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Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that function in posttranscriptional regulation as gene-specific regulators of RNA metabolism in plant organelles. Plant PPR proteins are divided into four classes: P, PLS, E and DYW. The E- and DYW-class proteins are mainly implicated in RNA editing, whereas most of the P-class proteins predominantly participate in RNA cleavage, splicing and stabilization. In contrast, the functions of PLS-class proteins still remain obscure. Here, we report the function of PLS-class PpPPR_31 and PpPPR_9 in Physcomitrella patens. The knockout (KO) mutants of PpPPR_31 and PpPPR_9 exhibited slower protonema growth compared to the wild type. The PpPPR_31 KO mutants showed a considerable reduction in the splicing of nad5 intron 3 and atp9 intron 1. The PpPPR_9 KO mutants displayed severely reduced splicing of cox1 intron 3. An RNA electrophoresis mobility shift assay showed that the recombinant PpPPR_31 protein bound to the 5' region of nad5 exon 4 and the bulged A region in domain VI of atp9 group II intron 1 while the recombinant PpPPR_9 bound to the translated region of ORF622 in cox1 intron 3. These results suggest that a certain set of PLS-class PPR proteins may influence the splicing efficiency of mitochondrial group II introns., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Two-component systems (TCSs) are signal transduction mechanisms for responding to various environmental stimuli. In angiosperms, TCSs involved in phytohormone signaling have been intensively studied, whereas there are only a few reports on TCSs in basal land plants. The moss Physcomitrella patens possesses several histidine kinases (HKs) that are lacking in seed plant genomes. Here, we studied two of these unique HKs, PAS-histidine kinase 1 (PHK1) and its paralog PHK2, both of which have PAS (Per-Arnt-Sim) domains, which are known to show versatile functions such as sensing light or molecular oxygen. We found homologs of PHK1 and PHK2 only in early diverged clades such as bryophytes and lycophytes, but not in seed plants. The PAS sequences of PHK1 and PHK2 are more similar to a subset of bacterial PAS sequences than to any angiosperm PAS sequences. Gene disruption lines that lack either PHK1 or PHK2 or both formed gametophores earlier than the wild-type, and consistently, more caulonema side branches were induced in response to light in the disruption lines. Therefore, PHK1 and PHK2 delay the timing of gametophore development, probably by suppressing light-induced caulonema branching. This study provides new insights into the evolution of TCSs in plants.

In land plant organelles, many transcripts are modified by cytidine to uridine RNA editing. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins via their sequence-specific RNA-binding motifs. In the moss Physcomitrella patens, all PPR editing factors have C-terminal E and DYW domains. To examine the contribution of E and DYW domains in RNA editing, we performed a complementation assay using mutated PpPPR_56 and PpPPR_71, which are responsible for mitochondrial editing sites. This assay showed that both E and DYW domains are required for RNA editing at the target sites, and that the conserved zinc-binding signature and the terminal triplet of the DYW domain are essential for editing. In addition, DYW domain-swapping experiments demonstrated that DYW domains are functionally different between PpPPR_56 and other mitochondrial PPR editing factors, and that residues 37-42 of the DYW domain are involved in site-specific editing. Our results suggest that PPR-DYW proteins specifically recognize their target editing sites via PPR motifs and the DYW domain.

Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcβ1Cer, Manβ4Glcβ1Cer, GlcNAcβ3Manβ4Glcβ1Cer, Galβ3Manβ4Glcβ1Cer, GalNAcα4Galβ3Manβ4Glcβ1Cer, GlcNAcβ3Galβ3Manβ4Glcβ1Cer, Galα4Galβ3Manβ4Glcβ1Cer and (GalNAcα4)1-4 GalNAcα4Galβ3Manβ4Glcβ1Cer. Lin-ear elongation of α4-GalNAc was observed at the non-reducing end of Galβ3Manβ4Glcβ1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcβ3Manβ4Glcβ1Cer, a characteristic GSL-glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis.

The moss Physcomitrella patens has 11 RNA editing sites in mitochondrial transcripts. We previously identified six DYW subclass pentatricopeptide repeat (PPR) proteins as RNA editing factors for nine out of 11 sites. In this study, we identified two novel DYW subclass PPR proteins, PpPPR_65 and PpPPR_98, as RNA editing factors. Disruption of the PpPPR_65 gene resulted in a complete loss of RNA editing at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirm this result, we further generated PpPPR_65 knockdown (KD) mutants by an inducible RNA interference (RNAi) system. The generated RNAi lines displayed reduced levels of RNA editing at both ccmFc-C103 and ccmFc-C122 sites. Next, we characterized the function of PpPPR_98 by constructing a KD mutant of PpPPR_98 expression. The KD mutant showed a 30% reduction in the level of atp9-C92 editing. When PpPPR_98 cDNA was introduced into the KD mutant, RNA editing levels were restored to the wild-type level. This indicates that PpPPR_98 is an editing factor for the atp9-C92 site. The recombinant PpPPR_98 protein bound to the upstream sequence of the editing site that was created by splicing of atp9 transcript. This suggests that atp9 RNA editing occurs after splicing of atp9 transcript. Our present and previous data provide the first evidence that all 11 known editing events require at least eight DYW subclass PPR proteins in the moss mitochondria.

