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.

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.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta.

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen.

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.

Analysis of NMR spectra of sugar chains of glycolipids by 1D homonuclear Hartmann-Hahn and NOE experiments.

We applied 1D homonuclear Hartmann-Hahn (1D-HOHAHA) and difference NOE experiments to determine the chemical structure of Forssman's antigen, a glycolipid purified from sheep red blood cells. The subspectra corresponding to the individual sugar components were extracted from overlapping proton resonances by selective excitation of the anomeric proton resonances, so that unambiguous assignments of the sugar proton resonances were accomplished. Then, difference NOE experiments were performed to determine the linkage of the sugar units. The present procedure was found to be useful for the structure determination of glycoconjugates and also reduces the amount of samples and machine time.

Analysis of NMR spectra of sugar chains of glycolipids by multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy.

We applied multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy to globoside, a glycolipid purified from human red blood cells. The subspectra corresponding to individual sugar components were extracted even from overlapping proton resonances by taking the cross sections of 2D spectra parallel to the F2 axis at anomeric proton resonances, so that unambiguous assignments of sugar proton resonances were accomplished.

Antagonism of the hypnotic effect of ethanol in mice by an alpha-1 adrenoceptor agonist.

A lipid soluble alpha 1-adrenoceptor agonist 2-(2-chloro-5-trifluoromethylphenylimino) imidazolidine (St 587) antagonized the hypnotic effect of ethanol in C57Bl/6 and CD-1 mice. In Swiss-Webster mice the effect of St 587 was weak and in BALB/c mice this drug potentiated ethanol hypnosis. St 587 did not enhance the elimination of ethanol. Cirazoline, an alpha 1-adrenoceptor agonist which is more potent than St 587, was relatively more effective in antagonizing the ethanol-induced hypnosis. Though it appears that St 587 exerted its ethanol antagonism by virtue of its alpha 1-adrenoceptor agonistic effect, other contributing factors may also have to be considered. St 587 may prove to be of value in understanding the mechanism of action of ethanol and in the treatment of acute ethanol intoxication.

An in vivo pharmacological method for the quantitative evaluation of the central effects of alpha 1 adrenoceptor agonists and antagonists.

A new in vivo pharmacological method for the quantitative evaluation of alpha 1-adrenoceptor agonists and antagonists has been developed. It consists of recording the myoclonic twitch activity (MTA) of the suprahyoideal muscle of rats anesthetized with urethane. In these animals, the isomers of amphetamine elicited myoclonic twitch activity; their effects were dose-related and the d-isomer was approximately 3.5 times more effective than the l-isomer. While pimozide did not block this response, the postsynaptic alpha 1-antagonist prazosin fully blocked the myoclonic twitch activity induced by d-amphetamine. Other postsynaptic alpha 1-antagonists, such as haloperidol, phenoxybenzamine and clozapine, were also effective in blocking this response to d-amphetamine. Since d-amphetamine elicited myoclonic twitch activity in rats pretreated with reserpine and alpha-methyl-p-tyrosine, it was concluded that d-amphetamine exerted a direct alpha 1-adrenoceptor stimulation. In rats pretreated with nialamide and pimozide, l-DOPA elicited myoclonic twitch activity which was dose-related. This effect of l-DOPA was promptly and fully blocked by prazosin. It was concluded that this response to l-DOPA resulted from stimulation of alpha 1-adrenoceptors. The relative potencies of four alpha 1-adrenoceptor stimulants, namely, cirazoline, St-587, (-)SKF 89748A and Sgd 101/75 were determined using this method. The results correlated very well with their relative potencies to increase the diastolic blood pressure of pithed rats. Evidence that myoclonic twitch activity is a centrally-mediated response has also been presented. It appears that the method is a simple, sensitive, versatile and easily quantifiable procedure for the evaluation of the central effects of alpha 1-adrenoceptor agonists and antagonists.

Studies on the interaction between ethanol and amfonelic acid.

Amfonelic acid (AFA), a non-amphetamine central stimulant dose-dependently reduced the hypnotic effect of ethanol in C57B1/6 mice. It did not enhance the elimination of ethanol. Amfonelic acid failed to modify the duration of pentobarbitone-induced hypnosis or the ethanol-induced hypothermia in these animals. Combined treatment with amfonelic acid and a lipophilic alpha 1-adrenoceptor agonist was not more effective than amfonelic acid alone in blocking ethanol hypnosis. The stimulation of locomotor activity by amfonelic acid in C57B1/6 mice was more sensitive to the blocking effect of ethanol than stimulation induced by d-amphetamine. The blocking effect of amfonelic acid, but not that of d-amphetamine, on the effects of ethanol developed tolerance. In pimozide-pretreated mice, amfonelic acid failed to reduce the ethanol-induced hypnosis. Hence it appears that dopamine (DA) released by amfonelic acid is responsible for its antagonism of ethanol. However, though amfonelic acid acted as a strong releaser of DA in Swiss-Webster, CD-1, DBA-2 and BALB/c mice, in these strains it failed to reduce the effect of ethanol. Moreover, methylphenidate, a dopaminergic stimulant, which acts by a mechanism similar to that of amfonelic acid was not effective in reducing the hypnotic effect of ethanol in C57B1/6 mice. For these reasons, additional mechanisms may have to be considered to explain this strain-dependent effect of amfonelic acid.

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.

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases.

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.0)/methanol (30/l, by volume) as a mobile phase. There was a linear relationship between the fluorescence emission (peak height), and the amount of each authentic disaccharide used for the coupling reaction. This method was applied to analyze commercially available chondroitin sulfates A and C, dermatan sulfate, and urinary glycosaminoglycans obtained from patients with mucopolysaccharidosis after digestion with chondroitinases. The data indicated that the present method is useful for the separation and quantitation of nmol-pmol levels of the unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases and can be used for their structural characterization.

Thrombin-inhibitory activity of whale heparin oligosaccharides.

Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed.

Further studies on the ethanol antagonism exhibited by 2(2-chloro-5-trifluoromethyl phenylimino) imidazolidine (St 587).

A lipid soluble alpha 1-adrenoceptor agonist 2-(2-chloro-5-trifluoromethyl phenylimino) imidazolidine (St 587) dose-dependently antagonized the hypnotic, hypothermic and respiratory depressant effects of ethanol in C57B1/6 mice. This effect was present whether St 587 was given before or after ethanol. St 587 did not block the pentobarbitone-induced hypnosis. It also did not influence the elimination of ethanol. Combined treatment with a subhypnotic dose of ethanol and St 587 resulted in marked hyperactivity in mice. This effect was completely abolished by pimozide pretreatment. It was inferred that the dopamine released from brain areas by this dose of ethanol together with the norepinephrine receptor activation offered by St 587 resulted in this hyperactivity. Cirazoline, a more potent alpha 1-adrenoceptor agonist than St 587 was relatively more effective than the latter in blocking the ethanol-induced hypnosis in mice. It seems that alpha 1-adrenoceptor stimulation is a major contributing factor to the ethanol antagonism exerted by St 587. This drug might prove to be useful in the treatment of acute ethanol intoxication and in understanding the mode of action of ethanol.

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.

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.

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.

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.

[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.