CXCL4 is a novel nickel-binding protein and augments nickel allergy.

BACKGROUND: Nickel (Ni) is the most frequent metal allergen and induces a TH1 -dependent type-IV allergy. Although Ni2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. OBJECTIVE: The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. METHODS: BALB/cA mice were sensitized with an i.p. injection of NiCl2 and LPS. Ten days after sensitization, mice were challenged with NiCl2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. RESULTS: Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni2+ . Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. CONCLUSIONS: These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo.

Lack of antibodies against the antigen domain 2 epitope of cytomegalovirus (CMV) glycoprotein B is associated with CMV disease after renal transplantation in recipients having the same glycoprotein H serotypes as their donors.

Cytomegalovirus (CMV) reinfection of seropositive individuals has been associated with adverse outcomes in organ transplantation and is a frequent cause of congenital infection. Previously we demonstrated that mismatching of CMV glycoprotein H (gH) serotypes was associated with CMV disease after renal transplantation. Because the antigen domain 2 (AD2) epitope of glycoprotein B (gB) is conserved among CMV isolates and is one of the known targets of neutralizing antibodies, in this study we investigated whether antibodies against the epitope contribute to protection from CMV reinfection in renal transplantation, irrespective of gH serological matching. For this purpose, the gB and gH serology and clinical outcomes were analyzed retrospectively for 77 transplant recipients in the donor positive/recipient positive setting, who were managed by preemptive strategy. We found that there was a good negative correlation between the numbers of antigenemia-positive cells and the levels of antibodies against gB AD2 in the CMV-gH antibody matched group, but not in the CMV-gH antibody mismatched group. None of the recipients with antibodies against both gB AD2 and strain-specific epitopes of gH have experienced CMV disease during 6 month after transplantation, while 28% of those who lacked either/both antibody response needed preemptive therapy. Because the outcome was statistically significant, antibodies against gB AD2 can be a useful indicator to predict emergence of CMV disease for preemptive therapy, in addition to antibodies against the mismatched gH types.

Comparison of oral falecalcitriol and intravenous calcitriol in hemodialysis patients with secondary hyperparathyroidism: a randomized, crossover trial.

BACKGROUND: Falecalcitriol is a novel vitamin D analog, which has a greater potential to suppress parathyroid hormone (PTH) and a longer half-life. There are few studies to compare clinical effects of oral falecalcitriol treatment with those of intravenous calcitriol treatment. METHODS: Twenty-one patients with moderate to severe SHPT were included in a random 2 x 2 crossover trial with the two vitamin D analogs (12 weeks for each treatment). The primary endpoint measure was a decrease in serum intact PTH (iPTH) level, and the secondary outcome measures included changes in serum calcium (Ca), phosphate (P), and metabolic bone marker levels. RESULTS: Both treatments decreased iPTH and whole PTH (wPTH) levels by similar degrees (iPTH, -200.1 +/- 107.0 with falecalcitriol vs. -200.8 +/- 114.9 pg/ml with calcitriol, p = 0.9895; wPTH, -137.1 +/- 73.1 with falecalcitriol vs. -120.4 +/- 81.1 pg/ml with calcitriol, p = 0.5603). Serum Ca, P, and Ca x P product levels at the end of each treatment were comparable and the frequencies of hypercalcemia and hyperphosphatemia were also similar during each treatment period. Although intravenous calcitriol treatment significantly changed intact osteocalcin and cross-linked N-telopeptide of type I collagen after 12 weeks, oral falecalcitriol treatment did not change any bone metabolic marker level. CONCLUSION: The present study showed that oral falecalcitriol treatment is effective for PTH suppression, and Ca and P metabolism in hemodialysis patients with moderate to severe SHPT, as well as intravenous calcitriol administration.

Changes in levator ani anatomical configuration following physiotherapy in women with stress urinary incontinence.