Plastid division is controlled by numerous nuclear genes. Arabidopsis thaliana CRUMPLED LEAF (AtCRL) is a plastid division-related gene, and the crl mutant exhibits a dwarf phenotype with abnormal cell division and a significant reduction in plastid numbers. However, the function of AtCRL is not fully understood. Here, we identified and characterized two AtCRL homologs, PpCRL1 and PpCRL2, in the moss Physcomitrella patens. PpCRL1 and PpCRL2 shared 77% amino acid identity with each other and 47% identity with AtCRL. Single PpCRL1 or -2 gene knockout (KO) mutants could not be distinguished from the wild-type mosses, but PpCRL1 and -2 double KO mutants displayed growth retardation of protonemata and gametophores and harbored approximately 10 large chloroplasts per cell. This indicates that PpCRL1 and PpCRL2 have redundant functions in chloroplast division and plant growth. Unlike the A. thaliana crl mutants, however, the PpCRL double KO mutants did not display abnormal orientation of the cell division plane. Complementation experiments showed that AtCRL partially rescued the defects in chloroplast size and number of the PpCRL double KO mutant. This suggests that PpCRL has a similar, but not identical, function to AtCRL. Time-lapse microscopic observation of the double PpCRL KO mutants revealed that some dumbbell-shaped chloroplasts failed to complete division at the late stage of plastid division; enlarged chloroplasts were thus generated. This strongly suggests that PpCRLs are involved in the complete separation of dividing chloroplasts.

The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and 'ii'(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.

Background: Bosentan is an oral dual endothelin receptor antagonist, which has been shown to be efficacious for preventing new digital ulcers in patients with systemic sclerosis (SSc) in two high-quality randomized controlled trials. However, its efficacy for nondigital ulcers in SSc remains unknown., Objectives: To evaluate the efficacy of bosentan on nondigital ulcers in patients with SSc., Methods: Bosentan was administered to five patients with SSc with pulmonary arterial hypertension, who also had nondigital ulcers refractory to conventional treatments. The efficacy of bosentan on nondigital ulcers and its association with clinical features of ulcers were analysed., Results: The nondigital ulcers refractory to conventional treatments were significantly improved by the administration of bosentan in cases surrounded with severe cyanosis. In contrast, nondigital ulcers without cyanosis were still refractory to bosentan therapy., Conclusions: Bosentan may be efficacious for accelerating the healing of nondigital ulcers with severe cyanosis, suggesting that nondigital ulcers caused by severely impaired peripheral circulation are highly responsive to this treatment., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

In plant organelles, RNA editing frequently occurs in many transcripts, but little is known about its molecular mechanism. Eleven RNA editing sites are present in the moss Physcomitrella patens mitochondria. Recently PpPPR_71, one member of 10 DYW-subclass pentatricopeptide repeat (PPR-DYW) proteins, has been identified as a site-specific recognition factor for RNA editing in the mitochondrial transcript. In this study, we disrupted three genes encoding a PPR-DYW protein-PpPPR_56, PpPPR_77, and PpPPR_91-to investigate whether they are involved in RNA editing. Transient expression of an N-terminal amino acid sequence fused to the green fluorescent protein (GFP) suggests that the three PPR-DYW proteins are targeted to mitochondria. Disruption of each gene by homologous recombination revealed that PpPPR_56 was involved in RNA editing at the nad3 and nad4 sites, PpPPR_77 at the cox2 and cox3 sites, and PpPPR_91 at the nad5-2 site in the mitochondrial transcripts. The nucleotide sequences surrounding the two editing sites targeted by a single PPR-DYW protein share 42 to 56% of their identities. Thus, moss PPR-DYW proteins seem to be site-specific factors for RNA editing in mitochondrial transcripts.

Trehalose dimycolate (TDM) is a major surface-exposed mycolyl glycolipid that contributes to the hydrophobic cell wall architecture of mycobacteria. Nevertheless, because of its potent adjuvant functions, pathogenic mycobacteria appear to have evolved an evasive maneuver to down-regulate TDM expression within the host. We have shown previously that Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av), replace TDM with glucose monomycolate (GMM) by borrowing host-derived glucose as an alternative substrate for the FbpA mycolyltransferase. Mycobacterium leprae (M.le), the causative microorganism of human leprosy, is also known to down-regulate TDM expression in infected tissues, but the function of its mycolyltransferases has been poorly analysed. We found that, unlike M.tb and M.av FbpA enzymes, M.av FbpA was unexpectedly inefficient in transferring alpha-branched mycolates, resulting in impaired production of both TDM and GMM. Molecular modelling and mutational analysis indicated that a bulky side chain of leucine at position 130 of M.le FbpA obstructed the intramolecular tunnel that was proposed to accommodate the alpha-branch portion of the substrates. Notably, even after a highly reductive evolution, M.le FbpA remained functional in terms of transferring unbranched acyl chains, suggesting a role that is distinct from that as a mycolyltransferase.

Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1.

