Exfoliation of Helicobacter pylori from gastric mucin by glycopolypeptides from buttermilk.

In the human stomach, Helicobacter pylori, an ulcer pathogenic bacterium, colonizes the gastric mucosal layer primarily. The ability of glycopolypeptides (GPP) prepared from buttermilk to exfoliate H. pylori bound to gastric mucin was investigated. The GPP were prepared from buttermilk by digestion with trypsin, papain, pancreatin, bromelain, or pepsin. Helicobacter pylori ATCC 43504T and 43579 adhered more strongly to all of the GPP tested than to whole buttermilk, the soluble fraction of buttermilk, gastric mucin prepared from mouse stomach, or commercial pig gastric mucin. The GPP digested with trypsin, papain, or pancreatin were significantly more adherent. When the GPP concentration was 10 mg/mL, bound H. pylori ATCC 43504T, 43579, and 5 clinical isolates were exfoliated markedly from immobilized porcine gastric mucin following treatment with GPP digested with trypsin or pancreatin. This ability of GPP did not correlate with sialic acid content, indicating that sialic acid content is not important in the exfoliation of this microorganism. Such an ability may depend on the structure or number of sugar chains, or the position of sialic acid. We conclude that GPP promote the exfoliation of H. pylori bound to gastric mucin and prevent the de novo adherence of this microorganism. As such, GPP are a promising food material for preventing H. pylori infection.

Comparative clinical study of cefcapene pivoxil and cefteram pivoxil in chronic respiratory tract infections by a double-blind method.

In a double-blind study, the efficacy and safety of the novel cephem antibiotic cefcapene pivoxil (CFPN-PI; 450 mg/day) was compared with cefteram pivoxil (CFTM-PI; 600 mg/day) in 171 patients with chronic respiratory tract infections. There was no significant difference between the clinical efficacy of the two drugs (80.2% for CFPN-PI versus 78.9% for CFTM-PI). There was no significant difference in the rate of elimination of the causative bacteria (60.5% for CFPN-PI versus 65.9% for CFTM-PI). Side-effects were observed in 6.0% of patients treated with CFPN-PI compared with 6.4% of patients treated with CFTM-PI. There were no significant differences in incidence of abnormal laboratory findings following treatment with the two drugs (13.9% for each), and none of the side-effects was severe. We conclude that CFPN-PI (450 mg/day) was as effective and as well tolerated as CFTM-PI (600 mg/day) in the treatment of chronic respiratory tract infections.

Histochemical evaluation for the biological effect of menatetrenone on metaphyseal trabeculae of ovariectomized rats.

To evaluate the biological effects of vitamin K2 (menatetrenone, MK-4) on ovariectomy (OVX)-induced bone loss, we have examined histological alterations of femoral metaphyses of sham-operated (sham group), ovariectomized (OVX group), and MK-4 dietary-supplemented OVX (MK-4 group; 50 mg/kg per day) female Fischer rats 1, 2, 5, and 8 weeks after OVX. In the first week, rats of the OVX and MK-4 groups showed discontinuous trabeculae compared with sham-operated rats. At 2 weeks after OVX, the OVX rats revealed many large tartrate resistant acid phosphatase (TRAP)-positive osteoclasts, while osteoclasts in the MK-4-treated rats were similar in size to those of the sham group. At 5 weeks, the OVX and MK-4 groups revealed fragmented trabeculae in femoral metaphyses. The cartilage matrix was partially exposed due to stimulated bone resorption in the OVX group, but not in the MK-4 group. After 8 weeks, the OVX rats had little metaphyseal trabeculae, whereas the MK-4-treated rats had maintained short trabeculae. Despite the presence of intense alkaline phosphatase-positive osteoblasts on trabeculae in the MK-4 group, TRAP-positive osteoclasts were flattened without developing ruffled borders. Therefore, MK-4 appeared to lessen the increase in osteoclastic bone resorption induced by OVX, as well as to maintain the accelerated osteoblastic activity. It is of importance to identify the target cells for MK-4 in bone. Autoradiography localized [3H]-labeled MK-4 mainly in osteoblasts and adjacent bone matrices, but not in osteoclasts, indicating that MK-4 targets osteoblasts. Thus, MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy.

