A new interpretation of eutectic behavior for distearoylphosphatidylcholine-cholesterol binary bilayer membrane

We investigated the thermotropic phase behavior of the distearoylphosphatidylcholine (DSPC)–cholesterol binary bilayer membrane as a function of the cholesterol composition (X_ch) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra, each of which has a single maximum, showed that the wavelength at the maximum intensity (λ_max) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L_β′ or L_β) and the liquid ordered (L_o) phases, ca. 470 nm for the ripple gel (P_β′) phase and ca. 490 nm for the liquid crystalline (L) phase, respectively. The transition temperatures were determined from the temperature dependences of the λ_max and endothermic peaks of the DSC thermograms. Both measurements showed that the pretransition disappears around X_ch = 0.035. The constructed temperature–X_ch phase diagram indicated that the phase behavior of the binary bilayer membrane at X_ch ≤ 0.15 is similar to that of general liquid–solid equilibrium for a binary system where both components are completely miscible in the liquid phase and completely immiscible in the solid phase. It was also revealed that the diagram has two characteristic points: a congruent melting point at X_ch = 0.08 and a peritectic-like point at X_ch = 0.15. The hexagonal lattice model was used for the interpretation of the phase behavior of the binary bilayer membrane. These characteristic compositions well correspond to the bilayer states in each of which cholesterol molecules are regularly distributed in the hexagonal lattice in a different way. That is, each composition of 0.035, 0.08 and 0.15 is nearly equal to that for the binary bilayer membrane which is entirely occupied with units, each composed of a cholesterol and 30 surrounding DSPC molecules within the next-next-next nearest neighbor sites (Unit (1:30): L_β(1:30)), with units, each of a cholesterol and 12 surrounding DSPC molecules within the next nearest sites (Unit (1:12): L_β(1:12)) or with units, each of a cholesterol and 6 surrounding DSPC molecules at the nearest neighbor sites (Unit (1:6): L_β(1:6)), respectively. Therefore, the eutectic behavior observed in the phase diagram was fully explainable in terms of a kind of phase separation between two different types of regions with different types of regular distributions of cholesterol. Further, the L_o phase was found in the higher X_ch-region (X_ch > 0.15). No endothermic peak over the temperature range from 10 to 80 °C at X_ch = 0.50 suggested that the single L_o phase can exist at X_ch > 0.50.     

Microbiological degradation of bile acids. Ring a cleavage and 7α,12α-dehydroxylation of cholic acid by Arthrobacter simplex

The metabolism of cholic acid by Arthrobacter simplex was investigated. This organism effected both ring a cleavage and elimination of the hydroxyl groups at C-7 and C-12 and gave a new metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid, which was isolated and identified through its partial synthesis. A degradative pathway of cholic acid into this metabolite is tentatively proposed, and the possibility that the proposed pathway could be extended to the cholic acid degradation by other microorganisms besides A. simplex is discussed. The possibility that the observed reactions in vitro could occur during the metabolism of bile acids in vivo is considered.     

Membrane association of a new inositol 1,4,5-trisphosphate binding protein, p130 is not dependent on the pleckstrin homology domain.

The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding protein from rat brain and was molecularly cloned to be found similar to phospholipase C-delta 1 (Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. and Hirata, M., 1992. Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol, J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. and Hirata, M., 1996. A new inositol 1,4,5-trisphosphate binding protein similar to phospholipase C-delta 1, Biochem. J. 313, 319-325). The 130-kDa protein and its deleted protein expressed in COS-1 cells were seen in both the membrane and the cytosol fractions. Truncation of 232 residues from the N-terminus, the protein molecule lacking the pleckstrin homology (PH) domain was also localized in the membrane fraction as much as seen with a full-length protein and other deleted proteins, thereby indicating that the PH domain is not primarily involved in the membrane localization. The addition of Mg2+ to homogenates of COS-1 cells caused the translocation of expressed proteins from the cytosol to the membrane fraction, yet further addition of AlF4- which induced the activation of GTP binding proteins did not cause a further translocation. The protein translocated to the membrane by the addition of Mg2+ was hardly extracted with Triton X-100. The inclusion of Ins(1,4,5)P3 or phosphatidylinositol 4,5-bisphosphate in cell homogenates caused the very small reduction in the amounts of membrane-associated proteins expressed by some constructs. These results indicate that (i) the PH domain is not primarily involved in the membrane localization of the 130-kDa protein, (ii) the activation of GTP binding protein does not appear to cause the translocation of the 130-kDa protein, and (iii) intrinsic phosphatidylinositol 4,5-bisphosphate present in the membrane appears to be involved in the membrane association of the 130-kDa protein to a very small extent, probably through the binding site in the PH domain.     