We isolated two major zwitterionic glycosphingolipids (ZGLs) from the phytopathogenic filamentous fungus Trichoderma viride. Structural analyses showed that the ZGLs (designated Tv-ZGL2 and Tv-ZGL3) were the same as the glycosphingolipids ZGL2 and ZGL4 from Acremonium sp., which are described in our previous paper. ZGLs have the following structure: Man(alpha1-6)GlcN(alpha1-2)Ins-P-Cer (Tv-ZGL2) and phosphocholine (PC)-->6Man(alpha1-6)GlcN(alpha1-2)Ins-P-Cer (Tv-ZGL3). To determine whether these ZGLs have functional roles in plant-fungus interaction, we tested to determine whether they would induce defense responses in cultured rice cells. We found that T. viride's ZGLs elicited expression of the PAL and PBZ1 genes, both of which are associated with pathogen resistance. Tv-ZGL2 induced cell death at a moderate rate. Tv-ZGL3, which contains a PC moiety, induced a high level of cell death in rice cells.

The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.

RNA editing in land plant organelles is a process primarily involving the conversion of cytidine to uridine in pre-mRNAs. The process is required for gene expression in plant organelles, because this conversion alters the encoded amino acid residues and improves the sequence identity to homologous proteins. A recent study uncovered that proteins encoded in the nuclear genome are essential for editing site recognition in chloroplasts; the mechanisms by which this recognition occurs remain unclear. To understand these mechanisms, we determined the genomic and cDNA sequences of moss Takakia lepidozioides chloroplast genes, then computationally analyzed the sequences within -30 to +10 nucleotides of RNA editing sites (neighbor sequences) likely to be recognized by trans-factors. As the T. lepidozioides chloroplast has many RNA editing sites, the analysis of these sequences provides a unique opportunity to perform statistical analyses of chloroplast RNA editing sites. We divided the 302 obtained neighbor sequences into eight groups based on sequence similarity to identify group-specific patterns. The patterns were then applied to predict novel RNA editing sites in T. lepidozioides transcripts; approximately 60% of these predicted sites are true editing sites. The success of this prediction algorithm suggests that the obtained patterns are indicative of key sites recognized by trans-factors around editing sites of T. lepidozioides chloroplast genes.

Pentatricopeptide repeat (PPR) proteins form a huge family in plants (450 members in Arabidopsis and 477 in rice) defined by tandem repetitions of characteristic sequence motifs. Some of these proteins have been shown to play a role in posttranscriptional processes within organelles, and they are thought to be sequence-specific RNA-binding proteins. The origins of this family are obscure as they are lacking from almost all prokaryotes, and the spectacular expansion of the family in land plants is equally enigmatic. In this study, we investigate the growth of the family in plants by undertaking a genome-wide identification and comparison of the PPR genes of 3 organisms: the flowering plants Arabidopsis thaliana and Oryza sativa and the moss Physcomitrella patens. A large majority of the PPR genes in each of the flowering plants are intron less. In contrast, most of the 103 PPR genes in Physcomitrella are intron rich. A phylogenetic comparison of the PPR genes in all 3 species shows similarities between the intron-rich PPR genes in Physcomitrella and the few intron-rich PPR genes in higher plants. Intron-poor PPR genes in all 3 species also display a bias toward a position of their introns at their 5' ends. These results provide compelling evidence that one or more waves of retrotransposition were responsible for the expansion of the PPR gene family in flowering plants. The differing numbers of PPR proteins are highly correlated with differences in organellar RNA editing between the 3 species.

Introduction: There have been limited multiregional studies in Asia examining the parameters of men's general and sexual health and quality of life in the general population vs. those in clinical cohorts of patients with erectile dysfunction (ED)., Aims: The aims of the Asian Men's Attitudes to Life Events and Sexuality (Asian MALES) study were to investigate the prevalence of ED, associated health conditions, and ED treatment-seeking patterns in the general male population in five regions of Asia (China, Japan, Korea, Malaysia, and Taiwan)., Main Outcome Measure: Standardized questionnaire previously used in a similar multiregional study and modified to ensure culturally appropriate content for Asia., Methods: Phase I of the study involved 10,934 adult men, aged 20-75 years, who were interviewed using the standardized questionnaire. Phase II of the study involved men with self-reported ED recruited from Phase I and via physician referral, invitations in general practitioner offices, and street interception (total Phase II sample, N = 1,209)., Results: The overall prevalence of self-reported ED in the Phase I study population was 6.4%. ED prevalence varied by region and significantly increased with age (P < 0.01). Men with ED reported significantly greater rates of comorbid illness (P < 0.0001) and a reduced quality of life (P = 0.0001), compared with men without ED. Phase II of the study revealed that fewer than half of men with self-reported ED had sought treatment for their problem. Men were more likely to seek help for erection difficulties from Western doctors than from traditional medicine practitioners (P = 0.0001). A man's partner/spouse was the most common influencer of treatment seeking in all regions except Malaysia., Conclusion: The findings confirm those of existing research on ED in both Asian and non-Asian males: ED is a prevalent condition; the prevalence of ED increases with age and is strongly associated with comorbid conditions; and the majority of men have never sought treatment for their condition. This study highlights a substantial need for the evaluation and treatment of ED in Asian men.

