Removal of airborne bacteria by filtration using a composite microporous membrane made of a pyridinium-type polymer showing strong affinity with microbial cells.

A composite microporous membrane made of poly(N-benzyl-4-vinylpyridinium chloride) that showed strong affinity with bacterial cells was prepared as a filter material for removing airborne bacteria. Thickness, pore diameter and porosity of the membrane were 0.72 mm, 14.5 microm and 63%, respectively. Electron micrographic analysis revealed that the membrane consisted of a very large number of connected beads of 1.4 microm in diameter made of the pyridinium-type polymer. Filtration using the membrane was performed easily at low flow rates with insignificant pressure drop across the membrane. Filtration at 63.7 cm/sec gave 99.98% and 99.996% removal (3.7 and 4.4 log10-unit reduction in concentration) of Escherichia coli and Pseudomonas aeruginosa, respectively. Staphylococcus aureus was not detected in filtrates. Since pores of the membrane were much larger than these bacteria, the efficient removal was best explained in terms of the affinity of the polymer with bacterial cells.

Identification of amine acceptor protein substrates of transglutaminase in liver extracts: use of 5-(biotinamido) pentylamine as a probe.

Transglutaminase is a calcium-dependent enzyme which catalyzes amine incorporation and cross-linking of proteins. To isolate the amine acceptor protein substrates of transglutaminase in mammalian livers, a biotin-labeled primary amine substrate of transglutaminase, 5-(biotinamido) pentylamine, was used for biotin labeling of proteins in the liver extracts by endogenous transglutaminase activity. The biotin-labeled proteins were isolated and recovered by biotin-avidin-affinity chromatography. The obtained proteins were separated by SDS-PAGE. Proteins with molecular masses of 15, 24, 35, 40, 44, 93, and 134 kDa were the main components of labeled proteins in mouse liver extract. In rat and guinea pig liver extracts, 32-, 38-, 40-, 44-, and 134-kDa proteins and28-, 40-, 44-, 55-, 60-, 91-, and 134-kDa proteins were the main components of labeled proteins, respectively.Using amino-terminal amino acid sequence analyses and sequence homology searches, the 38-kDa protein from rat liver was identified as a subunit of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and the 28-kDa protein from guinea pig liver was identified as a subunit of glutathione S-transferase (class theta) (EC 2.5.1.18). Both the glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle and glutathione S-transferase (class pi) from human placenta also could be amine acceptors in the amine incorporation catalyzed by guinea pig liver transglutaminase. These results suggest that these enzymes can be modified posttranslationally by cellular transglutaminase.

[Use of Caco-2 cells for isolation of influenza virus].

In this study we assessed the usefulness of Caco-2 cells, derived from a human colon carcinoma, to isolate an influenza virus. Throat washings collected from 30 patients with influenza-like illnesses in Miyazaki Prefecture in 1997 were inoculated in MDCK and Caco-2 cells, 17 influenza virus strains were isolated in MDCK cells, and 20 in Caco-2 cells. Of all the viruses isolated, only one strain was identified as influenza virus type B; other strains were identified as type A (H3N2). Furthermore, some influenza viruses were isolated in Caco-2 cells also from the specimens collected between 1991 and 1997. With Caco-2 cells, each type of influenza virus was isolated effectively without the supplement of trypsin in the culture medium. These facts indicate the usefulness of Caco-2 cells as a host to isolate influenza virus as shown to be suitable in the detection of many types of enteric viruses. Caco-2 cells will serve as a useful cell line for the surveillance of infectious disease because Caco-2 cells are sensitive to a wide range of virus.

[Detection of enterovirus 70 in acute hemorrhagic conjunctivitis by PCR-stringent microplate hybridization method].

