A component of polysaccharide peptidoglycan complex on Lactobacillus induced an improvement of murine model of inflammatory bowel disease and colitis-associated cancer.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signals play key roles in the pathogenesis of inflammatory bowel disease (IBD). We previously described that both intact cells and a cell wall-derived polysaccharide-peptidoglycan complex (PSPG) in a strain of lactobacillus [Lactobacillus casei Shirota (LcS)] inhibited IL-6 production in lipopolysaccharide (LPS)-stimulated lamina propria mononuclear cells (LPMCs) isolated from murine IBD. Diets with LcS improve murine IBD by suppression of IL-6 synthesis in LPMCs. Moreover, LcS supplementation with fermented milk ameliorates disease activity in patients with active ulcerative colitis. Here, we focused on the specific roles of PSPG in LcS concerning their anti-inflammatory actions. PSPG derived from LcS, and no other strain of lactobacilli, inhibited IL-6 production in LPS-stimulated murine IBD LPMCs. Purified PSPG-I from LcS inhibited IL-6 synthesis in LPS-stimulated murine IBD LPMCs through the inhibition of nuclear factor-kappaB. The anti-IL-6 action of LcS PSPG was abrogated by masking with monoclonal anti-PSPG-I. Furthermore, PSPG-I-negative L. casei strains (PSPG-I-negative mutant LcS: LC(DeltaPSPG-I), L. casei ATCC 334) did not inhibit IL-6 production. Finally, we confirmed the effects of PSPG-I on LcS in the models of both IBD and colitis-associated cancer (CAC). In the IBD model, ingestion of LcS improved ileitis and inhibited activation of IL-6/STAT3 signaling, while ingestion of the LC(DeltaPSPG-I) strain did not. In the CAC model, treatment with LcS, but not the LC(DeltaPSPG-I) strain, showed tumour-suppressive effects with an inhibition of IL-6 production in the colonic mucosa. These results suggested that a specific polysaccharide component in an L. casei strain plays a crucial role in its anti-inflammatory actions in chronic intestinal inflammatory disorders.

Participation of hepatic α/ß-adrenoceptors and AT1 receptors in glucose release and portal hypertensive response induced by adrenaline or angiotensin II.

It has been previously demonstrated that the hemodynamic effect induced by angiotensin II (AII) in the liver was completely abolished by losartan while glucose release was partially affected by losartan. Angiotensin II type 1 (AT1) and adrenergic (∝1- and ß-) receptors (AR) belong to the G-proteins superfamily, which signaling promote glycogen breakdown and glucose release. Interactive relationship between AR and AT1-R was shown after blockade of these receptors with specific antagonists. The isolated perfused rat liver was used to study hemodynamic and metabolic responses induced by AII and adrenaline (Adr) in the presence of AT1 (losartan) and ∝1-AR and ß-AR antagonists (prazosin and propranolol). All antagonists diminished the hemodynamic response induced by Adr. Losartan abolished hemodynamic response induced by AII, and AR antagonists had no effect when used alone. When combined, the antagonists caused a decrease in the hemodynamic response. The metabolic response induced by Adr was mainly mediated by ∝1-AR. A significant decrease in the hemodynamic response induced by Adr caused by losartan confirmed the participation of AT1-R. The metabolic response induced by AII was impaired by propranolol, indicating the participation of ß-AR. When both ARs were blocked, the hemodynamic and metabolic responses were impaired in a cumulative effect. These results suggested that both ARs might be responsible for AII effects. This possible cross-talk between ß-AR and AT1-R signaling in the hepatocytes has yet to be investigated and should be considered in the design of specific drugs.

pH-sensing nano-crystals of carbonate apatite: effects on intracellular delivery and release of DNA for efficient expression into mammalian cells.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

Selected base sequence outside the target binding site of zinc finger protein Sp1.