Small, regulatory, non-coding RNA (ncRNA) is involved in various cell functions in both prokaryotes and eukaryotes. However, information on ncRNA in cyanobacteria is still scarce. We studied ncRNA genes by computational screening to compare the intergenic regions of the Synechococcus elongatus PCC 6301 genome with the genomes of three freshwater cyanobacteria. We identified an ncRNA gene in S. elongatus, which has been previously described as yfr1 in marine cyanobacteria. The S. elongatus yfr1 gene is 65 nucleotides long and is positioned between guaB and trxA. We found a high conservation of the yfr1 gene in most cyanobacterial lineages. A yfr1-deficient mutant showed reduced growth under various stress conditions, e.g. oxidative stress and high salt stress conditions, and showed unusual accumulation of sbtA mRNA. A gel shift assay demonstrated interaction of the Yfr1 RNA with sbtA mRNA in vitro. This suggests that the sbtA transcript is a target RNA for the Yfr1 RNA.

RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

Burn injury often is associated with the abnormal lipid metabolism, including hyperlipidemia, desensitization to lipolytic responses to catecholamines, and reduction in the size of the white adipose tissue. Understanding the biological mechanisms for the decrease in fat mass despite desensitization to catecholamines is important both for the study of lipid metabolism and for the study of its relationship to concomitant insulin resistance. Using epididymal adipose tissue from adult male Sprague-Dawley rats after burn injury (n = 102) or sham-burn injury (n = 102), we tested the hypothesis that a whole-body burn injury causes apoptosis in that tissue. At 1, 3, and 7 days after 40% to 50% body burn injury to the rat, epidydimal adipose tissue was harvested and studied for apoptotic changes and lipolytic properties. For apoptosis, paraformaldehyde-fixed tissue sections were analyzed by in situ TdT-mediated dUTP-X nick-end labeling (TUNEL) staining, and tissue homogenates were also analyzed for DNA fragmentation by enzyme-linked immunoassay and ligation-mediated polymerase chain reaction ladder assay. Isolated adipocytes were stimulated with isoprotenerol, and glycerol production was measured as a reflector of effectiveness of lipolysis. Epididymal adipose tissue showed increased apoptosis manifested by the positive TUNEL staining and increased DNA fragmentation by enzyme-linked immunoassay at day 3 and 7 after burn injury. The DNA fragmentation was confirmed further by the ligation-mediated polymerase chain reaction ladder assay. This elevated DNA fragmentation persisted in the burned animals from day 3 until day 7 after burn injury, the end of observation period. Increase in apoptosis was correlated with decrease in DNA content and tissue weight in the epidydimis. At the functional level, a significant decrease in isoproterenol-induced lipolytic activity (glycerol production) was observed to almost 50% of control level at day 3 and 7 but was not decreased at day 1. Apoptosis of adipocytes may play a role in the altered lipid metabolism, including hyperlipidemia observed in burned subjects.

The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells.

Opinions on the basal relationship of land plants vary considerably and no phylogenetic tree with significant statistical support has been obtained. Here, we report phylogenetic analyses using 51 genes from the entire chloroplast genome sequences of 20 representative green plant species. The analyses, using translated amino acid sequences, indicated that extant bryophytes (mosses, liverworts, and hornworts) form a monophyletic group with high statistical confidence and that extant bryophytes are likely sisters to extant vascular plants, although the support for monophyletic vascular plants was not strong. Analyses at the nucleotide level could not resolve the basal relationship with statistical confidence. Bryophyte monophyly inferred using amino acid sequences has a good statistical foundation and is not rejected statistically by other data sets. We propose bryophyte monophyly as the currently best hypothesis.

The complete chloroplast DNA sequence (122 890 bp) of the moss Physcomitrella patens has been determined. The genome contains 83 protein, 31 tRNA and four rRNA genes, and a pseudogene. Four protein genes (rpoA, cysA, cysT and ccsA) found in the liverwort Marchantia polymorpha and the hornwort Anthoceros formosae are absent from P.patens. The overall structure of P.patens chloroplast DNA (cpDNA) differs substantially from that of liverwort and hornwort. Compared with its close relatives, a 71 kb region from petD to rpoB of P.patens is inverted. To investigate whether this large inversion and the loss of rpoA usually occur in moss plants, we analyzed amplified cpDNA fragments from four moss species. Our data indicate that the large inversion occurs only in P.patens, whereas the loss of the rpoA gene occurs in all mosses. Moreover, we have isolated and characterized the nuclear rpoA gene encoding the alpha subunit of RNA polymerase (RNAP) from P.patens and examined its subcellular localization. When fused to green fluorescent protein, RpoA was observed in the chloroplasts of live moss protonemata cells. This indicates that chloroplast RNAP is encoded separately by chloroplast and nuclear genomes in the moss. These data provide new insights into the regulation and evolution of chloroplast transcription.

The plastid genome of higher plants includes about 120 genes. We adopted genomic array technologies to the tobacco plastid genome. A microarray was constructed, consisting of 220 DNA fragments that cover the whole genome sequence. Each DNA fragment corresponds to a single known gene or an intergenic region. We evaluated reliability of this microarray by comparing the plastid RNA level in light- or dark-grown tobacco seedlings. The transcripts encoding photosynthetic subunits increased significantly in light-grown tissues as expected. Furthermore, we found unexpected signals in several intergenic regions, suggesting the existence of novel transcripts in tobacco plastids.

