Observation of everyday hand-washing behavior of Japanese, and effects of antibacterial soap.

People wash their hands only for a short time outside the home and when preparing meals at home. This may not be sufficient for those who prepare meals because of possible secondary contamination from food. Although washing with a placebo soap for a short period (lathering 3 s and rinsing 8 s) cleansed from hands about 95% of the total coliforms transferred from ground meat, an antibacterial soap further reduced the coliform count significantly (p < 0.01). To effectively avoid secondary contamination, it is recommended that people should more frequently wash their hands, using an antibacterial soap on the areas that have been in contact with raw meat, poultry, seafood, eggs, vegetables and other foods.

Entropic stabilization of the tryptophan synthase alpha-subunit from a hyperthermophile, Pyrococcus furiosus. X-ray analysis and calorimetry.

The structure of the tryptophan synthase alpha-subunit from Pyrococcus furiosus was determined by x-ray analysis at 2.0-A resolution, and its stability was examined by differential scanning calorimetry. Although the structure of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the alpha-subunit alone. The alpha-subunit from P. furiosus (Pf-alpha-subunit) lacked 12 and 6 residues at the N and C termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-alpha-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-alpha-subunit was essentially similar to that of the St-alpha-subunit in the alpha(2)beta(2) complex. The differences between both structures were discussed in connection with the higher stability of the Pf-alpha-subunit and the complex formation of the alpha- and beta-subunits. Calorimetric results indicated that the Pf-alpha-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-alpha-subunit. Rather, the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening of the polypeptide chain play important roles in extremely high stability in Pf-alpha-subunit.

Platelet-dependent thrombin generation in patients with unstable angina pectoris.

Platelet-dependent thrombin generation was assessed during both unstable and stable periods in 59 patients with unstable angina and at rest in 31 healthy controls. Thrombin generation during the unstable period was significantly greater than that at rest in the healthy control group (p < 0.0001) and that during the stable period (p < 0.0001). Changes in thrombin generation were related to the time after onset of unstable angina, not to degree of improvement in the severity of coronary stenosis.

Effects of biopsy on lung metastasis.

In order to clarify whether biopsy promotes lung metastasis, open or needle aspiration biopsy was performed 10 or 21 days after S-SLM osteosarcoma cells were transplanted subcutaneously in Fischer rats. The lungs were excised after six weeks and the lung weight and the number of metastatic nodules were measured. The mean weight was more in open than needle biopsy, and the number of nodules was significantly higher in open biopsy after 10 days, compared to the control. From these results we concluded that open is more likely to promote lung metastases compared to needle biopsy under the specific experimental conditions of this study.

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.

[Identification of PKC isozymes and effect of knockdown of PKC alpha by antisense oligodeoxynucleotide on iNOS expression via interleukin-1 receptor in vascular smooth muscle cells].

Protein kinase C (PKC) family, is now classified into three groups; conventional (cPKC), novel (nPKC) and atypical (aPKC), and to date, 10 members of isozymes have been identified. We have suggested that PKC is essential to interleukin-1 (IL-1)-triggered expression of inducible NO synthase (iNOS), and that by pharmacological analysis, cPKC is not involved in iNOS induction in rat vascular smooth muscle cells (VSMC). In the present study, we identified some PKC isozymes and investigated the effect of PKC alpha knockdown by antisense oligodeoxynucleotide (AS-ODN) strategy on iNOS expression and nuclear translocation of NF-kappa B in RASMC. Western blot analysis revealed the presence of cPKC (alpha), nPKCs (delta and epsilon) and aPKCs (tau and lambda). Short-time (10-20 min) treatment with phorbol 12-myristate 13-acetate (PMA) induced translocation of PKC alpha from cytosolic to particulate fraction. PKC alpha was completely downregulated by treatment with 100 nM PMA for 24 hours. Treatment with AS-ODN against PKC alpha mRNA depleted PKC alpha specifically, and had no detectable effect on the other PKCs. The production of iNOS mRNA, but not nuclear translocation of NF-kappa B, stimulated by IL-1 beta was decreased by PKC alpha knockdown. These results suggest that there are 5 PKC isozymes in RASMC, and that PKC alpha is involved in iNOS expression triggered by IL-1 beta, supporting our previous pharmacological conclusion.

Human platelet activation by thrombolytic agents: effects of tissue-type plasminogen activator and urokinase on platelet surface P-selectin expression.