Suppression of macrophage functions impairs skeletal muscle regeneration with severe fibrosis

When damaged, skeletal muscle regenerates. In the early phases of regeneration, inflammatory cells such as neutrophils/granulocytes and macrophages infiltrate damaged muscle tissue. To reveal the roles of macrophages during skeletal muscle regeneration, we injected an antibody, AFS98 that blocks the binding of M-CSF to its receptor into normal mice that received muscle damages. Anti-M-CSF receptor administration suppressed macrophage but not neutrophil infiltration. Histological study indicated that suppression of macrophages function leads to the incomplete muscle regeneration. In addition FACS and immunohistochemical study showed that the acute lack of macrophages delayed proliferation and differentiation of muscle satellite cells in vivo. Furthermore, mice injected with the anti-M-CSF receptor antibody exhibited not only adipogenesis, but also significant collagen deposition, i.e., fibrosis and continuous high expression of connective tissue growth factor. Finally we indicate that these fibrosis markers were strongly enriched in CD90(+) cells that do not include myogenic cells. These results indicate that macrophages directly affect satellite cell proliferation and that a macrophage deficiency severely impairs skeletal muscle regeneration and causes fibrosis.     

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis

The phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is thought to play an important role in cell regulation and signal transduction. However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined, and the phosphorylated forms of endogenous hnRNP K have not been biochemically characterized in detail. In this study, we extensively examined the phosphorylated forms of endogenous hnRNP K by direct protein-chemical characterization using phosphate-affinity electrophoresis followed by immunoblotting and MS. Phosphate-affinity electrophoresis enabled us to sensitively detect and separate the phosphorylated forms of hnRNP K. When we used 2-DE with phosphate-affinity SDS-PAGE in the second dimension, the nuclear fraction contained more than 20 spots of endogenous hnRNP K on the 2-D map. We determined that the multiple forms of hnRNP K were produced mainly by alternative splicing of the single hnRNP K gene and phosphorylation of Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the 2-D gel correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that the multiple forms of hnRNP K were differentially modulated in response to external stimulation with bacterial lipopolysaccharide or serum.     

Characterization of the aryl hydrocarbon receptor repressor gene and association of its Pro185Ala polymorphism with micropenis

BACKGROUND: Genetic background of a fetus contributes to the abnormal development after teratogen exposure. In rodents, in utero exposure to dioxins affects male external genital development. The effects of dioxins are mediated via the aryl hydrocarbon receptor (AHR) and its binding protein, aryl hydrocarbon receptor nuclear translocator (ARNT). In mice, aryl hydrocarbon receptor repressor (AHRR), which binds to ARNT in competition with AHR, plays a critical negative regulatory role in AHR signaling. We attempt to characterize the human AHRR gene and investigate the relationship between AHRR polymorphisms and the incidence of micropenis, a phenotype of undermasculinization. METHODS: We identified and characterized the human homolog of mouse AHRR, taking advantage of the publicly available draft version of the human genome sequence. After detecting an AHRR protein polymorphism by the direct sequencing of pooled human genomic DNA, we evaluated the association between the polymorphism and the presence or absence of micropenis (< -2.5 SD) in patients with micropenis and control subjects. RESULTS: The deduced sequence for human AHRR (715 residues) and the mouse AHRR protein exhibited 81% sequence homology to each other. The Pro185Ala polymorphism was identified between the PAS-A region and the highly conserved arginine/cysteine-rich RCFRCRL/VRC region. Forty-six percent (27/59) of patients with micropenis and 27% (22/80) of the controls were homozygous for 185Pro; this difference in frequencies was significant (P = 0.03). CONCLUSIONS: Homozygosity for the 185Pro allele of AHRR may increase the susceptibility of a fetus to the undermasculinizing effects of dioxin exposure in utero, presumably through the diminished inhibition of AHR-mediated signaling.     

Refinement of the locus for X-linked recessive chondrodysplasia punctata

Although the locus for X-linked recessive chondrodysplasia punctata (CDPX1) has been mapped to the region between PABX and DXS31 (the critical region is about 3 Mb long), the precise location within the critical region has not been determined. In this paper, we describe a boy with a 46,Y,der(X)t(X;Y)(p22.3;q11)mat karyotype and review the genotype-phenotype correlations in three male patients with the combination of apparent lack of clinical features of CDPX1 and a partial deletion of the critical region. The results suggest that the region defined by the two BssHII sites at 3180 and 3570 kb from the Xp telomere may be the target region for the CDPX1 locus.     

