Dual mode diffusion of NaCl in Japanese radish under cooking conditions.

Sorption and diffusion of NaCl in Japanese radish have been studied. The sorption isotherm was obtained at 98 degrees C by the conventional method. The concentration profile by the 1-dimensional diffusion of NaCl in Japanese radish from the 3.0% solution was measured at 98 degrees C with the FRITRUC method involving a foodstuff rod in a thin rubber casing. Fick's diffusion coefficient, D, calculated therefrom showed a threefold variation with a maximum. This variation was quantitatively interpreted by applying a dual-mode sorption and diffusion theory under an assumption that the rate determining step of the diffusion is that in the cell wall. Two thermodynamic diffusion coefficients, D(T)(p) and D(T)(L), where p and L are the species of NaCl sorbed by partition and Langmuir modes, respectively, a parameter, alpha, derived from the local equilibrium relations between the p and L species, and S, the concentration of the Langmuir adsorption site in the cell wall of the radish, were estimated. D(T)(p) was found to be smaller than D(T)(L). As an explanation of the larger D(T)(L), we invoked the higher hydration state of the adsorption site of the L species, being ascribed to residual anionic pectin in the radish than the local environment of the p species. The sorption isotherm showed a convex upward deviation from the linear relation. By using the parameters for the local equilibrium and some assumed parameters, the isotherm was found to be explainable. We suggest possible applications of the present method and interpretation to the diffusion study on the cooking systems comprising varieties of seasoning components and foodstuffs.

Inhibition of the binding of enterotoxigenic Escherichia coli Pb176 to human intestinal epithelial cell line HCT-8 by an extracellular protein fraction containing BIF of Bifidobacterium longum SBT2928: suggestive evidence of blocking of the binding receptor gangliotetraosylceramide on the cell surface.

The extracellular protein fraction (P100V) containing the protein BIF produced by Bifidobacterium longum SBT2928 (BL2928), which inhibits the binding of enterotoxigenic Escherichia coli Pb176 (ETEC) to the glycolipid binding receptor gangliotetraosylceramide (GA1) also inhibited the binding of ETEC to the human intestinal epithelial cell line HCT-8 (ATCC CCL 244) in a dose-dependent manner. ETEC-binding inhibitory experiments using crude colonization factor antigen (CFA)-II prepared from ETEC, rabbit anti-GA1 antiserum, medium containing GA1 and media containing lectins, as the binding-inhibitors, suggest that the interaction between the CFA-II antigen present on the cell surface of ETEC and GA1 expressed on HCT-8 cells plays a significant role in the adherence between them. It is strongly suggested that the P100V fraction works as a blocker for the ETEC receptor GA1 on HCT-8 cells.

Establishment of orally-administered Lactobacillus gasseri SBT2055SR in the gastrointestinal tract of humans and its influence on intestinal microflora and metabolism.

AIMS: To investigate the fate of a streptomycin-rifampicin-resistant variant of Lactobacillus gasseri SBT2055 (LG2055SR) and the influence of its oral administration on the composition and metabolism of the intestinal microflora. METHODS AND RESULTS: Intestinal passage of LG2055SR was monitored by a combination of selection with antibiotics and identification by a randomly amplified polymorphic DNA (RAPD)-PCR METHOD: Composition of intestinal microflora was analysed by the method developed by Mitsuoka et al. (1965, 1974). Establishment of orally-administered LG2055SR in the human intestine was confirmed in this study. LG2055SR ingestion specifically lowered faecal populations of Staphylococcus and faecal contents of p-cresol. CONCLUSION: LG2055SR and its parent strain, LG2055, are considered to be appropriate candidates for probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clarified that LG2055SR has the ability to establish in the human gastrointestinal tract and alters the composition and metabolism of the intestinal microflora and physical characteristics of faeces.

Intestinal transit of an orally administered streptomycin-rifampicin-resistant variant of Bifidobacterium longum SBT2928: its long-term survival and effect on the intestinal microflora and metabolism.