Human transcription factor Sp1 contains three contiguous repeats of the C2H2-type zinc finger motif and binds to the decanucleotide sequence 5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3' (GC box). In order to determine whether the three-zinc finger peptide Sp1(530-623) has selectivity for sequence outside the GC box, we used a selection and amplification of binding experiment. The high affinity sequence generated from this selection is 5'-GGGTGGGCGTGGC-3' (s-GC box), which is flanked by a novel conserved guanine triplet on the 5'-side of the core decanucleotide. Gel mobility shift assays reveal that Sp1(530-623) binds to the s-GC box with 2.3-fold higher affinity than to the wild-type GC box, 5'-GGGGCGGGGC-3' (c-GC box). DNase I and hydroxyl radical footprinting analyses show that the area of the s-GC box protected by binding of Sp1(530-623) is wider by 1 nt than that of the c-GC box. On the other hand, alkylation interference analyses demonstrate that Sp1(530-623) forms only one special base contact at the guanine triplet. With respect to cleavage of the c-GC and s-GC boxes by the 1,10-phenanthroline-copper complex (OP-Cu), binding of Sp1(530-623) has no effect on the cleavage pattern of the s-GC box, whereas OP-Cu actually enhances cleavage of the c-GC box. Additionally, the extent of cleavage of the s-GC box by DNase I and OP-Cu is clearly different from that of the c-GC box under peptide-free conditions. The results strongly indicate that: (i) the conformation of the s-GC box is evidently distinct from that of the c-GC box; (ii) Sp1(530-623) binds to the s-GC box without induction of a conformational change in DNA detectable by cleavage with OP-Cu. The present study provides useful information for the design of multi-zinc finger proteins with various sequence specificities.

Induction of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion.

Some strains of lactobacilli can stimulate macrophages and dendritic cells to secrete IL-12, which plays a key role in activating innate immunity. We examined the IL-12-inducing ability of 47 Lactobacillus strains belonging to 10 species in mouse peritoneal macrophages, and characterized the properties important for the induction of IL-12. Although considerable differences in IL-12-inducing ability were observed among the strains tested, almost all strains belonging to the Lactobacillus casei group (L. casei, Lactobacillus rhamnosus, and Lactobacillus zeae) or to Lactobacillus fermentum induced high levels of IL-12. Phagocytosis of lactobacilli was necessary for IL-12 induction, and the strains with strong IL-12 induction were relatively resistant to lysis in the macrophages. The sensitivity of Lactobacillus strains to in vitro treatment with M-1 enzyme, a member of the N-acetylmuramidases, was negatively correlated with IL-12-inducing ability. Using a probiotic strain, L. casei strain Shirota (LcS), we showed that the cell wall of LcS could be digested by long-term treatment with a high dose of M-1 enzyme and that the IL-12-inducing ability was diminished according to the duration of the enzyme treatment. The soluble polysaccharide-peptidoglycan complex released from the cell wall of LcS did not induce IL-12, whereas the insoluble intact cell wall of LcS induced IL-12. These results suggest that the intact cell wall structure of lactobacilli is an important element in the ability to induce IL-12 and that Lactobacillus strains having a rigid cell wall resistant to intracellular digestion effectively stimulate macrophages to induce IL-12.

Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry.

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.

Effects of sialic acid residues of transferrin on the binding with aluminum and iron studied by HPLC/high-resolution ICP-MS.

Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf = 1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf = 1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced.

Expression of kininogen, kallikrein and kinin receptor genes by rat cardiomyocytes.

To ascertain the existence of the kallikrein-kinin system in the heart, we have studied in vivo and in vitro whether rat cardiac tissue expresses kininogen, kallikrein and kinin receptor mRNAs. The reverse transcription-polymerase chain reaction demonstrated that the ventricular myocardium of adult male rats expressed mRNAs for T- and low-molecular-weight (L-) kininogens, tissue kallikreins such as true kallikrein and T-kininogenase, and bradykinin B2 receptor, but not those for high-molecular-weight kininogen and B1 receptor. Lipopolysaccharide (LPS; 0.5 mg/kg, i.v.) increased the levels of mRNA for T-kininogen at 12 h and the bradykinin B1 receptor at 24 h without affecting that for other components. All of these mRNAs for the kallikrein-kinin system were also detected in cultured cardiomyocytes derived from neonatal rat ventricles; dibutyryl cyclic AMP, LPS or inflammatory cytokines such as interleukin-1 and tumor necrosis factor, up-regulated mRNA expression of T-kininogen, T-kininogenase, or B1 receptor in these cells in vitro. These results suggest that there are two kinin-generating systems in rat myocardium comprising T-kininogen/T-kininogenase and L-kininogen/true kallikrein respectively, and that the former may be relatively important in inflammatory diseases or conditions in which cAMP levels increase in cardiomyocytes.

