Effects of ingesting milk fermented by Lactococcus lactis H61 on skin health in young women: a randomized double-blind study.

We conducted a randomized double-blind trial to evaluate the effects of fermented milk produced using only Lactococcus lactis strain H61 as a starter bacterium (H61-fermented milk) on the general health and various skin properties of young women. Healthy female volunteers (n=23; age=19-21r) received H61-fermented milk (10(10) cfu of strain H61/d) or conventional yogurt (10(10) cfu of both Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus per day), as a reference food, daily for 4 wk. Before and at the end of 4 wk, blood samples were taken, and skin hydration (inner forearms and cheek) and melanin content, elasticity, and sebum content (cheek only) were measured. Skin hydration at the inner forearm was higher at wk 4 than at wk 0 in both groups. Sebum content in cheek rose significantly after intervention in the H61-fermented milk group, but not the conventional yogurt group. Other skin parameters did not differ in either group. Serum analysis showed that total protein concentration and platelet count were elevated and reactive oxygen species decreased in both groups after the intervention. Although H61-fermented milk and conventional yogurt had similar effects on skin status and some blood characteristics of participants, an increase of sebum content in cheek is preferable to H61-fermented milk. As skin lipids contribute to maintaining the skin barrier, H61-fermented milk would provide beneficial effects on skin for young women.

Use of PCR-DHPLC with fluorescence detection for the characterization of the bacterial diversity during cassava (Manihot esculenta Crantz) fermentation.

Denaturing high-performance liquid chromatography (DHPLC) has been described as a suitable method to study DNA polymorphisms. Here, cassava (Manihot esculenta Crantz) fermentation liquor was examined using DHPLC analysis to characterize the bacterial diversity during the fermentation process. GC-clamped amplicons corresponding to a variable region of the bacterial community 16S rDNA were synthesized using polymerase chain reaction (PCR) and then resolved on a base-composition basis using preparative DHPLC. Eluate fractions were collected at random and used as a source of whole community DNA that could be used to determine the bacterial diversity. As a first approach, GC-clamps were removed from the eluted DNA fragments using PCR to avoid the possible bias these clamps could cause during the construction of clone libraries. As a second approach, a clone library of each eluate sample was constructed, preserving the GC-clamps of the DNA fragments. The first approach generated 132 bacterial rDNA sequences with an average size of 200 bp, 45% of which had similarity to unculturable or non-classified bacteria. The second approach produced 194 sequences identified as Proteobacteria (48%), uncultured or non-classified environmental bacteria (40%) and Firmicutes (12%). We detected a remarkably greater bacterial diversity using the first approach than the second approach. The DHPLC-PCR method allowed for the fast and non-laborious detection of a vast bacterial diversity that was associated with cassava fermentation, and we conclude that it is a promising alternative for the characterization of the overall microbial diversity in complex samples.

On-column ligand synthesis coupled to partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to a receptor.

This paper describes a two-step procedure whereby on-column ligand synthesis and partial-filling affinity capillary electrophoresis (PFACE) are sequentially coupled to each other to determine the binding constants of 9-fluorenylmethoxy carbonyl (Fmoc)-amino acid-D-Ala-D-Ala species to vancomycin (Van) from Streptomyces orientalis. In this technique four separate plugs of sample are injected onto the capillary column and electrophoresed. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and running buffer, respectively. The fourth sample plug contains an increasing concentration of Van partially-filled onto the capillary column. Upon electrophoresis the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the plugs of Van and Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Analysis of the change in the relative migration time ratio of the Fmoc-amino acid-D-Ala-D-Ala peptide relative to the non-interacting standards, as a function of the concentration of Van, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques.

Molecular genetic, biochemical, and clinical studies in three families with cardiac Fabry's disease.

The variant form of Fabry's disease, called cardiac Fabry's disease, which has left ventricular hypertrophy as its main clinical manifestation is not uncommon. Because there has been no pedigree analysis in families with cardiac Fabry's disease, we performed gene analyses, enzyme assays, and cardiac evaluations in 3 distinct families with cardiac Fabry's disease. Gene analyses were performed in all 18 members of 3 families including 3 male probands. Five hemizygotes and 6 heterozygotes were identified. Plasma alpha-galactosidase A activity was measured in all 18 family members. Echocardiography and electrocardiography were performed in the 5 hemizygotes and in 5 of the 6 heterozygotes. The proband and 3 heterozygotes from a pedigree with a mutation in exon 6 of the alpha-galactosidase A sequence leading to a Met296Ile substitution showed a decrease in alpha-galactosidase A activity. In a separate pedigree, a proband and his hemizygous brother, with a mutation in exon 2 leading to a Glu66Gln substitution, had a decrease in alpha-galactosidase A activity, whereas 3 heterozygotes had normal values. In the third pedigree, a decrease in alpha-galactosidase A activity was observed in 2 hemizygotes who have a mutation in exon 1 leading to an Ala2OPro substitution. Although all 5 hemizygotes exhibited left ventricular hypertrophy on echocardiography, all 5 heterozygotes lacked this finding. Because plasma alpha-galactosidase A activity was normal in some heterozygotes with cardiac Fabry's disease, gene analysis is essential for an accurate diagnosis. Patients with cardiac Fabry's disease thus show an x-linked form of hypertrophic cardiomyopathy.