AIMS: The objectives of this study are to investigate the fate of a streptomycin-rifampicin-resistant variant of Bifidobacterium longum SBT2928 (BL2928SR) and the influence of its oral administration on the composition and metabolism of the intestinal microflora. METHODS AND RESULTS: Intestinal passage of BL2928SR was monitored by a combination of selection with antibiotics and identification by a randomly amplified polymorphic DNA (RAPD)-PCR method. Intestinal microflora was analysed by the method developed by Mitsuoka et al. (1965, 1974). Long-term survival of orally administered BL2928SR in the human intestine was confirmed. BL2928SR ingestion specifically lowered faecal populations of Enterobacteriaceae and clostridia, including lecithinase-positive Clostridium spp. CONCLUSION: BL2928SR and its parent strain, BL2928, are considered to be appropriate candidates for probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clarified that BL2928SR has the ability for long-term survival in the human gastrointestinal tract, and alters the composition and metabolism of the intestinal microflora.

Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization.

A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized. Furthermore, we describe for the first time cloning and analysis of the gene encoding BSH (bsh) in a member of the genus Bifidobacterium. The enzyme has a native molecular weight of 125,000 to 130,000 and a subunit molecular weight of 35,024, as determined from the deduced amino acid sequence, indicating that the enzyme is a tetramer. The pH optimum of B. longum BSH is between 5 and 7, and the temperature optimum is 40 degrees C. The enzyme is strongly inhibited by thiol enzyme inhibitors, indicating that a Cys residue is likely to be involved in the catalytic reaction. The BSH of B. longum can hydrolyze all six major human bile salts and at least two animal bile salts. A slight preference for glycine-conjugated bile acids was detected based on both the specificity and the K(m) values. The nucleotide sequence of bsh was determined and used for homology studies, transcript analysis, and construction and analysis of various mutants. The levels of homology with BSH of other bacteria and with penicillin V acylase (PVA) of Bacillus sphaericus were high. On the basis of the similarity of BSH and PVA, whose crystal structure has been elucidated, BSH can be classified as an N-terminal nucleophile hydrolase with Cys as the N-terminal amino acid. This classification was confirmed by the fact that a Cys1Ala exchange by site-directed mutagenesis resulted in an inactive protein. Reverse transcription-PCR experiments revealed that bsh is part of an operon containing at least two genes, bsh and glnE (GlnE is glutamine synthetase adenylyltransferase). Two UV-induced BSH-negative mutants and one spontaneous BSH-negative mutant were isolated from B. longum SBT2928 cultures and characterized. These mutants had point mutations that inactivated bsh by premature termination, frameshift, or amino acid exchange.

Purification and characterization of a novel protein produced by Bifidobacterium longum SBT2928 that inhibits the binding of enterotoxigenic Escherichia coli Pb176 (CFA/II) to gangliotetraosylceramide.

A novel protein (BIF) which shows inhibitory activity on the binding of enterotoxigenic Escherichia coli Pb176 (ETEC with colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3) to gangliotetraosylceramide (asialo GM1 or GA1) was isolated from the culture supernatant fluid of Bifidobacterium longum SBT2928 (BL2928) at its stationary phase. The homogeneity of the final preparation of BIF was demonstrated by SDS-PAGE, polyacrylamide gel electrofocusing and N-terminal amino acid sequencing. The BIF was characterized as (i) a protein with an M(r) of approximately 104 kDa when chromatographed on a gel filtration column, and 52 kDa when separated on SDS-PAGE, and (ii) having an isoelectric point of 5.9. No change in size was produced by thiol reduction. These results suggest that BIF is a homodimer consisting of identical 52 kDa monomers. The purified BIF at the concentration of 25 micrograms protein ml-1 caused a 50% reduction in binding of the ETEC strain to GA1.