Small-angle neutron scattering and dynamic light scattering studies of N- and C-terminal fragments of ovotransferrin.

In order to rationalize the physicochemical heterogeneities between the N- and C-lobes of ovotransferrin (OTf), we have analyzed the structural characteristics of the isolated fragments corresponding to the N- and C-terminal halves of OTf (OTf/2N and OTf/2C) with and without iron by means of small-angle neutron scattering (SANS) using the contrast variation method with solvents of various D2O/H2O mixtures, and dynamic light scattering (DLS) measurements. The analyses of the internal structural characteristics from SANS data revealed that the radius of gyration (Rg) for both fragments decreased to the same extent with iron binding, and the structural distortion of OTf/2C was smaller than that of OTf/2N, decreasing with iron uptake. The DLS studies showed that the change in the diffusion coefficient induced by iron binding to OTf/2C was greater than that to OTf/2N. It was inferred that the OTf/2C molecule tends to become more compact on the whole by iron binding as compared to the OTf/2N molecule.

Kininogen expression by rat vascular smooth muscle cells: stimulation by lipopolysaccharide and angiotensin II.

To identify the presence of a local kallikrein-kinin system in vascular wall, we have studied whether rat vascular smooth muscle cells (VSMC) express kininogen in vitro and in vivo. Western blots using anti-T-kininogen antibody revealed the presence of T-kininogen in conditioned medium of cultured VSMC. T-Kininogen secretion by VSMC was markedly enhanced by the addition of lipopolysaccharide (LPS), angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) to the culture. Experiments using specific inhibitors for protein kinases and on the PMA-induced down-regulation of protein kinase C suggested that a protein kinase C-dependent or unidentified pathway is involved in AII or LPS action, respectively. The intravenous injection of LPS (0.5 mg/kg) resulted in an increase in T-kininogen mRNA levels in the vascular smooth muscle of rat aorta, peaking at 16 h. Polyacrylamide gel electrophoresis of cDNA products generated by reverse transcription-polymerase chain reaction (RT-PCR) from aortic mRNA using primers specific for either T- or low-molecular-weight kininogen revealed that rat vascular smooth muscle expressed T-kininogen gene but not low-molecular-weight kininogen gene, and that LPS exclusively stimulated T-kininogen expression. The mRNA for high-molecular-weight kininogen was undetectable in either aortic smooth muscle or cultured VSMC by means of RT-PCR analysis. RT-PCR using specific primers for rat tissue kallikrein genes showed that aortic smooth muscle expressed KLK1 (true kallikrein) mRNA, but not KLK10 (T-kininogenase) mRNA. These results demonstrated that rat VSMC are a source of T-kininogen but not of low-molecular-weight- or high-molecular-weight kininogen, in contrast to the expression of true kallikrein but not of T-kininogenase by these cells.

Spinocerebellar degenerations in Japan: a nationwide epidemiological and clinical study.