Effect of Japanese horseradish oil on methane production and ruminal fermentation in vitro and in steers.

The effects of alpha-cyclodextrin-horseradish oil complex (CD-HR) on methane production and ruminal fermentation were studied in vitro and in steers. In the in vitro study, diluted ruminal fluid (30 mL) was incubated anaerobically at 38 degrees C for 6 h with or without CD-HR, using cornstarch as substrate. The CD-HR was added at various concentrations (0, 0.17, 0.85 and 1.7 g/L). Treatment affected neither the pH of the medium nor the number of protozoa. Total VFA increased in a linear manner (P = 0.02), and NH3-N decreased quadratically (P = 0.04) as the concentration of CD-HR increased from 0.17 g/L to 1.7 g/L. Molar proportions of acetate decreased in a linear manner (P = 0.03), and propionate increased linearly (P = 0.008) with increasing concentrations of CD-HR. Production of methane was inhibited up to 90%, whereas accumulation of dihydrogen was increased 36-fold by 1.7 g/L of CD-HR supplementation relative to controls. The effect of CD-HR on methane production, ruminal fermentation and microbes, and digestibility was further investigated in vivo using four Holstein steers in a crossover design. The CD-HR supplement was mixed into the concentrate portion of a (1.5:1) Sudangrass hay plus concentrate mixture that was fed twice daily to the steers. Ruminal samples were collected 0, 2, and 5 h after the morning feeding. No effects of CD-HR supplementation on ruminal pH (P = 0.63) or protozoal numbers (P = 0.44) were observed. Molar proportion of acetate was decreased (P = 0.04) and propionate was increased (P = 0.005) by CD-HR treatment. Molar proportion of butyrate was increased (P = 0.05) in CD-HR-supplemented steers. Ruminal NH3-N was decreased (P = 0.05) by treatment. Blood plasma glucose concentration was increased (P = 0.02) and urea-N was decreased (P = 0.04) with CD-HR supplementation. Daily DMI was decreased (P = 0.04), and apparent digestibility of DM (P = 0.13), NDF (P = 0.14), and CP tended (P = 0.14) to be increased by treatment. Methane production was decreased (P = 0.03) by 19%, and the number of methanogens was also decreased (P = 0.03). Although N retention (P = 0.11), total viable bacteria (P = 0.15), and sulfate-reducing bacteria (P = 0.17) were not significantly altered by treatment, tendencies for increases were noted with CD-HR supplementation. The number of cellulolytic (P = 0.38) and acetogenic bacteria (P = 0.32) remained unchanged by treatment. These results indicate that CD-HR supplementation can be used to decrease methane production in steers.

Vitamin K2 (menatetrenone) inhibits bone loss induced by prednisolone partly through enhancement of bone formation in rats.

Vitamin K(2) (K(2), menatetrenone) has been reported to enhance bone formation and inhibit bone resorption in vitro. However, there is no evidence that K(2) enhances bone formation in vivo. The aim of this study was to characterize the effect of K(2) on bone formation in vivo. We carried out two experiments using a prednisolone (pred)-induced bone loss model in male (10-week-old) Fischer rats. Pred was orally administered three times a week. In experiment 1, we compared the degree of bone loss induced by a 4 week treatment (30 or 100 mg/kg) and an 8 week treatment (3, 10, or 30 mg/kg) with pred by peripheral quantitative computed tomography (pQCT). At 4 weeks, total bone mineral density (BMD) was decreased only with the 100 mg/kg pred treatment. At 8 weeks, total BMD was significantly reduced at >10 mg/kg pred. In experiment 2, we investigated the effect of K(2) on bone loss induced by 3 and 30 mg/kg pred. K(2) (15 mg/kg) was given to rats as a dietary supplement for 8 weeks. Intestinal calcium transport (S/M) and total, trabecular, and cortical BMD at the metaphysis and diaphysis were measured, and histomorphometry was performed in diaphysial cross sections. Pred treatment decreased total and trabecular BMD in the proximal metaphysis. A decrease in cortical BMD in the diaphysis was observed in the pred 30 mg/kg group. Pred treatment also reduced mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS). The decrease in total and trabecular BMD in the proximal metaphysis, and in cortical BMD in the diaphysis, was inhibited by K(2) treatment. K(2) treatment also inhibited the decrease in MS/BS and BFR/BS induced by 30 mg/kg pred. These results suggest that K(2) prevents bone loss partly through the enhancement of bone formation.