Supplementation of Bifidobacterium longum to a high-fat, low-calcium diet lowers cytolytic activity of fecal water in rats injected with 1,2-dimethylhydrazine dihydrochloride.

The effects of supplementing Bifidobacterium longum SBT 2928 and Lactobacillus acidophilus SBT 2062 to a high-fat, low-calcium diet on bile acid concentration, fatty acid concentration, cytolytic activity and intestinal alkaline phosphatase (ALP) activity of fecal water in rats injected with and without 1,2-dimethylhydrazine dihydrochloride (DMH) were examined. Male Wistar rats at 8 weeks of age were fed a diet containing 18% coconut oil, 2% corn oil and 0.1% calcium for 15 d. Lyophilized cultures were supplemented to test diets at a concentration of 1%. The feeding of a high-fat, low-calcium diet elevated the bile acid concentration, cytolytic activity and ALP activity of fecal water as compared to the AIN-76A diet, whereas the fatty acid concentration was not changed. None of the cultures had any effect on these parameters. Furthermore, 8 week-old rats were given a single subcutaneous injection of DMH at 40 mg/kg body weight, and fed the same diets for 15 d. The DMH injection had no effect on the bile acid concentration but increased the fatty acid concentration and cytolytic activity of fecal water. In contrast, ALP activity was lower in the DMH-treated rats than in the non-treated rats. The ingestion of B. longum lowered cytolytic activity but had no effect on the bile acids, fatty acids and ALP activity of fecal water. L. acidophilus had no effect on these parameters.

Proteinaceous factor(s) in culture supernatant fluids of bifidobacteria which prevents the binding of enterotoxigenic Escherichia coli to gangliotetraosylceramide.

We have examined the competitive binding of several species of Bifidobacterium and Escherichia coli Pb176, an enterotoxigenic E. coli (ETEC) strain, to gangliotetraosylceramide (asialo GM1 or GA1), a common bacterium-binding structure, and identified a factor(s) in the Bifidobacterium culture supernatant fluid that inhibits the binding of E. coli Pb176 to GA1. The ETEC strain we used expresses colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3. Competitive exclusion of ETEC from GA1 molecules by Bifidobacterium cells was found by an in vitro thin-layer chromatography overlay binding suppression assay. However, the ETEC cells were less effective in blocking the adherence of Bifidobacterium cells to GA1. These findings suggest that the two bacterial species recognize different binding sites on the GA1 molecule and that the mechanism of competitive exclusion is not due to specific blockage of a common binding site on the molecule. The neutralized culture supernatant fluids of Bifidobacterium species, including that of Bifidobacterium longum SBT 2928 (BL2928), showed remarkable inhibition of the ETEC binding to GA1. Our results suggest that the binding inhibitor produced by BL2928 is a proteinaceous molecule(s) with a molecular weight around or over 100,000 and a neutral isoelectric point. The binding inhibitor produced by BL2928 and other Bifidobacterium species is estimated to contribute to their normal anti-infectious activities by preventing the binding of pathogenic strains of E. coli to GA1 on the surface of the human intestinal mucosa.

A new screening method for the selection of Lactobacillus acidophilus group lactic acid bacteria with high adhesion to human colonic mucosa.

A hemagglutination (HA) assay was done for the screening of lectin-like components in surface layer protein (SLP) from Lactobacillus (L.) acidophilus A group strains. The new screening method, using polystyrene beads coated with rat-colonic mucin (RCM), which combines sugar chains similar to those of human colonic mucin, was also done. The results showed that the HA assay was not a good indicator for selecting strains having high adhesion to the human intestinal tract. The SLPs from 3 strains that strongly bound to RCM also bound well to carbohydrate portions of Carnoy's-fixed human colonic mucous layer. These results suggest that this method is a new promising screening technique for the L. acidophilus strains having high adhesion to the human intestinal tract.

Effect of Lactobacillus acidophilus on iron bioavailability in rats.