The surveillance of acute hemorrhagic conjunctivitis (AHC) caused by enterovirus 70 (EV70) is insufficient because of the difficulties of virus isolation using conventional methods. In this study, reverse transcription-polymerase chain reaction (RT-PCR) and stringent microplate hybridization (SMH) methods were assessed for the detection of EV70 in conjunctival swabs collected from the patients of AHC which broke out in Miyazaki Prefecture in 1990 and Okinawa Prefecture in 1994. Furthermore, with the use of the SMH method, we compared the genetic homogeneity of EV70 detected from the samples collected in 1990 and 1994 to the J670/71 strain isolated approximately 25 years ago. A similar investigation was performed between recent coxsackievirus A24 variant (CA24v) and EH24/70 strain, a standard strain of CA24v that was isolated approximately 25 years ago. As a result, RT-PCR products were detected in 26 of 34 conjunctival swabs collected in Miyazaki Prefecture in 1990. Likewise, RT-PCR products were detected in 9 of 10 conjunctival swabs collected in Okinawa Prefecture in 1994, and these RT-PCR products were all identified as EV70 by SMH method. Such a high level of virus recovery from specimen shows the evidence of usefulness of these techniques. Recent EV70 showed a reaction of about 60% to a probe synthesized with J670/71. Likewise, recent CA24v showed a reaction of about 30% to a probe synthesized with EH24/70. These finding are highly suggestive of the progress of viral mutation.

Depth perception in simple line drawings.

Three-dimensional interpretation of simple line drawings, composed of two triangles with a common side, was studied through the quantitative measurement of perceived orientation of the surface indicated by a stimulus figure. In a single triangle, depth perception is ambiguous and is not stable even if perceived. In two triangles with a common side, however, depth is stably perceived. Depth effect, defined as the magnitude of the angle formed by the two perceived surfaces, increased linearly as the magnitude of an angle at a vertex facing the common side became larger. The depth effect did not vary significantly for the change of a triangular from when the magnitude of the angle at the vertex facing the common side was constant. These results suggest that the depth effect changes systematically with variation in the triangle's form.

Developmental expression of cytochrome P450S within intrasplenically transplanted fetal hepatocytes.

Fetal hepatocytes were harvested at day 20 of gestation from spontaneously hypertensive rats (SHR) and then transplanted into recipient adult SHR spleens. Morphological examination of the recipient spleens revealed that, after 4 and 10 wk, large masses of hepatocytes were present in the red pulp with apparent cord-like structures. Larger batches of hepatocytes were observed in the spleens at 10 wk after than at 4 wk after transplantation. Of major significance was the fact that hepatocyte transplanted spleens were able to express several families of cytochrome P450 (cyto P450) proteins 2-10 wk after transplantation. Immunochemical determinations revealed that cytos P450 IA1, P450 IIB1, P450 p, P450 HLp, and P450 LA omega could be detected without any prior induction. All were intensely expressed 6 wk after transplantation; however, P450 IA1 and P450 IIB1 did not appear to be expressed by 2 wk after transplantation. Although cytos P450 p and P450 HLp did not appear to be expressed by 10 wk after transplantation, they were induced with dexamethasone at that time. Cyto P450 LA omega and peroxisomal acyl CoA oxidase were expressed 6 wk after transplantation in a 70% hepatectomized host. These results demonstrate that fetal hepatocytes can be successfully transplanted into the spleens of recipients and that the fetal hepatocytes appear to grow and develop cyto P450 metabolizing systems.

Intake ratio of water-insoluble dietary fiber to the water-soluble one in Japanese: an estimation on dietary records and the secular consideration on the national nutrition survey.

Thirty-four foods were analyzed in order to determine the content of water-soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). Using the results with the standard table for 227 foods, the intake ratio of IDF/SDF of an average Japanese was calculated for the period 1946-1990. The ratio was 3.22 in 1990 as calculated on the food intakes shown in the national nutrition survey, and the secular change was not detected since 1946 when the ratio was 3.30. The ratio was also shown to be well preserved between types of households including the age of the head. Using dietary records of 60 healthy city workers (average 42.8 years) for 4 weeks, however, the weekly average ratio for an individual was found to vary in the range of 2.25-5.13 although the total average for 60 individuals was 3.33. Thus, the well preserved IDF/SDF intake ratio for an average Japanese showed, on the contrary, a wide variation of food selection between each person.

Characterization of the SF agent, an Ehrlichia sp. isolated from the fluke Stellantchasmus falcatus, by 16S rRNA base sequence, serological, and morphological analyses.

