The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes.

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium.

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.

Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

[Arterial infusion of SMANCS-Lipiodol for advanced hepatocellular carcinoma].

Twenty-four patients were treated with arterial infusion of SMANCS dissolved in Lipiodol. Twenty of these patients had HCC with the main trunks of portal vein occluded by tumor, and four patients had severe cirrhosis and multiple HCC. The actual dose of SMANCS administered each patient ranged from 4 to 6 mg. Side effects occurred in 50%. Severe side effects such as shock and shivering-chilliness were observed in 18%. The differences between the values of hepatic functional serum indexes obtained before and after treatment with SMANCS were small and transient. With regard to the therapeutic response of the arterial infusion of SMANCS, the mean survival time was approximately 2.8 months. It was suggested that the more effective administration of SMANCS was combination of the arterial infusion of SMANCS-Lipiodol with TAE at the level of the right hepatic artery of left hepatic artery for multiple HCC.

HVJ-induced fusion of liposome to erythrocyte. Possible involvement of virus-induced activated state of erythrocyte membranes during fusion process.

HVJ-induced fusion between liposomes and erythrocytes was examined as a model of cell-cell fusion and the following results were obtained: Liposome-liposome fusion seldom occurred in the presence of HVJ. Liposomes free of receptors for viral HN protein could efficiently fuse with erythrocytes when the concentration of liposomes and erythrocytes was high. Direct interaction between HVJ and liposomes should be negligible in the present system. A distinct lag phase (10 min) was observed before the liposome-erythrocyte fusion occurred appreciably. Efficiency of the liposome-erythrocyte fusion decreased linearly up to 1 h after stopping the virus-erythrocyte fusion. The former fusion was almost negligible after 1 h but could be restored by further addition of HVJ. Efficiency of the liposome-erythrocyte fusion was suppressed significantly when erythrocytes were pretreated with N-ethylmaleimide (N-EM), a membrane-permeable sulfhydryl agent, whereas rho-chloromercuriphenylsulfonic acid (PCMBS), a membrane-impermeable sulfhydryl agent, had no appreciable effect on the fusion efficiency. The study suggests possible involvement of some HVJ-induced 'activated state' of the erythrocyte membrane during the process inducing the liposome-erythrocyte fusion. The structure including the membrane skeleton system may be responsible for producing an 'activated state' in the erythrocyte membrane. Such an 'activated state' was induced 10 min after the viral infection to erythrocytes, and thereafter decayed gradually.

[Distribution of enzymatically pathogenic bacteria from periodontal pocket in advancing periodontitis].

Production of 9 enzymatic activities of 527 strains freshly isolated from periodontal pockets in advancing periodontitis were investigated. Of these isolates, two strains showed lecithinase activity on egg yolk agar plate. Collagenase, plasmin and lipase were produced by 28 strains, 26 strains and 22 strains, respectively. Two lecithinase-producing strains were identified as Bacteroides intermedius. Nineteen strains of B. intermedius and 1 strain of Fusobacterium species produced lipase on egg yolk agar plate. All of the 28 collagenase-producing strains were B. gingivalis. B. gingivalis (20 strains) and non black-pigmented Bacteroides (6 strains) showed plasmin activity. These results indicate that Bacteroides species, mainly B. gingivalis and B. intermedius may exert an important influence on the exacerbation of the disease.

Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes.

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High-voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, kappa-chain, lambda-chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti-serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously.

Development of a thermoelectric cooling apparatus for high-voltage isoelectric focusing on a cellulose acetate membrane.

To develop an isoelectric focusing apparatus using a cellulose acetate membrane (Separax EF), we have designed a thermoelectric cooling isoelectric apparatus. This apparatus has two characteristics. Firstly, the cooling system was switched to a thermoelectric cooling system from an ice-cooling system. Secondly, the chamber lid of the electrophoretic apparatus was also devised so that samples could be applied without opening the chamber lid. With this apparatus we could perform the isoelectric focusing without worrying about room temperature and humidity in the laboratory. Applying 2000 V for an extra 5 min with our module cooling system, we achieved a much higher degree of resolution with three sheets of cellulose acetate membrane (Separax EF) overlaid for simultaneous electrophoresis. Thus, three types of information could be obtained from only one electrophoretic procedure.

[Oral condition of infantile acute lymphatic leukemia with microbisme substitute].

The oral condition of a 7 year old boy at the 12th and 13th remission induction therapy sessions since contracting acute lymphatic leukemia was investigated clinically and microbiologically. The findings obtained were as follows: 1) The appearance of gingivitis coincided with the decrease of white blood cells 7 to 9 days after administration of the anti-leukopenic drugs. 2) Gingivitis appeared on the same site of the oral cavity at both remission induction therapies sessions. 3) The bleeding from the gingival lesions corresponded to the decrease of blood platelet at both remission induction therapies sessions. Conversely, gingivitis disappeared with the improvement of peripheral blood conditions. 4) The total number of oral bacteria decreased after administration of the antibiotics, although the ratio of Candida in the oral microbial flora increased markedly. 5) Typical symptoms of oral candidiasis were not observed after the appearance of the microbisme substitute.

Phenotypic characteristics and DNA relatedness in Prevotella intermedia and similar organisms.

We have recently isolated from the gingival pockets, periapical lesions and saliva some anaerobic gram-negative, black-pigmented rods. Many of these isolates exhibited phenotypic characteristics similar to Prevotella intermedia (Bacteroides intermedius). However, several of these isolates, although resembling P. intermedia in most of the phenotypic expressions, were capable of fermenting lactose, a biochemical characteristic atypical of P. intermedia. These atypical clinical isolates (strains capable of fermenting lactose) and isolates exhibiting more typical phenotypic characteristics (i.e., lactose nonfermenting) were definitively identified as P. intermedia by DNA-DNA hybridization using a photoprobe biotin method. Quantitative hybridization of clinical isolates with labeled DNA of P. intermedia-type strains (ATCC 25611 and ATCC 33563) showed that almost all the clinical strains isolated from disease sites of adults belonged to the ATCC 25611 group, whereas strains isolated from the saliva of children belonged to the ATCC 33563 group. These data, together with the phenotypic characterization of the isolates, suggested that P. intermedia is a heterogeneous species both phenotypically and genetically.

[Relationship between clinical findings and subgingival microbial flora in periodontitis (1)].

The purpose of this study was to examine the relationship between clinical findings and subgingival microbial flora in periodontitis. The results obtained were as follows: 1. In a phase-contrast microscopic study, no correlation was found between the clinical findings, total bacteria or proportional distribution of spirochetes or motile rods in the periodontal pocket. 2. Anaerobic incubation revealed no correlation between clinical findings, total bacteria or proportional distribution of black-pigmented Bacteroides in the periodontal pocket.

[Relationship between clinical findings and subgingival microbial flora in periodontitis (2)].

The purpose of this study was to examine the relationship between clinical findings and subgingival relationship between clinical findings and subgingival microbial flora in periodontitis at the first medical examination and after initial preparation. The results obtained were as follows: 1. Clinical findings with the exception of plaque index showed improvement after initial preparation in comparison with the first medical examination. 2. In phase contrast microscopy, both total bacteria and incidence of spirochetes and motile rods decreased after initial preparation in comparison with the first medical examination. 3. Clinical findings with the exception of plaque index were related to the total bacteria and proportional distribution of spirochetes and motile rods in periodontal pockets, observed in phase contrast microscopy. 4. Total bacteria and proportional distribution of black-pigmented Bacteroides in periodontal pockets decreased after initial preparation in comparison with the first medical examination.