The effect of an amino acid mixture on the recovery from muscle fatigue after eccentric exercise (ECEX) training was examined in twenty-two male college students. The administration of 5.6 g of the amino acid mixture twice daily resulted faster recovery of muscle strength than that with a placebo. The oral administration of the amino acid mixture was proved to effective for muscle strength recovery after the eccentric exercise.

Abstract Two osteoarthritis patients had osteochondromatosis in the iliopectineal bursa which communicated with the hip joint space. They received surgical resection of the cystic mass and total hip arthroplasty and had good clinical outcomes. The authors consider that these patients' osteochondromatosis was a secondary development on the synovium of the iliopectineal bursa due to chronic inflammation caused by osteoarthritis.

Individual amino acid supplementation affects various types of athletic performance. However, little information on combinations of amino acids is currently available. This study evaluated an amino acid mixture containing L-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine to 3.6 g of total amino acids per dose. Twenty-three rugby players were given 3.6 g, twice, daily of the amino acid mixture for 90 days (June-August 1994) and blood samples were collected for analyses in September 1993, March 1994, September 1994, and September 1995. After 90 days of supplementation, almost all of the athletes reported improvement in vigor and earlier recovery from fatigue. Significant increases (P<0.05) were observed in hemoglobin, RBC count, hematocrit, and serum iron by amino acid supplementation. Significant increases (P<0.05) were also noted in total cholesterol and low-density lipoprotein along with decreased (P<0.05) alkaline phosphatase. All values reverted to original levels when measured after one year of continued training without supplementation.

Previous studies have shown that a mixture of amino acids, consisting of 9 essential amino acids and 3 non-essential amino acids was effective in facilitating muscle recovery from athletic activities. In this study, the objective was to determine whether this amino acid mixture improved the physical condition and associated blood parameters of athletes in training when administered for a prolonged period. Thirteen college middle- and long-distance runners were placed in a 6-month experiment and received the amino acid mixture at the dose of 2.2 g/day for one month, 4.4 g/day for one month, and 6.6 g/day for one month with washout periods between test periods. The physical condition was scored and blood samples were collected before and after each test period. When the subjects received 2.2 g of the amino acid mixture three times a day, the physical condition was significantly improved along with increases in red blood cell count, hemoglobin, hematocrit, serum albumin, and fasting glucose, and a decrease in creatine phophokinase (p<0.05), suggesting increased hematopoiesis and glycogenesis, and rapid alleviation of muscle inflammation by the amino acid mixture.

We have reported the existence of a triphosphonoglycosphingolipid, EGL-I, in the eggs of a sea gastropod, Aplysia kurodai [Yamada, S., Araki, S., Abe, S., Kon, K., Ando, S., and Satake, M. (1995) J. Biochem. 117, 794-799]. We have now isolated a novel glycosphingolipid, named EGL-II, from the eggs of Aplysia. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and proton magnetic resonance spectrometry, its structure was revealed to be as follows: Galalpha1-->3(GlcNAcalpha1-->2)Galalpha1-->3(3-O-MeGalalpha1-->2)Galalpha1-->3[6'-O-(2-aminoethylphosphonyl)Galalpha1-->2](2-aminoethylphosphonyl-->6)Galbeta1-->4(2-aminoethylphosphonyl-->6)Glcbeta1-->1ceramide. The major aliphatic components of the ceramide are palmitic acid, stearic acid, and anteisononadeca-4-sphingenine.

N2733, 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride, was examined for its effect on TNF-alpha production by human myeloid THP-1 cells stimulated with lipopolysaccharide (LPS). N2733 inhibited LPS-induced release of TNF-alpha from THP-1 cells with an IC50 of 11 microM. N2733 did not affect the cell viability at the concentration of 50 microM or 100 microM. This indicates that N2733 is a potent inhibitor for TNF-alpha production without severe cytotoxicity. N2733 was also studied in two murine endotoxin shock models induced with LPS. One model was DBA/2 mice injected with LPS (5.6 mg/kg, i.v.), which increased the serum level of TNF-alpha within 1 hr. Treatment of these mice with N2733 (100 mg/kg x 2, i.p.) decreased the serum level of TNF-alpha significantly. Another model was DBA/2 mice induced with LPS (30 mg/kg, i.v.), which reduced the survival rate to 30% during 7 days. Administrations of 30 mg/kg and 100 mg/kg N2733 (i.v.) restored the survival rates to 60% and 90% respectively. Our data demonstrate that N2733 inhibits LPS-induced TNF-alpha production, and this response is associated with an improvement in the survival rate of endotoxemic mice.

The outcome in 3225 patients with hepatocellular carcinoma (HCC) was studied in groups with equivalent prognosis treated with resection, transcatheter arterial embolization (TAE), and percutaneous transhepatic ethanol injection (PEI). Significant factors for better clinical background included a tumor diameter of < or = 30 mm, tumor number < or = 3 and (clinical) Stage I. In patients with Stage I disease having tumors of < or = 30 mm and < or = 3 in number, survival afer resection and PEI did not differ, while survival after TAE was significantly worse. In those patients with Stage II disease, survival after PEI was significantly better than after resection or TAE. In patients with Stage I or II disease having tumors > or = 31 in size and < or = 3 number, survival after resection was significantly better than after TAE. In patients with Stage I disease having tumors of > or = 31 mm and > or = 4 in number, survival after resection was significantly better than after TAE. Our conclusions are as follows. Firstly, resection or PEI is recommended for patients with Stage I disease having < or = 3 tumors all < or = 30 mm in size. Secondly, PEI is recommended for patients with Stage II disease having < or = 3 tumors all < or = 30 mm in size. Thirdly, for patients with Stage I disease having tumors 31 mm or larger in size, whatever the number of tumors, resection should be selected rather than TAE.