[Effect of combined treatment with vitamin K2 and 1 alpha-(OH)-vitamin D3 on bone loss in ovariectomized rats].

Different types of therapeutic agents for osteoporosis are often simultaneously prescribed for the same patient, but limited experimental findings indicate the significance of combined treatment. In the present study, the inhibitory effect of combined vitamin K2(K2) and 1 alpha-(OH)-vitamin D3(D3) treatment on bone loss was compared to that of K2 or D3 alone in ovariectomized rats. Female rats (20-week-old) were ovariectomized and divided into 4 groups as follows: they were treated for 8 weeks with vehicle, K2 (30 mg/kg), D3 (0.3 microgram/kg) and K2 and D3 (K2 + D3) at the respective doses. K2 was given as a dietary supplement and D3 was orally administered 3 times a week. Bone density of the femurs was measured by peripheral quantitative computed tomography. Ovariectomy resulted in decreased bone density in proximal metaphysis, especially in the trabecular region, and treatment with K2, D3 or K2 + D3 inhibited this decrease. Moreover, in the K2 + D3 group, bone density and mineral content in the trabecular region in proximal metaphysis and cortical bone width in diaphysis were significantly higher than those in the D3 group. Consistent with these observations, bone strength in the femoral midshaft tended to increase only in the K2 + D3 group compared to that in the vehicle group. These findings indicate that combined K2 and D3 treatment is more effective for bone loss than that with K2 or D3 alone.

Major metabolites of ginseng sapogenins formed by rat liver microsomes.

Incubation of ginseng sapogenins with microsomes from rat liver resulted in formation of their 20,24-epoxides as major metabolites. Identification of the metabolites was performed by HPLC, FAB-MS and EI-MS.

Screening of wound-responsive genes identifies an immediate-early expressed gene encoding a highly charged protein in mechanically wounded tobacco plants.

In order to identify genes that are temporally and spatially regulated during wound response, a cDNA population in mechanically wounded tobacco leaves was screened by the fluorescence differential display method. Of 28 clones initially identified to have altered levels of transcripts within 3 h of wounding, eight were characterized. Although each clone showed a unique pattern of transcript accumulation, one distinct clone was further characterized because of its immediate-early response. Its transcripts began to accumulate 10 min after wounding, reached a maximum level within 1 h and disappeared after 2 h. The response, which occurred repeatably and systemically, was observed by the treatment with propionic acid or erythrosin B, indicating that cytosolic acidification could be one of the signals for immediate-early response of this gene. The cDNA encodes a polypeptide of 513 amino acids with a relative molecular mass of 60,952. The putative polypeptide is rich in lysine (K), glutamic acid (E) and aspartic acid (D), which constitute up to 70% of total amino acids, and was therefore designated as KED. The KED polypeptide is composed of a highly hydrophilic N-terminal region and a relatively hydrophobic C-terminal region, suggesting that KED may function through electrostatic interactions with cellular components.

Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding.

An immediate-early, transiently activated wound-responsive gene was identified in tobacco by fluorescent differential display screening. The full-length cDNA encodes a polypeptide of 356 amino acids with a relative molecular mass of 39,082 Da. The deduced amino acid sequence shows two characteristic features; a leucine-zipper motif found in the more N-terminal region and a WRKY domain containing a zinc-finger motif located in the central region. The gene was designated as wizz (wound-induced leucine zipper zinc finger). Northern analysis showed that upon wounding wizz transcripts were locally and systemically accumulated within 10 min, reached a maximum level by 30 min, and decreased thereafter to the basal level. Analyses of a WIZZ-GFP fusion protein clearly indicated that WIZZ is a nuclear factor. WIZZ specifically binds to sequences containing two TTGAC core motifs that are separated by a spacer of appropriate length. The binding activity was dependent on bivalent cations, most probably zinc. In transient reporter assays, however, WIZZ did not show transactivation activity in tobacco suspension cells, suggesting that it functions together with other components. The results indicate that WIZZ is a new transcription factor which participates in early stages of the wound response.