PURPOSE: We quantified the effect of pelvic floor muscle training on the anatomical configuration of the levator ani using magnetic resonance imaging. MATERIALS AND METHODS: Five female participants with stress urinary incontinence underwent magnetic resonance imaging before and after participating in a pelvic floor muscle physiotherapy program. Axial T1-weighted images of the levator ani were taken with the participant in a supine position. Source images were then manually segmented and surface modeling was applied to build a 3-dimensional model of the levator ani. Models were then measured to determine the levator ani surface area as well as the encircled volume at rest and during voluntary contraction. The percentage of levator ani retraction and symphysis pubis movement during voluntary contraction before and after physiotherapy were also measured. RESULTS: After physiotherapy the levator ani surface area at rest was significantly smaller than before physiotherapy, decreasing from 677.11 +/- 45.00 to 620.48 +/- 36.14 mm(2) (p = 0.04). The relative reduction in volume encircled by the levator ani during contraction increased significantly from -11.66 +/- 7.42 to -26.02 +/- 13.52 mm(3) (p = 0.04). Levator ani surface retraction during a voluntary contraction increased significantly from 65.61% +/- 17.07% to 81.70% +/- 16.30% (p = 0.02). Symphysis pubis movement during pelvic floor muscle contraction decreased from 1.45 +/- 1.32 to 0.44 +/- 0.61 mm (p = 0.05). CONCLUSIONS: Findings from this preliminary study indicate that pelvic floor muscle training results in anatomical changes in the levator ani and reduction of pubic movement. These results provide insight into the possible anatomical mechanisms through which physiotherapy enables the pelvic floor muscle to minimize urine leakage.

Secretory production of Aspergillus oryzae xylanase XynF1, xynF1 cDNA product, in the basidiomycete Coprinus cinereus.

The signal peptide of Aspergillus oryzae endo-(1,4)-beta-xylanase XynF1 contains a C-terminal serine-arginine that directs efficient secretion of the enzyme into the culture medium. In the basidiomycete Coprinus cinereus, however, there is little secretion of XynF1 into the culture medium. Modification of the C-terminal sequence of the signal peptide to lysine-arginine resulted in efficient secretion of C. cinereus XynF1, suggesting the presence of a KEX2-like protease in this fungus.

Fragmentation reactions of optically active trisubstituted cyclopropylcarbinyl radicals.

Fragmentation reactions of optically active trisubstituted cyclopropylcarbinyl radicals and their application to the synthesis of natural products are described. Preparation of the optically pure substrates for radical fragmentation reactions was efficiently accomplished by lipase-mediated desymmetrization of sigma-symmetrical 3-substituted-1,2-cyclopropanedimethanols. In the presence of a radical stabilizing group, e.g., aryl, ester, or alpha,beta-unsaturated ester, the fragmentation occurs selectively to generate the radical on the alpha-carbon of the group and provide the optically pure alkene derivatives. These derivatives possess three chemically distinct functionalities, making them excellent chiral building blocks for the construction of biologically active molecules. The synthetic usefulness of the procedure developed here has been demonstrated by an application to the enantioselective synthesis of both enantiomers of the key intermediate, 4-(3,4-methylenedioxybenzyl)dihydrofuran-2(3H)-one (54), for the total synthesis of biologically active lignans.

A novel tandem [2 + 2] cycloaddition-Dieckmann condensation with ynolate anions. Efficient synthesis of substituted cycloalkenones and naphthalenes via formal [n + 1] cycloaddition.

A novel tandem [2 + 2] cycloaddition-Dieckmann condensation via ynolate anions is described. Ynolate anions are useful for the formation of reactive beta-lactone enolates via a pathway not involving the enolization of the corresponding beta-lactones. The [2 + 2] cycloaddition of ynolate anions with delta- or gamma-keto esters, followed by Dieckmann condensation, gives bicyclic beta-lactones, which are easily decarboxylated to produce synthetically useful 2,3-disubstituted cyclopentenones and cyclohexenones in one pot. This tandem reaction was applied to a novel, one-pot synthesis of highly substituted naphthalenes.

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant.

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.

The first tandem [2 + 2] cycloaddition--Michael reaction using ynolates: facile construction of substituted carbocycles.

[reaction: see text] A tandem [2 + 2] cycloaddition-Michael reaction using ynolate anions followed by decarboxylation produced polysubstituted five-, six-, and seven-membered cycloalkenes.

Glycine reduces novelty- and methamphetamine-induced locomotor activity in neonatal ventral hippocampal damaged rats.

The use of neonatal ventral hippocampal nVH lesioned rats is well established in animal models of schizophrenia. Moreover, the dysfunction of N-methyl-D-aspartate (NMDA) neurotransmission may play a crucial role in the pathophysiology of schizophrenia. To examine the effect of glycine (GLY) in this animal model, we compared the effects of GLY (0.8 and 1.6 g/kg, IP) on locomotor activity induced by a novel environment (NOVEL) and methamphetamine (MAP, 1.5 mg/kg, IP) in lesioned and sham-operated rats. Compared with sham rats, GLY significantly reduced NOVEL- and MAP-induced locomotor activity in lesioned rats (p <.001 and p <.05, respectively). It is suggested that GLY attenuated nVH-induced hyperactivity, and that this effect was evident both in the presence and absence of MAP. The nVH lesions may result in a form of hyperactivity that differs from normal locomotion in the degree to which it is highly sensitive to regulation by GLY.