We analyzed the clinical characteristics and survival of 185 patients with hepatitis B virus-related hepatocellular carcinoma (HBV group) and 1033 with hepatitis C virus-related hepatocellular carcinoma (HCV group) by multi center study. The patients in the HBV group (mean age 52.1 yr) were about 10 years younger than those in the HCV group (mean age 62.9 yr). Liver function, as measured by indocyanine green retention at 15 min, was better in the HBV group (17.5%) than in the HCV group (25.4%). A higher proportion of the HBV group (55%) than the HCV group (44%) had clinical stage I, T-factor differed significantly between the groups: 53% of the HBV group were T3-4 compared with 41% of the HCV group. Furthermore, a higher proportion of the HBV group were graded 2-3 for tumor thrombus in the portal vein (20.3%) and had poorly differentiated hepatocellular carcinoma (7%) compared with the HCV group (7.1% and 5% respectively). Univariate analysis identified poor prognostic factors for hepatocellular carcinoma as HBV, age < or = 50 yr, clinical stage II-III, a high AFP level, higher number of tumors, larger tumor size, tumor thrombus in the portal vein 2-3 and in the hepatic vein 2-3. On multivariate analysis, poor prognostic factors were a high AFP level, higher number of tumors, tumor thrombus in the portal vein 2-3 and in the hepatic vein 2-3, but not HBV, age, clinical stage or tumor size. These results suggest that HBV itself is not a stronger prognostic factor than HCV.

A 2.0-kbp Pst I DNA fragment of the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 genome contains two open reading frames (ORFs). The first ORF of 100 codons potentially encodes a polypeptide having 47% amino acid identity to Escherichia coli ribosomal protein S14, suggesting it as a ribosomal protein S14 gene (rps14). The second ORF of 351 codons is located 81 bp downstream of rps14 and its deduced amino acid sequence is in part similar to that of the Salmonella typhimurium oligopeptide permease membrane protein OppC. Northern blot analysis showed that rps14 is expressed as a 0.48-kb transcript whereas no transcript was detected from ORF351. Pulsed-field electrophoresis and blot hybridization analysis revealed that rps14 is a single-copy gene and is found within a 165-kbp region located upstream of rrnA on the circular genome.

We have previously characterized a tobacco cDNA encoding a novel type RNA-binding protein (RZ-1), which contains a zinc finger motif in addition to a consensus sequence-type RNA-binding domain and is localized in the nucleus. Here we isolated its genomic clone from a Nicotiana sylvestris genomic library. Southern blot analysis suggested that RZ-1 is coded for by a single locus per haploid genome. Comparison of the cDNA and genomic sequences indicated that the RZ-1 gene contains two introns, one in the coding region and another in the 3'-untranslated region. RT-PCR and ribonuclease protection analyses showed that splicing of RZ-1 pre-mRNA occurs efficiently. The RZ-1 protein is actively synthesized in rapidly dividing tobacco cells, as demonstrated by immunoblot analysis.

A part of the tRNA(Leu)(UAA) gene containing a 240-nucleotide group I intron was amplified by PCR from cyanobacterium Synechococcus PCC 6301 genomic DNA. The pre-tRNA synthesized from the cloned PCR product was efficiently self-spliced in vitro under physiological conditions. The gene encoding the tRNA(Leu)(UAA), trnL-UAA, was isolated from a Synechococcus PCC 6301 genomic library and the nucleotide sequence of a 2,167-bp portion was determined. The trnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intron and a 50-bp 3' exon. In addition, three open reading frames (ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regions of trnL-UAA. The predicted protein sequence of ORF3, which is located 74-bp upstream from trnL-UAA on the opposite strand, shows 66.2% amino acid identity to that of the Synechocystis PCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).

The nucleotide sequence of a 27,588-bp region of rice mitochondrial DNA was determined. This sequence contains putative genes that encode initiator methionine tRNA (trnfM), subunits III (nad3) and IV (nad4) of the NADH dehydrogenase complex, and ribosomal proteins S3 (rps3), S12 (rps12) and L16 (rpl16). An open reading frame that contains sequences homologous to parts of rps2 and atpA is also present. In addition to these regions, there are many short sequences with homology to fragments of mitochondrial DNAs from rice or other plants. These sequences may be remnants of multiple rearrangements of the genome and their presence seems to explain, in part, the large sizes of the mitochondrial genomes of higher plants.