The effect of Lactobacillus acidophilus on iron bioavailability in rats was examined by the hemoglobin regeneration method. For hemoglobin depletion, female Wistar rats were fed an iron-deficient diet at 3 weeks of age for 13 days. Rats were then assigned to one of four groups, such that average blood hemoglobin value and average body weight were similar among the groups. For hemoglobin regeneration, they were fed one of two ferrous sulfate-supplemented diets that contained the following iron levels (mg/kg): 13.7 for two groups; 21.7 for the other two groups. In the two groups fed the same diet, one group additionally received oral administration of 2 ml of skim milk fortified with 0.3% yeast extract once or twice a day for 7 days, and other rats were administered with skim milk fermented by L. acidophilus SBT 2062 in the same manner. Hemoglobin regeneration efficiency (HRE) was significantly higher in the fermented product-given rats than in the skim milk-supplied rats. There was no significant interaction in HRE between the dietary iron group and the oral administration group. These results indicate that L. acidophilus SBT 2062 is effective for increasing of iron bioavailability in rats.

Effects of skim milk and its fermented product by Lactobacillus acidophilus on plasma and liver lipid levels in diet-induced hypertriglyceridemic rats.

Effects of skim milk and its fermented product by Lactobacillus acidophilus on plasma and liver triglyceride and cholesterol levels were examined in diet-induced hypertriglyceridemic rats. Male Sprague-Dawley rats at 4 weeks of age were fed a hypertriglyceridemic diet that contained 20% coconut oil, 17.5% fructose, and 17.5% sucrose for 14 days. The test diet was supplemented with either 20% skim milk powder or 20% powder of skim milk fermented by L. acidophilus SBT 2062. Hypertriglyceridemia was observed in the control group, but plasma cholesterol levels were not increased. Skim milk suppressed the elevation of plasma triglyceride levels, while its fermented product had no significant effect. Both dairy products prevented the elevation of liver triglyceride and cholesterol levels, but had no effect on plasma cholesterol levels.

[Clinical study of anti-phospholipid antibody in patients with sarcoidosis].

Serum antibodies against five types of phospholipids were measured by enzyme-linked immunosorbent assay (ELISA) in 55 patients with sarcoidosis. In 21 cases (38%), either IgG antibodies or IgM antibodies were detected. These antibodies were thought to mainly be infective type. This positive rate was significantly higher than that (7%) of the control group (70 cases) (p < 0.01). As to the immunoglobulin classes, 5 cases had IgG antibodies only, 11 cases had IgM antibodies only, and 5 cases had both IgG and IgM antibodies. No correlation was observed between the occurrence of anti-phospholipid antibodies (APL-Ab) and disease activity of sarcoidosis. Significant correlations were found between the occurrence of APL-Ab and skin lesions, many extrathoracic organ lesions and the persistence of abnormal chest X-ray findings for over 2 years and 5 years. From these data, it is suggested that the presence of APL-Ab is associated with prolonged disease activity of sarcoidosis.

Identification of the replication region of Streptococcus thermophilus No. 29 plasmid pST1.

The replication region of the 2.77-kilobases (kb) plasmid pST1 from Streptococcus thermophilus No. 29 was identified. Deletion derivatives of pST1 were introduced into plasmid-free S. thermophilus No. 29 and examined for their ability to replicate autonomously. The nucleotide sequence had one open reading frame encoded for a 315 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacterial plasmids strongly suggest that the deduced protein (RepS) encoded by pST1 has a replicative role. pST1 also contains a DNA sequence similar to the origin nick sequences of pLP1 or pLAB1000, which initiate plasmid replication at the plus origin. In a maxicell system, E. coli CSR603 carrying pSTUC4 produced a protein that was considered to correspond to the product of the RepS gene. These results strongly suggest that pST1 is replicated following a rolling-circle mechanism via single-stranded DNA intermediates.

Establishment of a host-vector system in Lactobacillus helveticus with beta-galactosidase activity as a selection marker.