Defective control of apoptosis, radiosensitivity, and spindle checkpoint in ataxia telangiectasia.

We examined the regulation of apoptosis, radiosensitivity, and spindle checkpoint in response to DNA-damaging agents in ataxia telangiectasia (AT)-derived lymphoblastoid cell lines (AT-LCLs), which lack AT mutated (ATM) protein expression. In addition to the previous findings that AT-LCLs are defective in regulation of cell cycle at the G1, S, and G2-M checkpoints in response to X-ray irradiation (X-IR) and are highly sensitive to X-IR (J. Biol. Chem., 271: 20486-20493, 1996), we showed for the first time that AT-LCLs were defective in X-IR-associated spindle checkpoint control. The cells were also resistant to early apoptosis as much as LCLs derived from patients with Li-Fraumeni syndrome (LFS-LCLs). Terminal deoxynucleotidyl transferase-mediated nick end labeling assay of LCLs, however, demonstrated a significant increase in apoptotic cells among AT-LCLs cultured over a longer period after X-IR. These findings were in contrast to those of LFS-LCL, which showed very little increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive population, even in cells with hyperploidy. Thus, although early apoptosis and cell cycle controls in response to DNA damage are disrupted in both ATM and p53 mutations, cells from AT patients are much more susceptible to late-onset apoptosis than those of LFS. These differences may depend on the level of accumulation of DNA damage and/or threshold that triggers late-onset cell death in ATM or p53 mutations. Our findings allow a better understanding of the role of ATM in p53-dependent and independent signal transduction pathways in response to DNA damaging agents.

An atypical variant of Fabry's disease in men with left ventricular hypertrophy.

BACKGROUND: Fabry's disease is considered very rare. Left ventricular hypertrophy is one of the common manifestations in adults with classic hemizygous disease. Recently, several cases of an atypical variant of hemizygous Fabry's disease, with manifestations limited to the heart, have been reported. Therefore, we assessed the incidence of hemizygosity for Fabry's disease among male patients with left ventricular hypertrophy. METHODS: We measured plasma alpha-galactosidase activity in 230 consecutive male patients with left ventricular hypertrophy. Clinical manifestations were assessed, endomyocardial biopsies were performed, and the patients were screened for mutations in the alpha-galactosidase gene. RESULTS: Seven of the 230 patients with left ventricular hypertrophy (3 percent) had low plasma alpha-galactosidase activity (0.4 to 1.2 nmol per hour per milliliter; 4 to 14 percent of the mean value in normal controls). These seven unrelated patients, ranging in age from 55 to 72 years, did not have angiokeratoma, acroparesthesias, hypohidrosis, or corneal opacities, which are typical manifestations of Fabry's disease. Endomyocardial biopsy was performed in five patients and revealed marked sarcoplasmic vacuolization in all five. Samples from four patients were examined by electron microscopy and revealed typical lysosomal inclusions with a concentric lamellar configuration in all four. Two patients had novel missense mutations in exon 1 (Ala20Pro) and exon 6 (Met296lle). The remaining five had no mutations in the coding region of the alpha-galactosidase gene, but the amounts of the alpha-galactosidase messenger RNA were markedly lower than normal. CONCLUSIONS: Seven unrelated patients with atypical variants of hemizygous Fabry's disease were found among 230 men with left ventricular hypertrophy (3 percent). Fabry's disease should be considered as a cause of unexplained left ventricular hypertrophy.

Polyps of the prostatic urethra. Report of four cases.

We described 4 cases of polyps of the prostatic urethra. Three of them had adenomatous polyp with prostatic epithelium and 1 showed polypoid urethritis. These lesions were worth noting as a cause of hematuria, dysuria or hematospermia. Polyps or papillary lesions of the prostatic urethra appear to be more common than have been suspected. Careful attention should be paid to the prostatic urethra in patients with persistent painless hematuria.

An unusual profile of endocrine study in a patient with pre-Cushing's syndrome.