Enzymatic synthesis of N-acetylglucosaminyl-cyclodextrin by the reverse reaction of N-acetylhexosaminidase from jack bean.

Novel heterobranched cyclodextrins (CDs), N-acetylglucosaminyl-cyclodextrins (GlcNAc-CD), were synthesized from a mixture of GlcNAc and alpha, beta, or gamma CD by the reverse reaction of N-acetylhexosaminidase from jack bean. Optimum pH and temperature for the production of GlcNAc-alpha CD by N-acetylhexosaminidase were pH 4.9 and 50-70 degrees C, respectively. The maximum yield of GlcNAc-alpha CD was 17.5% (mol/mol) at the concentration of 1 M GlcNAc and 0.25 M alpha CD. The reverse reaction product, GlcNAc-alpha CD, was separated into two peaks by HPLC analysis on the ODS column. Their structures were identified as 6-O-beta-D-N-acetylglucosaminyl-alpha CD and 2-O-beta-D-N-acetylglucosaminyl-alpha CD by FAB-MS and NMR spectroscopies. N-Acetylhexosaminidase from jack bean also synthesized N-acetylgalactosaminyl-alpha CD from N-acetylgalactosamine and alpha CD.

[Interaction of warfarin and vitamin K2 on arterial thrombotic tendency using a rat aorta loop model].

Vitamin K2(K2), a therapeutic agent osteoporosis, is prohibited for patients with thrombosis who are receiving warfarin (WF). However, because some aged patients with thrombosis have osteoporosis, some patients treated with WF may be administered K2 concomitantly. We investigated here the interaction between K2 and WF on thrombosis in a rat aorta loop model. Administration of WF at 0.58, 0.82 and 1.16 mg/l in drinking water for 7 days decreased the thrombotic rate and increased the death rate, dose-dependently. Therefore in the following study, 0.80 mg/l of WF was used. After 2 days of WF-treatment, 1.5, 14 and 145 mg/kg of K2 was administered for 5 days. The blood coagulation time was markedly prolonged by WF treatment for 7 days and this effect was completely inhibited by all doses of K2. WF treatment significantly decreased the cumulative thrombotic rate for 5 days. Administration of 1.5 and 14 mg/kg of K2 did not influence the WF effect on thrombosis. The thrombotic rate in the 145 mg/kg K2 group was lower than that in the WF-control group, but similar to that in the WF-untreated group. These findings suggest that high dose of K2 reduces the effect of WF on thrombosis but does not enhance the occurrence of thrombosis more than that without WF treatment.

Novel antibiotic susceptibility tests by the ATP-bioluminescence method using filamentous cell treatment.

Antimicrobial susceptibility testing by the ATP-bioluminescence method has been noted for its speed; it provides susceptibility results within 2 to 5 h. However, several disagreements between the ATP method and standard methodology have been reported. The present paper describes a novel ATP method in a 3.5-h test which overcomes these deficiencies through the elimination of false-resistance discrepancies in tests on gram-negative bacteria with beta-lactam agents. In our test model using Pseudomonas aeruginosa and piperacillin, it was shown that ATP in filamentous cells accounted for the false resistance. We found that 0.5% 2-amino-2-methyl-1,3-propanediol (AMPD) extracted ATP from the filamentous cells without affecting normal cells and that 0.3 U of adenosine phosphate deaminase (APDase)/ml simultaneously digested the extracted ATP. We used the mixture of these reagents for the pretreatment of cells in a procedure we named filamentous cell treatment, prior to ATP measurements. This novel ATP method with the filamentous cell treatment eliminated false-resistance discrepancies in tests on P. aeruginosa with beta-lactam agents, including piperacillin, cefoperazone, aztreonam, imipenem-cilastatin, ceftazidime, and cefsulodin. Furthermore, this novel methodology produced results which agreed with those of the standard microdilution method in other tests on gram-negative and gram-positive bacteria, including P. aeruginosa, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, for non-beta-lactam agents, such as fosfomycin, ofloxacin, minocycline, and aminoglycosides. MICs obtained by the novel ATP method were also in agreement with those obtained by the agar dilution method of susceptibility testing. From these results, it was shown that the novel ATP method could be used successfully to test the activities of antimicrobial agents with the elimination of the previously reported discrepancies.

