Dinucleosome DNA of human K562 cells: experimental and computational characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.     

Protective effects of farnesol against oral candidiasis in mice

Farnesol is known as a quorum-sensing molecule for Candida albicans and is recognized to play pathogenic roles in Candida infection. To assess the possible role of farnesol in mucosal C. albicans infection, the effects of farnesol treatment against experimental oral candidiasis in mice were examined. Prednisolone-pretreated ICR mice were orally infected with C. albicans and 3, 24 and 30 hr later the animals were orally given farnesol. Forty-eight hr later they were killed for observation. Farnesol treatment in a dose ranging between 1.125 and 9 micromol/mouse showed a protective effect against oral candidiasis in a dose-dependent manner, at least as estimated by symptom scores of tongues. At 9 micromol/mouse it decreased bodyweight loss. Histological studies of 2.25 micromol/mouse farnesol-treated animals indicated that farnesol suppressed mycelial growth of C. albicans on the surface of tongues, but microbiological study did not prevent the change of CFU of C. albicans cells not only on tongues but also in feces, kidneys and livers. These results suggest that farnesol has very characteristic roles in protection against mucosal candidiasis.     

Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8

The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.     

Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.     

Characterization of the N-oligosaccharides attached to the atypical Asn-X-Cys sequence of recombinant human epidermal growth factor receptor

The extracellular domain of human EGF receptor (sEGFR) produced by CHO cells has been used in various biophysical studies to elucidate the molecular mechanism of EGF-induced receptor activation. We have found that the CHO sEGFR contains one oligosaccharide chain attached to an atypical N-glycosylation consensus sequence, Asn(32 )-X( 33 )-Cys(34 ). The oligosaccharide structure at Asn(32 ) is a mixture of the monosialo and asialo forms of a core fucosylated biantennary complex-type oligosaccharide. Deletion of this atypical glycosylation site by replacement of Asn(32 ) with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosylation at Asn(32 ) in CHO sEGFR was incomplete: 20% of Asn(32 ) remained unmodified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn(32 ), which may cause problems in biophysical studies. An attempt to remove the oligosaccharide at Asn(32 ) enzymatically did not succeed under nondenaturing conditions. Therefore, sEGFR with the mutation of Asn(32) -> Lys(32 )is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography.     

Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation

The sphingolipid metabolite sphingosine 1-phosphate (S1P) functions as a lipid mediator and as a key intermediate of the sole sphingolipid to glycerophospholipid metabolic pathway (S1P metabolic pathway). In this pathway, S1P is converted to palmitoyl-CoA through 4 reactions, then incorporated mainly into glycerophospholipids. Although most of the genes responsible for the S1P metabolic pathway have been identified, the gene encoding the trans-2-enoyl-CoA reductase, responsible for the saturation step (conversion of trans-2-hexadecenoyl-CoA to palmitoyl-CoA) remains unidentified. In the present study, we show that TER is the missing gene in mammals using analyses involving yeast cells, deleting the TER homolog TSC13, and TER-knockdown HeLa cells. TER is known to be involved in the production of very long-chain fatty acids (VLCFAs). A significant proportion of the saturated and monounsaturated VLCFAs are used for sphingolipid synthesis. Therefore, TER is involved in both the production of VLCFAs used in the fatty acid moiety of sphingolipids as well as in the degradation of the sphingosine moiety of sphingolipids via S1P.     

Cloning and expression of the exo-beta-D-1,3-glucanase gene (exgS) from Aspergillus saitoi

A gene of exo-1,3-beta-D-glucanase (exgS) was cloned from a koji mold, Aspergillus saitoi, genomic DNA using PCR. The exgS has an ORF comprising 2832 bp, which contains one intron of 45 bp, and encodes 945 amino acids. The deduced amino acid sequences showed that the ExgS has a non-homologous linker region consisting of 180 amino acids, which encompassed highly conserved regions observed in Exg homologues from filamentous fungi. A recombinant protein (ExgS) has been recovered from the cultural filtrate of an Aspergillus oryzae strain that carried an expression vector containing full length of the exgS. The N-terminal amino acid sequences of the recombinant exo-1,3-beta-D-glucanase (ExgS) were identical to that of native ExgS from A. saitoi.     

Functional Analysis of the α-1,3-Glucan Synthase Genes agsA and agsB in Aspergillus nidulans: AgsB Is the Major α-1,3-Glucan Synthase in This Fungus

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and ¹³C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.     

Studies on defense mechanisms against Candida albicans infection in congenitally athymicnude (nu/nu) mice

The defense mechanisms against Candida albicans infection were studied by using a mouse thigh lesion model in congenitally athymic nude (nu/nu) mice and their normal littermates (nu/+). Nu/nu mice were more resistant to C. albicans infection than nu/+ mice judging from the course of the thigh lesion, the results of CFUs (colony-forming units) of C. albicans in the lesion, and histopathological observations. Histopathological and serological studies revealed that granulocytic cellular infiltration was predominant, and there were few indications of development of cell-mediated immunity to protect Candida infection in Candida-infected nu/nu and nu/+ mice. These results confirmed that lower susceptibility of nu/nu mice to C. albicans infection as compared with nu/ + mice was due to accelerated non-specific defense mechanisms in nu/nu mice, and that cell-mediated or humoral immunity played a minor role in the defense against Candida infection in this experimental model.Furthermore, treatment with high titer of rabbit anti-C. albicans serum was effective to control the number of Candida cells in thigh lesions of BALB/c mice.Above experimental results seem to clearly indicate the great variability of defense manifestation according to the experimental model exployed.     

Bacteriophage-induced acidic heteropolysaccharide lyases that convert the acidic heteropolysaccharides of Rhizobium trifolii into oligosaccharide units

Acidic heteropolysaccharide lyases from lysates of phages 4S and BY15 grown on Rhizobium trifolii 4S and R. trifolii 0403, respectively, were used to analyze the capsular and excreted extracellular acidic polysaccharides of R. trifolii 0403. The activities of the enzymes as measured by viscometry were enhanced by the addition of calcium. The oligosaccharide products obtained by depolymerase digestion of the polysaccharides isolated from cells grown on agar plates for 5 days were isolated by gel filtration and had a glycosyl composition of glucose, galactose, glucuronic acid, and alpha-linked 4-deoxy-L-threo-hex-4-enopyranosyluronic acid in an approximate molar ratio of 5:1:1:1. This latter component was identified by 1H-nuclear magnetic resonance spectroscopy and confirmed by UV spectroscopy, ozonolysis, and its reactivity with thiobarbituric acid. The oligosaccharide had glucose as the reducing terminus, 4-deoxy-L-threo-hex-4-enopyranosyluronic acid as the enzymatically generated nonreducing terminus, and galactose as the terminus of the branched chain. The noncarbohydrate components of the oligosaccharides were acetate, ketal-linked pyruvate, and ether-linked 3-hydroxybutyrate. The mode of action of the enzymes was by beta-elimination from a uronic acid residue with concomitant loss of the glycosyl component substituted at C-4. The 235-nm absorbing properties of the resulting terminal unsaturated sugar were used to study the kinetics of depolymerization of the capsular and excreted extracellular acidic polysaccharides, using the enzyme from phage BY15. The two substrates exhibited different kinetics of depolymerization, and the oligosaccharide products differed in the amount of noncarbohydrate substituents, indicating that the acidic capsular and excreted extracellular polysaccharides from 5-day-old cultures of R. trifolii 0403 were different.