A unilateral adrenal tumour was incidentally detected in a 39-year-old woman with no characteristic features of Cushing's syndrome. Basal levels of glucocorticoids were within normal limits. However, abnormal pattern of plasma cortisol and ACTH was observed. The dexamethasone suppression test and the metyrapone test showed also abnormal response. Adrenocortical scintigram demonstrated high accumulation of the radiopharmaceutical in the tumour region alone. Final diagnosis was "pre-Cushing's syndrome" and a solitary adenoma was removed from the left adrenal gland.

Membranous lipodystrophy (Nasu's disease): a rare cause of neuropathic urinary incontinence.

A 37-year-old man with membranous lipodystrophy (Nasu's disease) was urodynamically studied, to elucidate his urinary incontinence. Although medication failed to improve this unfavorable condition, urodynamic assessment of this patient clearly demonstrated the existence of an uninhibited overactive bladder.

[Possible mechanisms of hematoma formation in the urinary tracts in a patient with idiopathic thrombocytopenic purpura].

A 30-year-old female was admitted to our hospital complaining of hematuria and right flank pain in September, 1987. She had been diagnosed idiopathic thrombocytopenic purpura in 1980, and had similar symptoms before. Hematoma in the right ureter was demonstrated by retrograde pyelography and CT-scanning, and these symptoms improved within one month. Each activity of plasma clotting factors was within normal limits. Enzymatic studies of the urine revealed low values of plasmin-, urokinase-, and kallikrein-like activities in both excerbation and remission. These hemorrhagic tendencies might have been the result of marked thrombocytopenia: After bleeding into the urinary tracts began, the bleeding would tend to form hematoma because of elevated clotting activity; then hematoma would grow due to decreased urine fibrinolytic activities. This suggested that a decline of fibrinolysis in urine might have a promoting effect on the process of hematoma formation.

Serotonin and tryptamine metabolism in the acute hepatic failure model: changes in tryptophan and its metabolites in the liver, brain and kidney.

When heat-killed Propionibacterium acnes, a gram-positive anaerobe, is intravenously injected into mice followed by an intravenous injection of gram-negative lipopolysaccharide (LPS) 7 days later, most of the mice die of massive hepatic cell necrosis within 24 hours of LPS injection. Using this experimental model, acute hepatic failure was induced in mice, and the tryptophan metabolism in the liver, brain and kidney was studied. As a result, the tryptophan level was remarkably high in all three organs, and the metabolism of both the tryptamine pathway and serotonin pathway was induced. However, in the brain, the tryptamine metabolism was more induced compared to the serotonin, suggesting that the metabolites of tryptamine, may be involved in hepatic encephalopathy.

Effects of bile acids on liver cell injury by cultured supernatant of activated liver adherents cells.

When heat-killed Propionibacterium acnes (P. acnes) is intravenously injected into mice followed by an intravenous injection of a small amount of lipopolysaccharide (LPS) 7 days later, most of the mice die of massive hepatic cell necrosis within 24 hours of LPS injection. In addition, when the liver adherent cells including Kupffer cells are separated from the mice 7 days after P. acnes injection and incubated in vitro with LPS, remarkable activity of the cytotoxic factor is found in the culture supernatant. This cytotoxic factor is thought to cause liver injury. Using this experimental model, the effects of various bile acids on liver cell injury were studied. As a result, ursodeoxycholic acid and dehydrocholic acid suppressed liver cell injury induced by the cytotoxic factor. However, cholic acid, deoxycholic acid and chenodeoxycholic acid did not have any hepatocytoprotective effects.

Determination of serum cholestatic factor level by ELISA in drug-induced allergic hepatitis.

We have shown that intrahepatic cholestasis often observed in drug-induced allergic hepatitis may be induced by a kind of lymphokine, the cholestatic factor (CF). In this study, we measured the CF level in the serum of patients with jaundice by ELISA using anti-CF monoclonal antibody. As a result, CF was detected in the serum of most of the patients at the peak of jaundice, but it was not detected when the patients were recovering from jaundice. When the changes in the serum CF level were followed during the clinical course of a patient, it reached its maximum level before that of the serum total bilirubin level and quickly decreased thereafter. These results also suggest that intrahepatic cholestasis in drug-induced allergic hepatitis may be induced by CF.

Tryptophan metabolism in D-galactosamine-induced liver injury.

We have reported that in rats with D-galactosamine-induced liver injury, the serum level of indoleacetic acid (IAA), a metabolite of tryptophan (TRP), increases before the increase in serum transaminase activity. To determine whether this IAA is derived from hepatocytes, isolated hepatocytes were treated with D-galactosamine and loaded with TRP, and the changes in TRP and IAA levels in the culture supernatant of the isolated hepatocytes were measured at various time intervals. As a result, IAA level in the culture supernatant of hepatocytes treated with D-galactosamine and loaded with TRP significantly increased in a time-dependent manner. This indicates that in D-galactosamine-induced liver injury, a metabolic pathway which produces IAA from TRP through tryptamine is present in hepatocytes.