Twenty indole derivatives were investigated for their ability to reverse multidrug resistance (MDR), for their ability to compete with [3H]azidopine in binding to P-glycoprotein (P-gp) and for their hydrophobicity. Six derivatives almost completely reversed the resistance to vincristine (VCR) in multidrug-resistant KB-C2 cells, and other derivatives partially overcame the resistance. The ability of the derivatives to enhance vincristine cytotoxicity did not significantly correlate with the inhibition of [3H]azidopine binding to P-gp or with their hydrophobicity. However, all the derivatives that inhibited > 50% of the photolabeling completely reversed VCR resistance. The 2-pyridyl group with a basic nitrogen atom attached at position 3 of indole in an appropriate spatial orientation seems to be an important feature for the interaction of the indole derivatives with P-gp. One of the derivatives, 1, which has low cytotoxicity and hydrophobicity, completely reversed the resistance of KB-C2 cells to Adriamycin, actinomycin D and VCR. Our data indicate that MDR-reversing indole derivatives with low cytotoxicity and hydrophobicity exist. These characteristics will surely be profitable for clinical use.

A group of proteins containing a conserved ribonucleoprotein consensus sequence (RNP-CS)-type RNA-binding domain (CS-RBD) of approximately 80 amino acids is present in eukaryotic cells and binds specifically to a wide variety of RNA molecules. We have isolated 12 kDa single-stranded DNA binding proteins from the unicellular cyanobacterium Synechococcus 6301. The amino-terminal sequence was determined and two distinct genomic clones were isolated from a Synechococcus 6301 genomic library. Sequence analysis revealed that two closely related proteins contain a single CS-RBD of 82 amino acids and are named as 12RNP1 and 12RNP2. Both of the CS-RBDs share the highest amino acid identity with those of chloroplast ribonucleoproteins (40-51%). The 12RNP proteins were expressed in Escherichia coli bearing plasmids encoding glutathione S-transferase/12RNP fusion proteins and subjected to in vitro nucleic acid-binding assay. Both 12RNP1 and 12RNP2 bind to RNA homopolymers poly(U) and poly(G), indicating that they might be RNA-binding proteins. This is the first example of such proteins in prokaryotes. The 12RNP1 and 12RNP2 genes are transcribed as monocistronic mRNAs and the steady-state mRNA level of 12RNP1 is over 20-fold than that of 12RNP2. Due to the easiness of genetic manipulations the cyanobacterium will provide an excellent system to analyze the function of not only cyanobacterial but also plant RNA-binding proteins.

Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed.

An HLA-DR restricted T cell clone (26G11) which recognized a lymphoid cell-derived autoantigen associated with DR4 molecule was shown to induce not only autologous but also allogenic DR4+ B cells to produce large amounts of antibodies of the IgG and IgM classes. Using the helper activity of this clone, we investigated the mechanism of anti-DNA antibody production in DR-matched patients with systemic lupus erythematosus (SLE). When cultured with 26G11 cells, B cells from DR-matched normal control subjects produced large amounts of IgM anti-DNA antibody, but did not produce IgG anti-DNA antibody which is thought to have a pathological role in SLE. In contrast, B cells from DR-matched patients with active SLE spontaneously produced a fairly large amount of IgG anti-DNA antibody, and the production was augmented by the T cell clone. Little IgG anti-DNA antibody was produced by the B cells of patients with inactive SLE in either the presence or absence of T cell clone. We next fractionated B cells into low density B (LD-B) and high density B (HD-B) cells by centrifugation on discontinuous Percoll density gradients. IgG anti-DNA antibody was spontaneously produced by LD-B cells of active SLE patients but not by those either of inactive SLE patients or normal controls. On the other hand, although IgG anti-DNA antibody was not spontaneously produced by the HD-B cells of both active and inactive SLE patients, it could easily be induced by their culture with the T cell clone. Our results clearly show the existence of IgG anti-DNA antibody-producing B cells in the peripheral blood of SLE patients irrespective of their disease activity and suggest that autoreactive T cells may play a pathogenic role in SLE through the induction of autoantibody production.

Publication bias is a recognized phenomenon, i.e. studies with statistically significant results are more likely to be published than those finding no difference between the groups studied. Summarized odds ratio calculated from odds ratios of published studies in a meta-analysis may be overestimated because of publication bias. This is a significant problem in research areas involving weak associations between causes and results. The magnitude of publication bias in a given research area cannot be determined directly. The present study enables us to calculate the summarized odds ratio of hypothetical unpublished studies from odds ratios of published studies indirectly, employing a moment method by assuming the natural logarithm value of the odds ratio to be distributed normally. We can then estimate summarized odds ratio in all studies, which include not only those published but also those unpublished. When these studies are homogeneous in quality and their odds ratios homogeneous in quantity, the method can eliminate publication bias.

A series of glycosphingolipids containing 2'-aminoethylphosphoryl(----6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl----6GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc alpha 1-4GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, Gal beta 1-3GalNAc alpha 1-4GalNAc-beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, and GlcNAc beta 1-3Gal-beta 1-3GalNAc alpha 1-4GalNAc beta 1-4 (2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc-beta 1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.

We have previously identified three chloroplast ribonucleoproteins and characterized their cDNAs. Here we present the genomic organization, sequence and expression of one of their genes. The 31 kd ribonucleoprotein (cp31) from tobacco (Nicotiana sylvestris) chloroplasts is coded for by a single-copy nuclear gene. This gene was isolated and its sequence was determined. The gene contains four exons and three introns. The position of its first intron is conserved among the genes for the maize abscisic acid-induced glycine-rich protein, the human hnRNP A1 protein and cp31. The transcription start site was determined to be 168 bp upstream from the translational initiation codon in both leaf and root tissues. No alternatively spliced transcripts was detected, suggesting that a diversity of chloroplast ribonucleoproteins is generated probably by gene amplification rather than alternative splicing.