The organism designated the SF agent was originally isolated in Japan in 1962 from Stellantchasmus falcatus metacercaria parasitic on gray mullet fish. The SF agent resembles members of the genus Ehrlichia morphologically and exhibits weak antigenic cross-reactivity with Ehrlichia sennetsu. This organism causes mild clinical signs in dogs, but severe splenomegaly and lymphadenopathy in mice. This suggests that the SF agent may be similar to either Neorickettsia helminthoeca, an intracellular parasite of a fluke and the cause of salmon poisoning disease in dogs, or E. sennetsu, the causative agent of human sennetsu ehrlichiosis in Japan and Malaysia. In order to determine the phylogenetic relationship between the SF agent and other ehrlichial species, the 16S rRNA gene was amplified by the PCR and sequenced. The SF agent sequence was most closely related to the sequences of Ehrlichia risticii (level of sequence similarity, 99.1%), the causative agent of Potomac horse fever, and E. sennetsu (level of sequence similarity, 98.7%). The next most similar sequence was that of N. helminthoeca, but the level of sequence similarity was only 93.7%. E. sennetsu, E. risticii, the SF agent, and N. helminthoeca formed a distinct cluster that was separated from all other ehrlichial species. As determined by immunofluorescence labeling, antiserum against the SF agent cross-reacted strongly with E. sennetsu, E. risticii, and N. helminthoeca. When three genetically distinct ehrlichial isolates obtained from horses with Potomac horse fever were compared with the SF agent, we found that the SF agent was most closely related to Ohio isolate 081, followed by IllinoisT (T = type strain) and a Kentucky isolate. We observed strong antigenic cross-reactivities and similarities in Western blot (immunoblot) reaction profiles when we compared the SF agent, E. risticii, and E. sennetsu; however, weaker antigenic cross-reactivity was observed when the SF agent and N. helminthoeca were compared. Our results indicate that the SF agent is antigenically more closely related to E. risticii and E. sennetsu than to N. helminthoeca. The biological and antigenic characteristics and the 16S rRNA sequence data suggest that the SF agent is a new species that belongs to the genus Ehrlichia.

Site-directed mutation in conserved anionic regions of guinea pig liver transglutaminase.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl) lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. There are conserved anionic regions in transglutaminases, some of which are thought to be possible calcium-binding sites. By site-directed mutagenesis, three mutant forms of recombinant guinea-pig liver transglutaminase, in which some acidic amino acid residues in two conserved regions became nonionic, were expressed in Escherichia coli: TGM1, with Asp-231 and -232 changed to Asn; TGM2, with Glu-445, -448, -449, -450, and -452 changed to Gln; and TGM3, with the mutations of both TGM1 and TGM2. The size and level of synthesis of the mutant proteins were unchanged when monitored by immunoblotting. All mutants retained enzyme activity, and their apparent Km values for substrates during histamine incorporation into acetyl alpha s1-casein were similar to those of the wild-type enzyme, but their Vmax values were smaller. The deamidation rate of glutamine residues in the acetyl alpha s1-casein was unaffected, but the rate of protein cross-linking catalyzed by these mutants was very low. All mutations caused with the enzyme a decrease in the sensitivity to activation by calcium and an increase in the sensitivity to inhibition by GTP. These results indicated that the negative charges of some acidic amino acid residues in the two conserved anionic regions of transglutaminase are not essential for its activity but the loss of their negative charges affects some catalytic properties.

Meisenheimer rearrangement of azetopyridoindoles, VI. Synthesis of 12-carbaeudistomin and related compounds.

For structure-activity relationship investigation of eudistomins 1, 12-carbaeudistomin 3, its 1,10-trans isomer 4, and 11,12-didehydro-12-carbaeudistomin 5 have been synthesized. The [2,3]-Meisenheimer rearrangement of the corresponding N-oxide of the 2-vinylazetopyridoindole 12a bearing a benzenesulfonyl group as a protective group of the indole nitrogen atom afforded the oxazepino ester 14, which was easily isomerized to 20a. Compounds 3 and 4 were synthesized from 14 and 20a, respectively, according to the following reaction sequences [hydrogenation of the double bond (Pd-C/H2), desulfonylation (Mg in MeOH), hydrolysis (AlBr3-EtSH), and Curtius rearrangement (a mixed anhydride method using NaN3), followed by debenzylation (Pd-C/H2)]. The Curtius reaction of the carboxylic acid 27 using DPPA gave the carbamate 29, which was subjected to debenzylation (AlBr3-EtSH) followed by desulfonylation (LiAlH4) to afford 5. Evaluation of anti-influenza virus activities of the amino compounds 3, 4, and 5 revealed that 12-carbaeudistomin 3 possesses a specific activity against influenza virus B.

Stannum (IV) chloride stimulates vanadium (IV) catalyzed oxidation of NADH.