Methods for analysis of urinary glycosaminoglycans.

Methods for the analysis of urinary GAGs that can be used for or are applicable to routine assays are described. The most popular method for isolation of GAGs from a urine sample is CPC precipitation, in spite of the fact that it is time-consuming. To identify the different types of GAGs excreted, separation by one-dimensional cellulose acetate electrophoresis followed by staining with alcian blue or toluidine blue may suffice for routine purposes. Solvents such as barium acetate, calcium acetate, barbital buffer and pyridine-formic acid are used for the separation. However, the separation of the seven types of GAGs by conventional one-dimensional electrophoresis is difficult, and a discontinuous electrophoretic method with barium acetate buffer and barium acetate buffer containing ethanol has proved effective for the separation. HPLC separation methods are used for assaying the profiles of enzymatic digestion products of GAGs. Advanced HPLC methods for separating intact GAGs of different types are currently unavailable. Unsaturated disaccharides produced with heparitinase and/or heparinase from heparan sulphate and oligosaccharides produced by hyaluronidase digestion of hyaluronic acid can be separated by HPLC. For chondroitin sulphate isomers, unsaturated disaccharides produced by digestion of the samples with chondroitinase ABC or chondroitinase AC are separated by HPLC and determined by their UV absorbance or by fluorescence labelling. Highly sensitive quantitation of chondroitin sulphate isomers is possible by these methods, which are also efficient for the investigation of the constituents of GAG polymers. Some of these methods have been applied to urine samples from patients with, e.g., mucopolysaccharidoses.

A comparative 1H NMR study of mouse alpha(1-53) and beta(2-53) epidermal growth factors.

The three-dimensional structure of the mouse epidermal growth factor (EGF) in solution was studied by comparison of the 1H NMR spectra of alpha EGF (1-53) and beta EGF (2-53, des-asparaginyl 1 form). Using pH dependence of chemical shifts and a two-dimensional difference spectrum, the effect of the N-terminal deletion was investigated based on the complete assignment of the proton resonances. The affected residues were all found to be located exactly in the triple-stranded, beta-sheet core in the N-terminal domain of the EGF molecule.

Influence of D-1 receptor system on the D-2 receptor-mediated hypothermic response in mice.

The hypothermia induced by apomorphine, a mixed dopamine (DA) agonist in male Swiss-Webster mice, was not blocked by the selective D-1 antagonist SCH 23390 but was completely blocked by the selective D-2 antagonists haloperidol, sulpiride and YM-09151-2. The selective D-1 agonist SKF 38393 did not elicit hypothermic response but the selective D-2 agonist quinpirole caused a marked lowering of rectal temperature. D-2 antagonists blocked this response to quinpirole. SCH 23390 enhanced and SKF 38393 attenuated the hypothermia induced by quinpirole. Ineffective doses of haloperidol and SKF 38393, when given together, completely blocked the effect of quinpirole. It was concluded that hypothermia is a D-2 receptor mediated response but modulated by the D-1 receptor system. In another series of experiments the influence of neuroleptics and antidepressants on the hypothermic effect of apomorphine and quinpirole was investigated. The hypothermic effect of a low dose (1 mg/kg) of apomorphine was blocked by the D-2 receptor antagonists, but not by classical antidepressants. However, the response to a high dose (10 mg/kg) of apomorphine was blocked by both classical antidepressants and D-2 antagonists (except haloperidol). These drugs did not show similar effect on quinpirole-induced hypothermia. It is clear that the hypothermic response, especially that of quinpirole, is not a suitable model for testing either neuroleptics or antidepressants.

The protective effect of cyclosporin A on experimentally-induced acute hepatic injury in mice.

When heat-killed Propionibacterium acnes (P. acnes) and a small amount of endotoxin lipopolysaccharide (LPS) were intravenously injected into mice at a week's interval, most of them died of massive hepatic cell necrosis. This experimentally-induced acute liver injury was significantly inhibited by cyclosporin A (CsA), resulting in a remarkable improvement of the survival rate. This protective effect of CsA on acute liver injury was also histopathologically confirmed. To study the mechanism by which CsA protected the mice from fatal hepatic injury, adherent cells prepared from the murine liver 7 days after P. acnes injection were incubated with LPS in the presence of CsA, and the effect of CsA on the production of the cytotoxic factor from the adherent cells was estimated. As a result, CsA inhibited the activation of liver adherent cells and suppressed the release of the cytotoxic factor.