The mechanisms that underlie reocclusion during thrombolytic therapy have not yet been clarified. The purpose of this study was to investigate the activating effects of tissue-type plasminogen activator and urokinase and the inhibitory effects of acetylsalicylic acid by measuring platelet surface P-selectin as a marker of platelet activation. After addition of urokinase (final concentration 192 U/ml, 1920 U/ml, or 19,200 U/ml) or tissue-type plasminogen activator (final concentration 120 U/ml, 1200 U/ml, or 12,000 U/ml) to platelet-rich plasma from 12 healthy persons, platelet surface P-selectin expression was measured by means of flow cytometry with an anti-CD62 monoclonal antibody. The presence of urokinase and tissue-type plasminogen activator increased platelet surface P-selectin expression in a concentration-dependent manner. In the next step, either 160 mg/day (n = 6) or 660 mg/day (n = 6) acetylsalicylic acid was administered to the 12 healthy persons, and venous blood samples were collected after 7 days of treatment. Platelet surface P-selectin expression was measured with the method used earlier and after addition of tissue-type plasminogen activator or urokinase. Although the effect of acetylsalicylic acid at 160 mg/day on P-selectin expression was minimal, a dose of 660 mg/day suppressed platelet P-selectin expression and inhibited the platelet activating effects of tissue-type plasminogen activator and urokinase in a statistically significant way. Platelets were activated by tissue-type plasminogen activator or urokinase, and this platelet activation was suppressed with administration of acetylsalicylic acid at 660 mg/day.

A novel adrenaline derivative, AZ002, and its hypoglycemic action in yellow KK mice.

AZ002 (L-threo-(3,4-dihydroxy phenyl)-N-methyl serine methyl ester) is a newly synthesized adrenaline derivative. AZ002 caused relaxation of rat jejunum (beta 3-receptors) (ED50 = 18 microM), but did not affect the atrial rate (beta 1) or tracheal relaxation (beta 2) at a concentration of 0.3 mM. The pA2 values for propranolol in inhibiting the isoproterenol- and AZ002-stimulated relaxation of rat jejunum were 6.27 and 6.33, respectively. Thus, AZ002 is a selective agonist for beta 3-adrenoceptor. AZ002 stimulated lipolysis (ED50 = 10 microM) and glucose uptake (ED50 = 1 microM) in rat adipocytes. In both cases, stimulation was antagonized by high concentrations of the beta-adrenoceptor antagonist propranolol, but not by the alpha-adrenoceptor antagonist phentolamine. The effect of AZ002 on glucose uptake was synergistic with that of insulin. AZ002 was also assessed in vivo by using genetically obese mice (KK/Ay strain) with hyperglycemia. Administration of AZ002 in the diet for a week decreased blood glucose and non-esterified fatty acids.

Environmental differences of a pyridine derivative (AU-1421)-access sites between (H+,K+)-ATPase and (Na+,K+)-ATPase.

We investigated the environmental differences of a pyridine derivative-access sites between hog gastric (H+,K+)-ATPase and dog kidney (Na+,K+)-ATPase. The environmental differences are inferred from the marked contrasts in inhibition properties of (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine hydrochloride (AU-1421) between the two adenosine triphosphatases (ATPases). AU-1421 is a hydrophobic pyridine derivative which inhibits (H+,K+)-ATPase and (Na+,K+)-ATPase competitively with respect to K+. AU-1421 inhibition of (Na+,K+)-ATPase activity is preincubation time-dependent, pH-independent (pH 6.4-8.4), and hardly reversible by dialysis. These features are opposite to those found with the gastric (H+,K+)-ATPase. Furthermore, in the presence of dilute sodium dodecyl sulfate (30-120 microM), the inhibitory potency of AU-1421 for (Na+,K+)-ATPase is increased, whereas the inhibitory potency for (H+,K+)-ATPase is decreased. These results favor proposals that protonated species of AU-1421 interact with its access-site of (H+,K+)-ATPase at the exterior surface of the enzyme molecule by ionic interaction, whereas AU-1421 interacts with its access-site of (NA+,K+)-ATPase at the interior domain of the enzyme molecule predominantly by hydrophobic interaction. The differences indicate the existence of a different feature of K(+)-access sites between the two ATPases.

