced, it is a good animal model for the study of both somatic and germline stem cells. To clarify the features of stem cells in regeneration, we investigated the proliferation and lineage of stem cells in E. japonensis. Neoblasts, which have the morphological characteristics of undifferentiated cells, were found to firmly adhere to the posterior surface of septa in each trunk segment. Also, smaller neoblast‐like cells, which are designated as N‐cells in this study, were located dorsal to the neoblasts on the septa. By conducting 5‐bromo‐2′‐deoxyuridine (BrdU)‐labeling‐experiments, we have shown that neoblasts are slow‐cycling (or quiescent) in intact growing worms, but proliferate rapidly in response to fragmentation. N‐cells proliferate more actively than do neoblasts in intact worms. The results of pulse‐chase experiments indicated that neoblast and N‐cell lineage mesodermal cells that incorporated BrdU early in regeneration migrated toward the autotomized site to form the mesodermal region of the blastema, while the epidermal and intestinal cells also contributed to the blastema locally near the autotomized site. We have also shown that neoblasts have stem cell characteristics by expressing Ej‐vlg2 and by the activity of telomerase during regeneration. Telomerase activity was high in the early stage of regeneration and correlated with the proliferation activity in the neoblast lineage of mesodermal stem cells. Taken together, our results indicate that neoblasts are mesodermal stem cells involved in the regeneration of E. japonensis.     

The TLR4-TRIF pathway protects against H5N1 influenza virus infection

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.     

Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme-substrate complex is a reactive species

Heterotetrameric sarcosine oxidase is a flavoprotein that catalyses the oxidative demethylation of sarcosine. It is thought that the dehydrogenated substrate is the anionic form of sarcosine. To verify this assumption, the rate of flavin-adenine dinucleotide (FAD) reduction (k(red)) was analysed using protiated and deuterated sarcosine (N-methyl-d(3)-Gly) at various pH values using stopped-flow method. By increasing the pH from 6.2 to 9.8, k(red) increased for both substrates and reached a plateau, but the pK(a) value (reflecting the ionization of the enzyme-substrate complex) was 6.8 and 7.1 for protiated and deuterated sarcosine, respectively, and the kinetic isotope effect of k(red) decreased from approximately 19 to 8, indicating deprotonation of the bound sarcosine. The k(red)/K(d) (K(d), sarcosine dissociation constant) increased with increasing pH and reached a plateau. The pK (reflecting the ionization of free enzyme or free sarcosine) was 7.0 for both substrates, suggesting deprotonation of the βLys358 residue, which has a pK(a) of 6.7, as the pK(a) of the free sarcosine amine proton was determined to be approximately 10.1. These results indicate that the amine proton of sarcosine is transferred to the unprotonated Lys residue in the enzyme-substrate complex.     

Comparable ages for the independent origins of electrogenesis in African and South American weakly electric fishes

One of the most remarkable examples of convergent evolution among vertebrates is illustrated by the independent origins of an active electric sense in South American and African weakly electric fishes, the Gymnotiformes and Mormyroidea, respectively. These groups independently evolved similar complex systems for object localization and communication via the generation and reception of weak electric fields. While good estimates of divergence times are critical to understanding the temporal context for the evolution and diversification of these two groups, their respective ages have been difficult to estimate due to the absence of an informative fossil record, use of strict molecular clock models in previous studies, and/or incomplete taxonomic sampling. Here, we examine the timing of the origins of the Gymnotiformes and the Mormyroidea using complete mitogenome sequences and a parametric bayesian method for divergence time reconstruction. Under two different fossil-based calibration methods, we estimated similar ages for the independent origins of the Mormyroidea and Gymnotiformes. Our absolute estimates for the origins of these groups either slightly postdate, or just predate, the final separation of Africa and South America by continental drift. The most recent common ancestor of the Mormyroidea and Gymnotiformes was found to be a non-electrogenic basal teleost living more than 85 millions years earlier. For both electric fish lineages, we also estimated similar intervals (16-19 or 22-26 million years, depending on calibration method) between the appearance of electroreception and the origin of myogenic electric organs, providing rough upper estimates for the time periods during which these complex electric organs evolved de novo from skeletal muscle precursors. The fact that the Gymnotiformes and Mormyroidea are of similar age enhances the comparative value of the weakly electric fish system for investigating pathways to evolutionary novelty, as well as the influences of key innovations in communication on the process of species radiation.