Stannum has been found to be an essential ultratrace element, however, the biological functions of stannum remain to be clarified. We found that stannum (IV) chloride stimulates vanadium (IV) catalyzed free radical chain oxidation of NADH. Stannum (IV) chloride, per se, did not catalyze the NADH oxidation. Superoxide, H2O2, and OH. are known to be the key species in vanadium catalyzed NADH oxidation. The inhibition of the vanadium (IV) catalyzed NADH oxidation in the presence of stannum (IV) chloride by catalase, superoxide dismutase (SOD), and hydroxyl radical scavengers indicated that the stannum (IV) chloride stimulated NADH oxidation consisted of almost the same reaction steps as that in the absence of stannum (IV) chloride. The results of inhibition studies on the NADH oxidation with SOD and catalase suggested that the reaction mixture containing stannum (IV) chloride contained a greater amount of H2O2 and a lower amount of O2- than that containing only vanadium (IV). Hydrogen peroxide is the precursor of OH. in the free radical chain reaction. The stimulation of NADH oxidation by stannum (IV) chloride is due to the stimulation of H atom abstraction by OH.. Stannum (IV) chloride might stimulate the generation of OH. by producing H2O2.

Three-dimensional interpretation of quadrilaterals.

The three-dimensional interpretation of two-dimensional images was studied by using quadrilateral patterns, and some relationships between their structure, depth, and shape interpretations were analyzed under various viewing conditions. We defined rectangular and nonrectangular viewing conditions as follows: the viewing condition in which the quadrilateral, including parallel sides, could be a projection from a rectangle and the viewing condition in which it could not be so. For 9 subjects, 10 measurements were made in each experiment. Analysis showed that a pair of parallel sides of a quadrilateral were viewed as parallel to the viewer's forehead when the sides were horizontal in the image plane and were seen as slanting in depth if they were slanted in the image plane. The quadrilateral composed of parallel and nonparallel sides was perceived as rectangular when viewed with foveal vision even though under the nonrectangular viewing condition, if that viewing condition was not so different from the rectangular viewing condition. The quadrilateral did not appear to be rectangular when viewed in peripheral vision even though the rectangular viewing condition was used.

Removal of micro-organisms by filtration through unwoven cloth coated with a pyridinium-type polymer.

Unwoven cloth coated with 32 mg/g of a copolymer of N-benzyl-4-vinyl-pyridinium chloride and styrene was found to be effective in removing micro-organisms from water. In experiments demonstrating removal of Escherichia coli by filtration through ten sheets of the unwoven cloth, the rate of removal was 99.99% at a filtration rate of 2.6 cm/h, and remained at 99% even at a high filtration rate of 300 cm/h and a low influent concentration of the bacterial cells such as 10(3) cells/ml. The rate of removal tended to increase with a decrease in the influent bacterial concentration. Seven other bacteria and two yeasts were effectively removed by filtration through the unwoven cloth. Filtration through the unwoven cloth was also effective in removing spores of fungi from water but was not very effective in removing bacteriophage T4 from aqueous solution.

Disambiguating ambiguous figures by a model of selective attention.

Ambiguous figures are visual stimuli which are interpreted multiply by the human visual system. A model is proposed which disambiguates the ambiguous figures. The model was formulated based on the characteristics of visual information processing, accompanied with selective attention. In the ambiguous figure "my husband and my father-in-law", it was necessary to simulate visual information processing so that attention was directed to the multiple features in the figure to disambiguate the ambiguous figure. Pictures, obtained from the model, were examined as to whether they were interpreted unambiguously or not. Results show that the model, simulated selective attention, can disambiguate the ambiguous figures. This suggests that the image per se, viewed through selective attention, becomes unambiguous before the figure is interpreted in the higher level. Results also show that the computer simulation of selective attention would make it possible to examine factors affecting the initial interpretation of the figure.

Purification and characterization of a new NAD(+)-dependent enzyme, L-tartrate decarboxylase, from Pseudomonas sp. group Ve-2.

A new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarboxylation on NAD+ suggests that the decarboxylation involves redox reactions of the substrate. The enzyme catalyzes NAD(+)-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40,000). The apparent Michaelis constants for L-tartrate and NAD+ are 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomonas sphaeroides, and tartrate dehydrogenase from P. putida.

Effect of L-cysteine on plasma concentration and urinary excretion of 1-(tetrahydro-2-furanyl)-5-fluorouracil metabolites.