Endothelin converting enzyme of bovine carotid artery smooth muscles.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme in vascular smooth muscle. Like cultured endothelial cells, noncultured vascular smooth muscle homogenate of bovine carotid arteries, converts human big ET- 1 to ET-1 at pH 3.0, pH 5.0 and pH 7.0, and the apparent ratio of these three activities is about 6:5:1, respectively. Peptides generated during incubation of the homogenate and big ET- 1 at the three pHs were identified as ET- 1 by radioimmunoassay and high performance liquid chromatography. The two acid enzymes are in the cytosol (103,000xg sup) and are inhibited by pepstatin A, while the neutral enzyme is sensitive to EDTA or phosphoramidon; 73% of the neutral enzyme activity was membrane-bound and the remainder (27%) cytosolic.

Endothelin-converting enzyme and its in vitro and in vivo inhibition.

We investigated endothelin (ET)-converting enzyme and its localization in the vasculature. The membrane and cytosol fractions of cultured endothelial cells of bovine carotid artery contain phosphoramidon-sensitive ET-converting enzymes, and their molecular weights are about 100 and 540 kDa, respectively. The specific conversion of big ET-1 by these enzymes proceeds at pH 7.0 +/- 0.5, and it is inhibited by EDTA, o-phenanthroline, and phosphoramidon. Big ET-3 is converted by the membrane enzyme at a rate about one-tenth that of big ET-1, but it is not converted by the cytosol enzyme. Big ET-1 (but not ET-1)-induced hypertension in rats was remarkably suppressed by pretreatment with phosphoramidon, and big ET-1 (but not ET-1)-induced contraction of isolated coronary arteries, either with or without the endothelium, was substantially suppressed by phosphoramidon. These results suggest an essential role of phosphoramidon-sensitive enzyme(s) in the vascular conversion of big ET-1, and the existence of such enzymes also in nonendothelial cells. We found three converting enzymes operating at different optimal pH values in noncultured vascular smooth muscle cells; two pepstatin-sensitive, cytosolic acid proteinases and a phosphoramidon-sensitive neutral enzyme(s) in the membrane and cytosol. All of these findings strongly suggest the importance of phosphoramidon-sensitive neutral enzymes in the vascular conversion of big ET-1.

(Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), calcium ions and ethylenediamine as the K(+)-site directed probe for Na+/K(+)-ATPase.

Recently, we have shown that a hydrophobic amine (AU-1421) produces an irreversible inactivation of Na+/K(+)-ATPase activity. This inactivation was prevented by K+ and its congeners. In this study, we examined the possibility of Ca2+ or ethylenediamine as a probe of the K+ occlusion center of Na+/K(+)-ATPase. The inactivation by AU-1421 was prevented by Ca2+ with an apparent high affinity (approximately 0.1 mM). Ca2+ protection was cancelled by high concentrations of ATP, ADP or Mg2+. Ca2+ and K+ were similar in these respects. Kinetic analyses of the above data indicated the presence of two AU-1421 occlusion sites on the enzyme, either one of which is susceptible to Ca2+ occlusion. Ethylenediamine also prevented the inactivation by AU-1421 or by C12E8 solubilization of the enzyme, suggesting that ethylenediamine, like K+, stabilized the enzyme. However, an apparent affinity of ethylenediamine (approximately 1.4 mM) was one order of magnitude lower than that of K+ (approximately 0.2 mM), and the protective manner did not show a simple competition. In addition, ethylenediamine binding was unaffected by ATP or ADP at a low affinity site, and antagonized K+ binding. From these results we concluded that ethylenediamine does not act like K+ or Ca2+ in protecting AU-1421 inactivation, since it can't stabilize the enzyme conformation as an E2 (K(+)-bound form).

Influence of pregnancy on the course of malaria in mice infected with Plasmodium berghei.

The course of malarial infection was compared in pregnant mice inoculated with Plasmodium berghei at different stages of gestation. When 12-14 wk old, pregnant BALB/c mice were inoculated with 1 x 10(6) of P. berghei NK65-infected red cells at gestation day 0, 2, 4, 6, 8, 10, 12, 14 or 16, the mice inoculated on gestation days 6-12 expired 6.5 days after inoculation compared to 9.5 days in non-pregnant mice. Parasitemia in these pregnant mice increased rapidly on day 4 after inoculation and anemia also developed earlier on day 5. However, the degree of parasitemia and anemia in the terminal stage of infection in these pregnant mice was milder than that of non-pregnant controls. Blood urea nitrogen increased at the terminal stage although the degree of increase in mice inoculated on gestation days 6-10 was comparatively small. Pregnant malarial mice died earlier with less physiological changes than non-pregnant controls. It was concluded that pregnancy makes the host susceptible to physiological changes caused by malaria.