Cloning and sequence analysis of the basidiomycete Lentinus edodes ribonucleotide reductase small subunit cDNA and expression of a corresponding gene in L. edodes.

We have previously isolated the uck1 gene encoding UMP-CMP kinase from the basidiomycete Lentinus edodes (Kaneko et al., 1998). It was shown to be most actively transcribed in hymenophores of mature fruiting bodies of L. edodes. The reduction of NDPs produced by the nucleoside monophosphate kinase to dNDPs has been known to be catalyzed by ribonucleotide reductase (RNR) which consists of a heterodimer of large and small subunits. So we attempted to isolate the L. edodes cDNA(s) of RNR and study the expression in L. edodes of the corresponding gene(s), resulting in an isolation of the small subunit cDNA from a mature fruiting-body cDNA library of the fungus. This cDNA, named Le.rnr2c, was shown to encode a 418 amino acids (aa) protein, named Le.RNR2, of which the deduced aa sequence shows an overall identity of 71.9% to that of Schizosaccharomyces pombe RNR small subunit. The Le.rnr2 gene was found to be most actively transcribed in hymenophores of mature fruiting body of L. edodes. The in situ RNA-RNA hybridization analysis showed the presence of markedly large amount of the Le.rnr2 transcript in both hymenium and outer region of trama in the hymenophore. The same experiment was done for the uck1 gene, obtaining a similar result. The hymenium contains many basidia in which fusion of two nuclei, meiosis, replication, etc. essential for production of basidiospores occur. The outer region of trama is the region branching out into subhymenium. These imply that Le.rnr2 gene (and uck1 gene) play a role mainly in the nucleotide biosynthesis essential both for production of basidiospores and for divergence of trama cells into subhymenium cells in the hymenophore.

Decreased zinc ion accumulation by the basidiomycete Lentinus edodes over-expressing L. edodes PriA gene.

The information that the deduced expression product of Lentinus edodes priA gene consists of N-terminal hydrophobic sequence, putative zinc-binding motifs and C-terminal membrane-binding-promoting unique sequence led us to analyze its function in L. edodes. Here L. edodes monokaryotic cells over-expressing priA gene were found to exhibit a remarkably decreased accumulation of zinc ion, indicating the involvement of the priA gene in regulation of the intracellular zinc concentration.

Neurotoxic effect of high dose methamphetamine administration on the hippocampal formation of adult mice: morphometric study using image analyzer.

The volume of the hippocampal formation was measured after repeated methamphetamine (MAP) administration. MAP (30 mg/kg, i.p.) or an equivalent volume of saline (SAL) was administered once daily for 5 days to adult male BALB/c mice. The animals were perfused 7 days after the last injection, and brain sections were stained with cresyl violet and studied with a computer-assisted image analyzer. The volume of the molecular layer at the ventral position of the dentate gyrus of MAP-treated animals was significantly decreased (77% of control, p < 0.001). In contrast, the volumes of the molecular layers at the dorsal and midseptal positions of the dentate gyrus did not change after MAP administration. Similarly, repeated MAP treatment did not affect the volumes of the granular layer and hilus at the dorsal, midseptal or ventral positions of the dentate gyrus. The present results are the first to document a persistent neurotoxic effect of high dose MAP administration on the hippocampal volume of adult mice.

Effects of phencyclidine on behavior and extracellular levels of dopamine and its metabolites in neonatal ventral hippocampal damaged rats.