Two neutral glycosphingolipids having large straight oligosaccharide chains with eight and nine sugars, provisionally named COS and CNS, were isolated and purified from larvae of the green-bottle fly, Lucilia caesar, as the only two remaining unidentified significant neutral glycolipids in this organism. From the results of sugar analysis, permethylation, negative-ion fast atom bombardment mass spectroscopy (FAB-MS), and 1H-NMR studies, the structures of the two glycolipids are proposed to be: COS, GalNAc beta 1-3GlcNAc beta 1-3Gal beta 1-3GalNAc alpha 1-4GalNAc beta 1-4GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer; and CNS, Gal beta 1-3GalNAc beta 1-3GlcNAc beta 1-3Gal beta 1-3GalNAc alpha 1-4GalNAc beta 1-4GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer. The fatty acid and long-chain base compositions of the above glycolipids were very similar, and were dominated by arachidic acid, and tetradeca- and hexadeca-4-sphingenines. The great similarity between the compositions of their ceramide moieties suggests that COS may be a precursor in the glycosylation reaction yielding CNS.

The neutral glycosphingolipids from larvae of the green-bottle fly, Lucilia caesar, were analyzed. Thin-layer chromatograms showed that the larvae contained at least seven major glycolipid components. Of them, the four components with shorter sugar chains consisting of one to four sugar units, were purified by Iatrobeads column chromatography. The structures were identified by partial acid hydrolysis, sequential enzymatic hydrolysis, chromium trioxide oxidation and methylation analysis as: Glc beta (1-1)Cer, Man beta (1-4)Glc beta (1-1)Cer, GlcNAc beta (1-3)Man beta (1-4)Glc beta (1-1)Cer and GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc(1-1)Cer. These glycolipids altogether comprised 38.3% of the total neutral glycolipid fraction. Unlike common vertebrate glycosphingolipids, the larval ones appear to be quite unique in having the sugar structures, -GlcNAc beta (1-3)Man- and -GalNAc beta (1-4)GlcNAc beta (1-3)Man-, which are novel finding in the natural systems examined so far. The ceramide moieties were composed of normal fatty acids (16:0-22:0) with a small amount of branched acids (16 and 18), and tetradeca- and hexadeca-4-sphingenines as the long-chain bases.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.

Three novel glycosphingolipids containing five to seven sugars in their saccharide chains have been isolated from larvae of the green-bottle fly, Lucilia caesar. The structures of these glycolipids were identified by sequential enzymatic degradation with exoglycosidases, methylation analysis, and partial acid hydrolysis to be: Gal-NAc alpha (1-4)GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc-Cer, Gal alpha (1-4)GalNAc alpha (1-4)GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc-Cer, and GlcNAc beta (1-4)Gal alpha (1-4)Gal-NAc alpha (1-4)GalNAc beta (1-4)GlcNAc beta (1-3)Man beta (1-4)Glc-Cer. It should be noted that one N-acetylgalactosamine in their saccharide chains is alpha-glycosidically linked to another N-acetylgalactosamine by a 1-4 bond and that this is the first example of the presence of an alpha (1-4) linked galactosamine unit in nature. The major fatty acids were normal saturated fatty acids with carbon numbers of 16 to 22. The long-chain bases were dominated by C14 and C16-4-sphingenine homologues.

Three globosides were isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii by mild alkaline hydrolysis, acetone precipitation, Unisil column chromatography and preparative thin-layer chromatography. These globosides are unique in their sugar chains, since they contain mannose instead of galactose found so far in all globosides of mammals and other animals examined. The main globoside in the spermatozoa was characterized as Glc-NAcbeta(1 leads to 2)Manbeta(1 leads to 3)Manbeta(1 leads to 4)Glcbeta(1 leads to 1)-ceramide by partial acid hydrolysis, analysis of its anomeric configuration with chromium trioxide, methylation analysis and enzymatic hydrolysis. The globoside contained normal saturated fatty acids ranging in length from C16 to C21, palmitic and stearic acids being predominant. Its main long-chain was octadeca-4-sphingenine.

The spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii, had a complex spectrum of neutral glycosphingolipids. CDS, CTS, CTeS were isolated as minor components (together they accounted for 3% of the total neutral glycolipid fraction), while CMS and the glycolipids (Lipid I, II, and III) with larger saccharide chains that were previously characterized (1--4) occurred as major ones. The chemical structures were determined by employing exoglycosidases, partial acid hydrolysis and permethylation analysis. The proposed structures are as follows: Man alpha(1-3)Man beta(1-4)Glc-Cer for CTS, and Man alpha(1-3)[Xyl beta(1-2)]Man beta(1-4)Glc-Cer for CTeS. CTeS was the first example of a tetraglycosyl ceramide containing xylose. In another fraction, two types of diglycosyl ceramides were obtained as a mixture, and identified as Man beta(1-4)Glc-Cer and Gal beta(1-4)Glc-Cer, respectively. The long-chain base was predominantly composed of sphingosine, and the acyl groups were mainly of palmitate and stearate.