A nationwide survey of patients in Japan with spinocerebellar degenerations (SCD), including SDS and SND, was conducted from 1988 to 1989. The survey consisted of two parts. The first revealed that the estimated total number of patients with SCD in Japan was 5,050 (range: 4,100-6,000) with an estimated prevalence of 4.53 per 100,000 in 1987. The second part investigated the neurological and functional status of patients with SCD. The percentages of those belonging to each subtype of SCD were: OPCA; 34.4%, LCCA; 15.2%, MHCA; 12.6%, HHCA; 7.5%, SDS; 7.0%, HSP; 3.9%, DRPLA; 2.5%, FA; 2.4%, MJD; 2.0% and SND; 1.5%. Compared with European epidemiological studies Japan had a higher proportion of non-hereditary types of SCD. Various clinical features of SCD subtypes were compared grouped by pathological lesion and heredity. HHCA and LCCA: cerebellar ataxia predominated in all stages, and neurological signs other than cerebellar ataxia were rare. MHCA, DRPLA and MJD: in the early phase ataxia was the most common symptom in MHCA, the AC form of DRPLA and MJD, but ataxia was less common and chorea or epilepsy were often observed in ME and PH forms of DRPLA. Other frequently observed clinical features were parkinsonian rigidity in MHCA, abnormal movements and posture in DRPLA and MJD, and disturbances of eye movements in MHCA, the AC form of DRPLA and MJD. OPCA, SDS and SND: dominant clinical features were cerebellar ataxia in OPCA, autonomic disturbance in SDS, and parkinsonian rigidity in SND. FA and HSP: both were rare in Japan. Clinical features related to supra-supinal lesions were frequently observed in FA. Functional status of SCD: the severity of illness was significantly associated with the level of independence in each item of ADL. Activities not requiring dynamic balance were performed independently for a longer period than those requiring dynamic balance. Among SCD subtypes, functional prognosis was poorest in non-hereditary, multi-systemic types (OPCA, SDS and SND) followed by hereditary multi-systemic types (MHCA, DRPLA and MJD), and better in spinal types (FA and HSP) and cerebellar types (HHCA and LCCA).

Metabolic aspects of the 1 beta-proton and the 19-methyl group of androst-4-ene-3,6,17-trione during aromatization by placental microsomes and inactivation of aromatase.

Aromatase catalyzes the conversion of androst-4-ene-3,17-dione to estrogen through sequential oxygenations at the 19-methyl group. Androst-4-ene-3,6,17-trione (AT) is a suicide substrate of aromatase, and the mechanism of inactivation of aromatase has been postulated to involve enzymatic oxygenation at the 19-position. [1 beta-3H,4-14C]-, [19-3H3,4-14C]-, and [1 beta-3H,19-14C]ATs, with high specific activities, were synthesized to study metabolic aspects and the inactivation mechanism. Incubation of the labeled AT with human placental microsomes yielded the 19-oxygenated derivatives, 19-hydroxy-AT and 19-oxo-AT, as well as the aromatization products, 6-oxoestrone and 6-oxoestradiol. A stereospecific 1 beta-proton elimination occurred during the aromatization of [1 beta-3H,4-14C]AT, and a marked tritium isotope effect was observed in the first hydroxylation at C-19 of [19-3H3,4-14C]AT. After incubation of the three double-labeled ATs, the solubilized proteins were subjected to SDS-PAGE and the 3H/14C ratio of the aromatase-bound metabolite in a 46-69 kDa fraction was analyzed. A marked decrease of the 3H/14C ratio of the metabolite was observed in the experiment using [19-3H3,4-14C]AT, compared with that of the labeled AT used, but there were no significant changes in the other experiments, indicating that the adduct retains the 1 beta-proton, the 19-carbon, and one of the three 19-methyl protons of AT. Thus, we conclude that further oxygenation of 19-oxo-AT produced by the two initial hydroxylations of AT at C-19 yields not only 6-oxoestrogen (by a mechanism similar to that involved in the aromatization of the natural substrate) but also a reactive electrophile that immediately binds to the active site in an irreversible manner, resulting in inactivation of aromatase.

Structure of 6-deoxytalose-containing polysaccharide from the cell wall of Bifidobacterium adolescentis.

The isolation and analysis of the cell wall and the polysaccharide-glycopeptide complexes of Bifidobacterium adolescentis YIT4011 are presented. Polysaccharide-glycopeptide complexes, PS-GP1 and PS-GP2, were solubilized from the cell wall by treatment with N-acetylmuramidase. PS-GP1 and PS-GP2 were found to be composed of glucose, 6-deoxytalose and a small amount of glycopeptide. The products of Smith degradation of the PS-GPs had no glucose-containing fraction, but were composed of 1,2/1,3-linked 6-deoxytalose. Furthermore, a second Smith degradation of this fraction yielded trisaccharide-glyceraldehyde. These results and methylation analysis led to the conclusion that PS-GP1 or 2 has a repeating unit of----3)6dTal(beta 1----3)6dTal(beta 1----3)6dTal(beta 1----2)-6dTal(alpha 1----2)6dTal(alpha 1----2)6dTal(alpha 1-, and that glucose residues are linked to position C-3 of the 2-O-substituted 6-deoxytalose residues.

