Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (1) and PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFalpha-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.     

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.     

Identification and characterization of the ER/lipid droplet-targeting sequence in 17beta-hydroxysteroid dehydrogenase type 11

17β-Hydroxysteroid dehydrogenase type 11 (17βHSD11) is mostly localized on the endoplasmic reticulum (ER) membrane under normal conditions and redistributes to lipid droplets (LDs) when the formation of LDs is induced. In this study, confocal microscopy analyses of the subcellular localization of the mutated 17βHSD11 proteins in cells with or without LDs revealed that both an N-terminal hydrophobic sequence and an adjacent sequence that has a weak homology with the PAT motif are independently necessary and both parts together (28 amino acid residues in total) are sufficient for the dual localization of 17βHSD11. Mutation analyses suggest that the PAT-like motif in 17βHSD11 will not be functionally similar to the canonical PAT motif. Hsp60 was identified as a possibly interacting protein with the PAT-like motif, and biochemical and microscopic analyses suggest that Hsp60 may be partly, but not necessarily involved in recognition of the PAT-like part of the targeting sequence of 17βHSD11.     

Arg97 at the heme-distal side of the isolated heme-bound PAS domain of a heme-based oxygen sensor from Escherichia coli (Ec DOS) plays critical roles in autoxidation and binding to gases, particularly O2

The catalytic activity of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) on cyclic di-GMP is markedly enhanced upon binding of gas molecules, such as O2 and CO, to the heme iron complex in the sensor domain. Arg97 interacts directly with O2 bound to Fe(II) heme in the crystal structure of the isolated heme-bound sensor domain with the PAS structure (Ec DOS-PAS) and may thus be critical in ligand recognition. To establish the specific role of Arg97, we generated Arg97Ala, Arg97Glu, and Arg97Ile mutant Ec DOS-PAS proteins and examined binding to O2, CO, and cyanide, as well as redox potentials. The autoxidation rates of the Arg97Ala and Arg97Glu mutant proteins were up to 2000-fold higher, while the O2 dissociation rate constant for dissociation from the Fe(II)-O2 heme complex of the Arg97Ile mutant was 100-fold higher than that of the wild-type protein. In contrast, the redox potential values of the mutant proteins were only slightly different from that of the wild type (within 10 mV). Accordingly, we propose that Arg97 plays critical roles in recognition of the O2 molecule and redox switching by stabilizing the Fe(II)-O2 complex, thereby anchoring O2 to the heme iron and lowering the autoxidation rate to prevent formation of Fe(III) hemin species not regulated by gas molecules. Arg97 mutations significantly influenced interactions with the internal ligand Met95, during CO binding to the Fe(II) complex. Moreover, the binding behavior of cyanide to the Fe(III) complexes of the Arg mutant proteins was similar to that of O2, which is evident from the Kd values, suggestive of electrostatic interactions between cyanide and Arg97.     

Characterization of microsatellite loci from the Japanese eel Anguilla japonica

The Japenese eel, Anguilla japonica, is generally assumed to be composed of a single population with wide distribution range, and some genetic studies using allozyme or mitochondrial DNA methods supported this population model. However, one genetic study suggested the existence of multiple populations in this species, and thus, more detailed studies on the population structure is needed. Here we characterized a total of 11 microsatellite markers of the Japanese eel. These will serve as powerful tools for detailed population study for the Japanese eel, though two of them showed the significant departure from the Hardy–Weinberg expectations.     

Identification of a novel unconventional myosin from scallop mantle tissue.

We isolated a cDNA encoding a novel unconventional myosin from scallop mantle tissue (scallop unconventional myosin: ScunM) and determined the nucleotide sequence. It comprises 2,739 bp with 5' and 3'-noncoding sequences and has an open reading frame of 2,334 bp that encodes 778 amino acids. While ScunM has a motor domain and a short tail domain without having light chain-binding IQ motifs like myosin XIV, the deduced amino acid sequence exhibits low homology, 30-36%, to known myosins. Phylogenetic analysis of the motor domain suggested that ScunM belongs to a novel unconventional myosin class. ScunM has an insertion of 67 amino acids in the putative actin-binding site (loop2 site). Western blot analysis with an antibody produced against the N-terminal region revealed that ScunM was strongly expressed in the mantle and mantle pallial cell layer of scallop.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: An organ culture model for the urodele retinal regeneration

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented “neuron‐like cells” with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU‐labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron‐specific proteins; HPC‐1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite‐like processes. Numerous lightly pigmented cells with neuron‐like morphology showed HPC‐1 immunoreactivity. Fibroblast growth factor‐2 (FGF‐2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron‐like cells and HPC‐1‐like immunoreactive cells in a dose‐dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue‐intrinsic factors responsible for newt retinal regeneration. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 209–220, 2002; DOI 10.1002/neu.10031     

Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis.

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.