RATIONALE: The use of neonatal hippocampal lesioned rats is well established in animal models of schizophrenia. Moreover, the dysfunction of N-methyl-D-aspartate (NMDA) neurotransmission may play a crucial role in the pathophysiology of schizophrenia. OBJECTIVE: To examine the role of NMDA neurotransmission in the neonatal ventral hippocampal damaged rat. METHODS: In initial experiments, we compared the effects of mild environmental stress (HAB) and the injection of saline and methamphetamine (MAP, 1.5 mg/kg, IP) in lesioned and sham-operated rats. We also examined the effects of a single injection of phencyclidine (PCP, 10 mg/kg, IP) on locomotor activity and extracellular levels of dopamine (DA) and its metabolites in the nucleus accumbens (NAc) of lesioned and sham-operated rats using an in vivo brain microdialysis method. RESULTS: Compared with sham-operated controls, the lesioned rats showed increased locomotor activity at postnatal day 56 (PD56) but not at PD35 after HAB and MAP administration. Similarly, the lesioned rats showed increased locomotor activity at PD56 but not at PD35 after PCP administration. Unexpectedly, the increase in DA levels was significantly greater in the sham-operated rats than in the lesioned rats. CONCLUSIONS: The results suggest that neonatal hippocampal lesioned rats are accompanied by the dysfunction of NMDA neurotransmission. They also suggest that hyperresponsiveness to PCP following neonatal hippocampal lesions does not depend on the extracellular DA concentration in the NAc.

Structure and function of a pyrimidine/purine-biased sequence from the 5'-flanking region of the basidiomycete Lentinus edodes gene priA.

The priA gene of the basidiomycete Lentinus edodes possesses a pyrimidine (CT)-rich stretch (26 bp) that includes a short (6-bp) repeat, the elements of which form a mirror repeat at and near the transcriptional initiation sites. A DNA fragment that included this sequence was inserted into pBR322, and the resulting plasmids were introduced into Escherichia coli. Analysis of the susceptibility of these pBR322 derivatives to cleavage by S1 nuclease, following isolation from E. coli, indicated the formation of an open, S1-sensitive structure within and just downstream of the CT/AG-biased sequence. Replacement of two dTMP residues in one of the repeat elements by dGMP resulted in the elimination of the S1-cleavable open structure from the plasmids. To analyze the effect of the CT/AG-biased sequence from priA in the basidiomycete Coprinus cinereus, the integrating vectors pLC2 and pLC2mutCT were used; these contained the wild-type priA promoter and the mutant priA promoter with the aforementioned mutation in the mirror repeat, respectively. The Streptomyces-derived bialaphos resistance gene (bar) was fused downstream of the promoters, and the resulting plasmids, pLC2-bar and pLC2mutCT-bar, were introduced into C. cinereus. Transformants carrying pLC2mutCT-bar grew significantly more slowly on bialaphos-containing agar plates and contained a noticeably lower level of the bar transcript when compared with the transformants obtained with pLC2-bar. These results suggest that an unusual structure induced by the CT/AG-biased sequence is required for efficient gene expression from the priA promoter.

Basidiomycete fungal gene encoding a regulatory subunit A homologue of protein phosphatase 2A.

A gene, Le.paa, encoding a regulatory subunit A (PR65) homologue of protein phosphatase 2A was isolated from the basidiomycete mushroom Lentinus edodes. The deduced Le.paa gene product (Le.PR65) had the highest sequence similarity to the Schizosaccharomyces pombe PR65 protein (54.1% similarity). The Le.paa gene was shown to be transcribed more actively during the late stages of fruiting development of the fungus. Gill tissue in which basidiospores are formed contained abundant Le.paa transcript as compared with gill-depleted pileus and stipe.

Increased heavy metal sensitivity of Escherichia coli producing the expression product of priA gene derived from the basidiomycete Lentinus edodes.

We have previously isolated a developmentally regulated novel gene, priA, from the basidiomycete Lentinus edodes. The deduced PRIA protein contains the two set of motifs similar to a 'zinc finger' typified by transcription factor TFIIIA and the motif of a 'zinc cluster' observed in metallothioneins. It also contains a hydrophobic N-terminal sequence. Here Escherichia coli cells producing PRIA were found to show a remarkable sensitivity to zinc ion and other heavy metal ions such as nickel and cadmium. Deletion analysis of PRIA revealed that the zinc-binding motifs and the hydrophobic N-terminal sequence are responsible for conferring the heavy metal sensitivity on the host cells.

Analysis of mfbA alleles of the eubasidiomycete Lentinus edodes.

A fruiting-body-specific mfbA cDNA derived from Lentinus edodes FMC2 has been shown to encode a high-molecular-weight protein, MFBA, containing the cell-adhesion-promoting Arg-Gly-Asp (RGD) sequence. Southern-blot analysis showed that all L. edodes strains tested have the mfbA gene (homologue). Nucleotide sequence analysis of the 1-kb mfbA fragments containing the RGD-coding sequence showed that each L. edodes strain has two types of mfbA homologues. It was found in FMC2 that two mfbA homologues are derived from different nuclei and these mfbA alleles are transcribed with similar frequencies in the fruiting bodies.