A host-vector system was established in Lactobacillus helveticus with beta-galactosidase activity as a selection marker. Plasmid pBG10 was constructed by joining the beta-galactosidase gene from L. bulgaricus, the promoter region of the erythromycin resistance gene from pAM beta 1, the replication region of pBR329, and the replication region of the L. helveticus cryptic plasmid pLJ1. L. helveticus SBT2195 (Lac- mutant), transformed with pBG10, was selected on skim milk plates. The structural gene of alpha-amylase (1536 bp) from Bacillus licheniformis, inserted downstream of the promoter region of the erythromycin resistance gene of pBG10, was expressed in L. helveticus SBT2195. Plasmid pBG10 is a food-grade and expression vector in L. helveticus.

Localization of metallothionein in hair follicles of normal skin and the basal cell layer of hyperplastic epidermis: possible association with cell proliferation.

Metallothionein is a low-molecular-weight metal-binding protein. Although it is inducible by a variety of agents and ubiquitously present in many tissues, its physiologic functions are still not clear. The present study was undertaken to determine the possible functions of metallothionein in both the proliferation and differentiation of epidermal keratinocytes. Metallothionein was detected immunohistochemically in hair matrix cells of the bulb and cells of the outer root sheath of anagen hair follicles, but not in dermal papillae in normal skin in the back of mice. In hyperplastic epidermal tissue, induced by either a phorbol ester tumor promoter or cholera toxin, the basal cells of the interfollicular epidermis stained strongly for metallothionein. Elevated expression of mRNA of the metallothionein gene was also demonstrated when the skin was stimulated by agents that induced hyperplasia. Papillomas produced by two-stage carcinogenesis protocols also stained for metallothionein. These observations suggest that metallothionein is involved in the proliferation of epidermal keratinocytes.

[Interleukin-2 receptor expression in pulmonary granulomatous diseases].

Interleukin-2 receptor expression (IL-2R) on monocytes and alveolar macrophages (AM) was determined in patients with sarcoidosis and pulmonary tuberculosis. In sarcoidosis and tuberculosis, IL-2R on monocytes was detectable, while it was undetectable in healthy controls. IL-2R on AM in sarcoidosis and tuberculosis was significantly increased as compared to healthy controls. IFN-gamma, which has been shown to be increased in sarcoidosis and tuberculosis as compared to healthy controls, induced IL-2R on monocytes in healthy controls, suggesting that IFN-gamma is at least in part responsible for the induction or enhancement of IL-2R on monocytes or AM in sarcoidosis and tuberculosis. Phorbol myristate acetate which is known to be protein kinase C (PKC) activator induced IL-2R on monocytes, and PKC inhibitor, H7, inhibited IFN-gamma-induced IL-2R on monocytes in healthy controls. Calcium ionophore, A23187, induced IL-2R on monocytes and calmodulin antagonist, W7, inhibited IFN-gamma-induced IL-2R on monocytes. Based on these results, it seems that not only the PKC pathway but also the calcium-calmodulin pathway is involved in IFN-gamma-induced IL-2R.

[Serum soluble IL-2 receptor level in patients with sarcoidosis].

Serum levels of soluble IL-2 receptors (sIL-2R) by an ELISA method in 28 patients with sarcoidosis and 16 healthy controls were studied, and the source of sIL-2R was further examined. sIL-2R in serum was significantly higher in sarcoidosis than in controls. In sarcoidosis sIL-2R in serum significantly correlated with serum ACE level, and was significantly higher in stage II or III patients than in stage O patients. sIL-2R in supernatants of cultured monocytes (Mo) and alveolar macrophages (AM) was significantly higher in sarcoidosis than in controls. sIL-2R in supernatants of cultured T lymphocytes obtained from peripheral blood or BALF was barely detectable in sarcoidosis, while it was undetectable in controls. Furthermore, sIL-2R in serum was significantly correlated with sIL-2R in supernatants of cultured Mo and AM. These results indicate that sIL-2R in serum is an useful index of the disease activity of sarcoidosis, and may be mainly derived from IL-2R on Mo and AM.