[Seasonal changes of laboratory data in patients with Japanese cedar pollinosis].

Japanese cedar pollen (JCP) causes a significant seasonal allergic rhinitis in Japan during the early spring. Blood samples were collected monthly from October 1993 through October 1994 from 11 patients with Japanese cedar pollinosis. The patients were segregated into two categories based on specific IgE (RAST): single positive RAST to JCP only and multiple positive RAST to JCP, house dust (H1) and mite (D1). These two populations differed in levels of total serum IgE, numbers of eosinophils, basophils and neutrophils in peripheral blood and clinical symptoms. Seasonal increase of JCP-specific IgE was observed after pollen season in both groups. In the single positive group, but not in the multiple positive group, seasonal increase of the number of eosinophils in peripheral blood was observed with post seasonal fall and the level of total serum IgE was increased in the same manner as that of the JCP specific IgE. Although it was not significant, there was a broad seasonal increase of serum nitrate anion, a metabolite of nitric oxide.

Processing of cathepsins L, B and D in psoriatic epidermis.

Proteinase activity is increased in psoriatic epidermis. To elucidate the involvement of enzymes in psoriatic epidermis, the expression of cathepsins, L, B and D was investigated by Western blotting and immunohistological studies. Normal epidermis contained abundant inactive precursors (39 kDa) of cathepsins L and B and an inactive intermediate form (45 kDa) of cathepsin D. Cathepsin L in psoriasis was processed to a variable extent from the precursor to a single-chain form (30 kDa) and a mixture of single- and heavy-chain (25 kDa) forms of the active mature enzyme, corresponding to the immunohistological staining patterns 'diffuse dense', 'small granular', and unevenly distributed 'condensed granular'. Cathepsin B showed a mixture of precursor form (39 kDa) and single-chain (30 kDa) forms and was expressed as a 'diffuse dense' staining pattern in the mid-spinous layer and as a 'condensed' pattern in the upper spinous and granular layers. Cathepsin D was processed to the heavy-chain (31 kDa) form of activated mature enzyme with small granular staining and a mixture of heavy-chain and degraded protein (28 kDa) with larger and more condensed granular staining. The distribution patterns of 'small granular' cathepsin L, and of cathepsins B and D expression in suprabasal keratinocytes were very similar to that of involucrin. After complete clinical resolution of psoriasis by 8-methoxypsoralen plus UVA treatment, the expression of the three cathepsins was normalized. These results suggest that cathepsins L, B and D in forms activated to a variable extent may be involved in the pathology of psoriasis.

The utility of volatile hydrocarbon analysis in cases of carbon monoxide poisoning.

A new approach to investigate the circumstances relating to carbon monoxide intoxication by analysing volatile hydrocarbons in the blood of cadavers is reported. Headspace gas chromatography/mass spectrometry was used to demonstrate the hydrocarbons. The results can be characterized into four categories depending on the compounds detected. In construction fire cases where no accelerants were found at the scene benzene, toluene and styrene were detected in the blood. In cases where gasoline was found in the fire debris surrounding the victim, high levels of benzene, toluene, ethylbenzene, xylene isomers, n-hexane and n-heptane were detected in the blood. In cases where kerosene was found in the fire debris around the victim, benzene, toluene, ethylbenzene, xylene isomers, C9-aromatics(n-propylbenzene, trimethyl-benzene isomers), n-octane, n-nonane and n-decane were detected in the blood. In cases where the victim was found inside a gasoline-fuelled automobile filled with exhaust gas, benzene, toluene, ethylbenzene, xylene isomers, C9-aromatics were found, but no aliphatic hydrocarbons such as components of petroleum. The analyses of the combustion gases of inflammable materials, exhaust gas, gasoline vapours and kerosene vapours were also performed to evaluate the results of the blood analyses. Consequently, some compounds are proposed as indicators to discriminate between inhaled gases i.e. styrene in common combustion gas, n-hexane and n-heptane as well as benzene, toluene and C9-aromatics in gasoline cases, n-nonane and n-octane as well as benzene, toluene and C9-aromatics in kerosene cases, and benzene, toluene, C9-aromatics but no aliphatic hydrocarbons in exhaust gas cases.