Effects of L-cysteine (CySH) on the plasma concentrations and the urinary excretion of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) and its metabolites were studied by high performance liquid chromatography in rats. Significantly higher plasma concentrations of FT, 5-fluorouracil (5-FU) and cis-4'-OH-FT were obtained after an oral administration of FT (500 mg/kg) combined orally with CySH (500 mg/kg) when compared to FT alone. The urinary excretions of 5-FU, trans-3'-OH-FT, cis-4'-OH-FT, trans-4'-OH-FT and 4',5'-dehydro-FT significantly decreased up to 12 h but that of alpha-fluoro-beta-alanine significantly increased up to 24 h by the combined administration of CySH. Furthermore, the plasma concentration of 5-FU significantly increased at 0.5 h and its urinary excretion significantly decreased up to 4 h after an intraperitoneal administration of 5-FU (10 mg/kg) combined orally with CySH (500 mg/kg) when compared to 5-FU alone. The urinary pH significantly changed to acidic and the urinary volume significantly increased by the combined administration of CySH, so it was thought that the reabsorption of 5-FU through renal tubules from urine could increase and the increment of the urinary excretion of alpha-fluoro-beta-alanine was caused by this. Then it was suggested that the increase of the plasma concentrations of 5-FU and cis-4'-OH-FT could be attributed to the decrease of their urinary excretions after an administration of FT combined with CySH when compared to FT alone.

Use of monoclonal antibodies against Rickettsia tsutsugamushi Kawasaki for serodiagnosis by enzyme-linked immunosorbent assay.

Monoclonal antibodies (MAbs) against Rickettsia tsutsugamushi Kawasaki were prepared. The crossreactivity tests of the MAbs performed by using antigenically distinct strains of R. tsutsugamushi in immunofluorescence and immunoblotting analyses indicated that the Kawasaki strain contains a strain-specific epitope and also contains a common epitope on the 56-kDa polypeptide cross-reactive with the Gilliam strain, group- and subgroup-specific epitopes on the 46-kDa polypeptide, and a subgroup-specific epitope on the 25-kDa polypeptide. By using the strain-specific MAb for serodiagnosis of tsutsugamushi disease (or scrub typhus fever), we have established a method which was designated the inhibition enzyme-linked immunosorbent assay. The principle of the method is to measure the percentage of inhibition of antigen absorption on a MAb-coated plate by antibody-positive sample sera which were mixed with the antigen suspension. The advantages of this test for practical use are that (i) crude antigen can be used, i.e., purification of the antigen is not required; (ii) the test is more sensitive than immunofluorescence; (iii) the final judgment of plus or minus is clear-cut; and (iv) rickettsial antigenic types in the patients can be distinguished by this test.

Removal of pathogenic human viruses by insoluble pyridinium-type resin.

Cross-linked poly(N-benzyl-4-vinylpyridinium bromide) (BVP resin) was found to be very efficient in removing pathogenic human viruses from aqueous solution. In batch removal experiments using 50 g/l of BVP resin at 35 degrees C, the level of infectivity in suspensions of enterovirus, herpes simplex virus, poliovirus, and human immunodeficiency virus was reduced 1000-100,000 fold during a 2 h period. Those of coxsackievirus and echovirus were reduced 60-600 fold during 1 h contact. The haemagglutination titres of solutions of human rotavirus, influenza virus, human adenovirus, and Japanese encephalitis virus were reduced 16-256 fold during 30 min of contact. In removal experiments by a continuous flow column method for poliovirus, enterovirus, and coxsackievirus with initial infectivities of less than 10(5)/ml, the infectivity of these viruses was no longer detectable in the effluent solution. For poliovirus, coxsackievirus, and echovirus with initial infectivities higher than 10(6), 99.8-99.9998% of the input viruses was removed as indicated by the reduction of infectivity.

Effect of L-cysteine on distribution volume of 1-(tetrahydro-2-furanyl)-5-fluorouracil.

Higher plasma concentrations of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) were obtained after administration of FT (100 mg/kg, i.v.) combined with L-cysteine (CySH, 500 mg/kg, p.o.). The volume of distribution (Vd) and body fluid volumes significantly decreased. These results suggest that the increase of the plasma concentrations of FT can be attributed to the decrease of the Vd of FT, which is considered to be based on the decrease of body fluid volumes by the combined administration of CySH.