Structural studies of cell wall polysaccharides from Bifidobacterium breve YIT 4010 and related Bifidobacterium species.

The chemical compositions of the cell walls obtained from 8 strains in 5 species of Bifidobacterium were analyzed. These cell walls were shown to be composed of peptidoglycan and polysaccharide moieties. Some variations with respect to contents of neutral sugars and content of phosphorus were observed with some cell wall preparations from the same species. The neutral polysaccharides in cell walls of 4 strains of Bifidobacterium (B. bifidum YIT 4007, B. breve YIT 4010, B. infantis YIT 4025, and B. longum ATCC 15707) were purified and their chemical structures were analyzed. One of these polysaccharides, obtained from B. breve YIT 4010, was analyzed in detail by GLC, 1H- and 13C-NMR spectroscopic analyses, methylation, Smith degradation and acetolysis, and the results suggested the following structure for the repeating unit of the polysaccharide: (Formula: see text).

Kinetic studies on the plasminogen activation by the staphylokinase-plasmin complex.

A pure complex of staphylokinase and plasmin was prepared by affinity chromatography with lysine-Sepharose, which enabled the simple analysis of the mechanism of plasminogen activation by staphylokinase. We used a truncated staphylokinase (SAK), which lacks the 10 amino acid residues at the NH2 terminal of native staphylokinase. The purity of this complex was confirmed by the native PAGE profile. Image analysis of the SDS-PAGE profile revealed that the molar ratio of plasmin and SAK in the complex was about 1:1. Using this SAK-plasmin complex, the kinetic parameters for the activation of Glu- or Lys-plasminogen were determined. The kinetic constant, kcat/Km, obtained when Lys-plasminogen was used as a substrate was approximately 10 times higher than that obtained when Glu-plasminogen was used. This plasminogen activation property of the SAK-plasmin complex was comparable to that of other plasminogen activators, such as streptokinase, urokinase, and tissue-type plasminogen activator (t-PA). This SAK-plasmin complex will simplify the elucidation of plasminogen activation by SAK. Through kinetic studies, the fibrin specificity and participation of plasminogen activator inhibitor will be clarified.

Structure of polysaccharide-peptidoglycan complex from the cell wall of Lactobacillus casei YIT9018.

The isolation and analysis of the polysaccharide-peptidoglycan complexes of Lactobacillus casei YIT9018 are presented. Two polysaccharide-peptidoglycan complexes, PS-PG1 and PS-PG2, were solubilized from the heat-killed cell by treatment with N-acetylmuramidase. PS-PG1 was composed of glucose, rhamnose, and small amount of galactose and glucosamine. PS-PG2 was composed of glucose, rhamnose, galactosamine, and glucosamine. The ratio by weight of these fractions was about 1:8. PS-PG2 was analyzed in detail. Smith degradation and deamination of this complex yielded oligosaccharide units. The results of methylation analysis of these units and intact PS-PG2 led to the most probable structure of PS-PG2: (formula; see text)

Structure of acidic polysaccharide from cell wall of Propionibacterium acnes strain C7.

The structure of polysaccharide prepared by lysozyme digestion from the cell wall of Propionibacterium acnes strain C7 was examined. The polysaccharide fraction was composed of glucose, galactose, mannose, galactosamine, and diaminomannuronic acid in a molar ratio of 1:1:0.3:1:2. By Smith degradation of the polysaccharide, diaminouronic acid-containing fractions were obtained, and the configuration of diaminouronic acid was identified as 2,3-diacetamido-2,3-dideoxymannuronic acid [Man(NAc)2A] by means of 1H-NMR and 13C-NMR spectroscopic analyses. The results of analyses involving methylation and partial acid hydrolysis led to the conclusion that the polysaccharide has the repeating unit----6)Gal(alpha 1----4)Man(NAc)2A(beta 1----6)Glc(alpha 1----4)Man(NAc)2A (beta 1----3)GalNAc(beta 1--. In addition, a portion of the galactose residues were substituted at C-4 by alpha 1----2 linked mannotriose.