A newly synthesized pyridine derivative, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine hydrochloride (AU-1421), as a reversible gastric proton pump inhibitor.

A newly synthesized pyridine derivative, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine hydrochloride (AU-1421), was found to inhibit the gastric proton pump in vitro and in vivo. In the leaky membrane vesicles of hog gastric mucosa, AU-1421 inhibited the K(+)-stimulated H, K-ATPase activity competitively with respect to K+, with a Ki value of 0.18 microM at pH 7.4 and 0.10 microM at pH 6.4. This inhibition was not affected by the length of the preincubation time of the enzyme with AU-1421, and activity was restored by dialysis. In the intact hog gastric membrane vesicles, which developed a pH gradient in the presence of valinomycin, AU-1421 also inhibited the gastric proton pump activity as measured by the quench of acridine orange fluorescence. In the isolated rabbit gastric glands, AU-1421 inhibited the histamine-, dibutyryl cAMP- and K(+)-stimulated uptake of [14C]aminopyrine into gastric glands with similar IC50 values of about 1 microM. Furthermore, AU-1421 suppressed gastric acid secretion in acute fistula rats and Heidenhain-pouch dogs with the same potency as cimetidine. These results suggest that AU-1421 is an antisecretory agent which reversibly inhibits the H,K-ATPase.

A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase.

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.

Synthesis and biological activity of some lipophilic derivatives of 1-thio-muramoyl-L-alanyl-D-isoglutamine.

2-N-Octadecanoyl derivatives of 1-S-acetyl-, 1-S-octadecanoyl-, and of 6-O-octadecanoyl-1-S-octadecanoyl-1-thiomuramoyl-L-ala nyl-D-isoglutamine were synthesized from benzyl 3,4,6-tri-O-acetyl-2-deoxy-2-(octadecanoylamino)-beta-D-glucopy ran oside. Their immunoadjuvant activities were examined in guinea-pigs.

Synthesis of (N-acetyl-1-O-acylmuramoyl)-L-alanyl-D-isoglutamines, and their immunoadjuvant activities.

1-O-Acyl derivatives of N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP) have been synthesized from 2-acetamido-1-O-benzoyl-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl) ethyl]-alpha-D-glucopyranose. Their immunoadjuvant activities were examined in guinea-pigs.

Synthesis and biological activities of N-acetyl-1-thiomuramoyl-L-alanyl-D-isoglutamine and some of its lipophilic derivatives.

N-Acetyl-1-thiomuramoyl-L-alanyl-D-isoglutamine and some lipophilic analogs were synthesized from benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl)ethyl ]- alpha-D-glucopyranoside (1). O-Debenzoylation of 2, derived from 1 by oxidation, gave 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl)ethyl ]-D-glucopyranose (3). Condensation of the alkoxy-tris(dimethylamino)phosphonium chloride (4), formed from 3 by the action of carbon tetrachloride and tris(dimethylamino)phosphine, with potassium thioacetate afforded 2-acetamido-1-S-acetyl-2-deoxy-4,6-O-isopropylidene-3-O-[ D-1-(methoxycarbonyl)ethyl]-1-thio-beta-D-glucopyranose (8). Coupling of the acid 9, obtained from 8 by hydrolysis and subsequent S-acetylation, with the methyl ester of L-alanyl-D-isoglutamine gave N-[2-O-(2-acetamido-1-S-acetyl-2,3-dideoxy-4,6-O- isopropylidene-1-thio-beta-D-glucopyranose-3-yl)-D-lactoyl]-L-alan yl-D- isoglutamine methyl ester (10), which was converted, via O-deisopropylidenation, S-deacetylation, and de-esterification, into the N-acetyl-1-thiomuramoyl dipeptide. Condensation of 11 (derived from 10 by S-deacetylation) and of 12 (obtained from 10 by S-deacetylation and de-esterification) with various acyl chlorides yielded the corresponding 1-S-acyl-N-acetylmuramoyl-L-alanyl-D-isoglutamine derivatives, which were converted into the desired, lipophilic 1-thiomuramoyl dipeptides by cleavage of the isopropylidene group. Condensation of 11 with the alkyl bromides yielded the 1-S-alkyl derivatives, which were also converted, via O-deisopropylidenation and de-esterification, into the corresponding 1-S-alkylmuramoyl dipeptides. The biological activities were examined in guinea-pigs and mice.