Isolation and characterization of novel heterogeneous branched cyclomalto-oligosaccharides (cyclodextrins) produced by transgalactosylation with alpha-galactosidase from coffee bean.

Transgalactosylated derivatives of cyclomalto-hexaose (alpha CD), -heptaose (beta CD), and -octaose (gamma CD) were synthesized by alpha-galactosidase from coffee bean using melibiose as a donor and alpha CD, beta CD or gamma CD as an acceptor. Mono- and di-O-alpha-D- galactosylated CDs were isolated and purified by HPLC. Their structures were elucidated by fast-atom bombardment mass spectrometry (FABMS) and 13C NMR spectroscopy. For structural determination of positional isomers of 6(1),6n-di-O-alpha-D-galactosyl-CDs, digestion products with cyclodextrin glucanotransferase were analyzed by HPLC and FABMS.

In vivo administration of IL-1 beta accelerates silk ligature-induced alveolar bone resorption in rats.

The effects of recombinant human interleukin-1 beta (rhIL-1 beta) on alveolar bone resorptive activity in rats were examined. Continuous administration of rhIL-1 beta or phosphate-buffered saline (PBS) was given via osmotic pumps for 3, 7 and 14 days to rats with silk ligatures around second maxillary molars. Other animals without ligatures received insertion of pumps containing rhIL-1 beta or remained untreated. Sections were subject to three different stains:--hematoxylin and eosin (H-E) for histology, acid phosphatase (ACPase) activity for osteoclast detection, and immunohistochemistry using anti-rat monocyte/macrophage monoclonal antibody (ED 1). In addition, body weight, plasma calcium and phosphorus levels were monitored. The mean body weight of rats receiving rhIL-1 beta was significantly lower (P < 0.05 to P < 0.01) compared with untreated rats throughout the experimental period. On Day 7, plasma calcium and phosphorus levels were significantly lower in rats receiving rhIL-1 beta than in rats receiving PBS only (P < 0.05). Sections revealed a moderate inflammatory cell infiltrate reaching near the alveolar crest in both groups with ligatures on Day 3. Only rats receiving rhIL-1 beta exhibited enhancement of inflammatory cell invasion on Days 7 and 14. In rats receiving rhIL-1 beta with ligatures, numerous resorption lacunae containing ACPase-positive multinucleated giant cells (MNGCs), coinciding with ED1-positive cells, were located on the mesial side of the septum where extensive bone resorption had occurred throughout the experimental period. In animals receiving rhIL-1 beta without ligatures, compared with untreated rats, increased ACPase-positive cells were observed on the mesial side of the septum on Day 3. In animals receiving PBS only, a few ACPase-positive cells were observed confined to the mesial regions where slight bone resorption occurred on Days 7 and 14. These results indicate that the administration of rhIL-1 beta accelerated alveolar bone destruction in ligature-induced periodontal tissue inflammation over a two-week period.

Relaxation effect of an audiovisual system on dental patients. Part 2. Palus-amplitude.

Dental care services include relaxation of patients to reduce their displeasure at and tension during treatment. For this purpose, it is common to use audio or audiovisual programs, that is, to let them listen to music or watch TV. However, the former utilizes only the auditory sense, and the latter is disadvantageous in that a TV set is a rather large-scale unit. In the present study, we determined the relaxation effect of an audiovisual instrument on dental patients. The instrument, which gives virtual vision, was composed of a lightweight eyeset of the snow goggle type that has a built-in TV screen, with earphone and accessories. The relaxation effect was determined using a polygraph.