Suggestive evidence for functionally distinct, tumor-suppressor genes on chromosomes 1 and 11 for a human fibrosarcoma cell line, HT1080.

One approach for identifying chromosomes which carry putative tumor-suppressor genes is the introduction of specific chromosomes into the tumor cells of interest. We examined the ability of human chromosomes derived from normal fibroblasts to suppress or modulate tumorigenicity in nude mice and the in vitro properties of HT1080, a human fibrosarcoma cell line. We first isolated mouse A9 cells containing a single human chromosome (1, 2, 7, 11, or 12) integrated with pSV2neo plasmid DNA. Following fusion of microcells from these A9 cells with the HT1080 cells, clones that were resistant to G418 were isolated and karyotypically analysed. Three of 4 microcell-hybrids with an introduced chromosome 1 were non-tumorigenic (#1-7, -8 and -13), whereas the parental HT1080 cells were highly tumorigenic. The other microcell-hybrid clone (#1-1) formed tumors, the cells of which had lost one copy of chromosome 1. Two clones from the #1-1 cells were isolated; one contained an extra copy of chromosome 1, and the other one did not. The former was non-tumorigenic and the latter was tumorigenic. The introduction of chromosome 11 also suppressed the tumorigenicity of HT1080 cells, while the introduction of other chromosomes, i.e., 2, 7, or 12, had minimal or no effect on the tumorigenicity of these cells. Cells from tumors formed by microcell-hybrids with the introduction of chromosome 2, 7, or 12 still contained the introduced chromosome. Interestingly, only the microcell-hybrids with an introduced chromosome 1 had an alteration in cellular morphology and modulation of in vitro transformed properties, i.e., cell-growth and saturation density in a medium containing 10% calf serum and cell-growth in soft-agar. Thus, the results indicate the presence of putative tumor-suppressor genes for HT1080 cells on chromosomes 1 and 11, and further suggest that the genes on these chromosomes control different neoplastic phenotypes.

Normal human chromosome 1 carries suppressor activity for various phenotypes of a Kirsten murine sarcoma virus-transformed NIH/3T3 cell line.

In order to identify chromosomes that carry putative tumor-suppressor genes for the various phenotypes of Kirsten sarcoma virus-transformed NIH/3T3 (DT) cells, we performed microcell-mediated chromosome transfer into DT cells. We first isolated mouse A9 clones, containing a single human chromosome 1, 11 or 12 tagged with pSV2-neo plasmid DNA. Then, chromosome 1, 11 or 12 was transferred from the A9 clones into DT cells by microcell fusion. The growth rate, colony-forming ability in soft agar and tumorigenicity of the DT cells were controlled by chromosome 1, but not by chromosome 11 or 12, indicating that normal human chromosome 1 carries a putative tumor-suppressor gene(s) that affects various transformed phenotypes of DT cells.

Transformation of Lactobacillus helveticus subsp. jugurti with plasmid pLHR by electroporation.

Lactobacillus helveticus subsp. jugurti was transformed with plasmid, pLHR (8.5 kilobases), by electroporation. The plasmid, pLHR, consists of a cryptic plasmid, pLJ1, from L. helveticus subsp. jugurti, the Escherichia coli vector pBR329, and the erythromycin resistance gene of pAM beta 1 from Enterococcus faecalis. Maximum transformation efficiency of 1.3 x 10(4) transformants per microgram of DNA was obtained by exposure to a pulse with an exponential decay waveform at 4 kV/cm with 25 microF capacitance. The presence of glycine in the growth medium was essential for transformation. Plasmid DNA isolated from transformants had not undergone detectable rearrangements or deletions. In addition, it was found that L. helveticus subsp. jugurti has a restriction and modification system.