A Novel Bipartite Double-Stranded RNA Mycovirus from the White Root Rot Fungus Rosellinia necatrix: Molecular and Biological Characterization,Taxonomic Considerations,and Potential for Biological Control

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5′ (24 nt) and 3′ (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5′ untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3′-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ∼50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.Viruses are found ubiquitously in major groups of filamentous fungi (40), and an increasing number of novel mycoviruses are being reported (3, 36). Mycoviruses with RNA genomes are now classified into 10 families, of which four accommodate double-stranded RNA (dsRNA) viruses and the remaining six comprise single-stranded RNA (ssRNA) viruses (23). While many ssRNA mycoviruses, like hypoviruses and endornaviruses, do not produce particles, dsRNA virus genomes, whether undivided (the family Totiviridae) or divided (11 or 12 segments for the family Reoviridae, 4 segments for the family Chrysoviridae, and 2 segments for the family Partitiviridae), are encapsidated in rigid particles. Most mycoviruses are considered to cause cryptic infections, while some cause phenotypic alterations that include hypovirulence and debilitation. However, the lack of artificial introduction methods for most mycoviruses has greatly hampered progress in exploring mycovirus-host interactions (23, 40). Thus, a virus etiology of altered fungal phenotypes was established only for a limited number of examples, including hypovirus-C. parasitica and mycoreovirus-C. parasitica.White root rot is one of the most devastating diseases of perennial crops worldwide, particularly highly valued fruits in Japan like apple, Japanese pear, and grapevine. The causal fungus, Rosellinia necatrix, is an ascomycete with a wide range of host plants of >197 species spanning 50 families (31) and is difficult to control by conventional methods, as is often the case for soilborne pathogens. Fungicide application, though it may be effective, is labor-intensive and raises environmental concerns, while cultural practices may not be effective. Successful biocontrol of chestnut blight disease in Europe with hypovirulent strains (25, 38) inspired a group of Japanese researchers to conduct an extensive search of a large collection of >1,000 field fungal isolates for mycoviruses that might serve as virocontrol agents. Virocontrol or virological control refers to one form of biological control utilizing viruses that infect organisms pathogenic to useful organisms (23). Approximately 20% of the collected isolates of R. necatrix were found to be dsRNA positive and presumed to be infected by mycoviruses (4, 29). Agarose gel profiles of dsRNAs suggested infections by members in the families Totiviridae, Partitiviridae, Reoviridae, and Chrysoviridae, as well as unassigned viruses (S. Kanematsu and A. Sasaki, unpublished results). Among those dsRNAs, the genomic segments of Mycoreovirus 3 (MyRV3) (55) and Rosellinia necatrix partitivirus 1 (RnPV1) (44) were well characterized. However, many other dsRNAs remain uncharacterized.Artificial virion introduction protocols, which are often unavailable for mycoviruses, have been developed for specific viruses infecting the white root rot fungus. Using a polyethylene glycol (PEG)-mediated method, as established for MyRV1 and MyRV2 infecting C. parasitaca (27, 28), RnPV1 and MyRV3 were shown to be infectious as particles (45, 46). Subsequently, the cause-effect relationship was established: MyRV3 was demonstrated to confer hypovirulence (attenuated virulence) on an isogenic strain and a few vegetatively incompatible virulent strains of R. necatrix (33, 45), while RnPV1 was shown to be associated with symptomless infection. Protoplast fusion is also available for introduction of partitiviruses and uncharacterized viruses into recipient fungal strains that are vegetatively incompatible with virus-containing ones (A. Sasaki, unpublished results). Furthermore, DNA transformation systems are available for foreign gene expression in R. necatrix (33, 42). These technical advances have made the R. necatrix-mycovirus systems attractive for studies of virus-host interactions and virocontrol (23, 37).R. necatrix strain W779 was isolated by Ikeda et al. (29, 30) from soil in Ibaraki Prefecture as a dsRNA-positive strain that had yet to be characterized. Here we describe the purification and biological and molecular properties of a novel virus isolated from W779. Particles ∼50 nm in diameter isolated from strain W779 consist of two dsRNA elements termed dsRNA-1 and -2 of approximately 9 and 7 kbp and a major protein of 135 kDa encoded by one of two open reading frames (ORFs) on dsRNA-1. Importantly, purified virus particles were shown to be infectious and confer hypovirulence on vegetatively incompatible fungal strains. The two dsRNA segments share the conserved terminal sequences at both ends, and both possess extremely long (>1.6 kb) 5′ untranslated regions (UTRs) similar to each other, two ORFs, and relatively short 3′ UTRs. The 3′-proximal ORF of dsRNA-1 encodes an RNA-dependent RNA polymerase (RdRp) showing low levels (22 to 32%) of sequence identity to those of members of the families Totiviridae and Chrysoviridae. A phylogenetic analysis with RdRp sequences revealed that the W779 virus is placed into a separate clade from the recognized virus families. These attributes indicate that dsRNA-1 and -2 represent the genome segments of a novel bipartite virus, designated Rosellinia necatrix megabirnavirus 1 (RnMBV1), with virolocontrol agent potential. We propose the establishment of a new family, Megabirnaviridae, to accommodate RnMBV1 as the type species.