Yasuhiro Kanemasa

Colloid titration of heparin using Cat-Floc (polydiallyldimethyl ammonium chloride) as standard polycation

Abstract The positive polymer, Cat-Floc, was used for colloid titration. Colloid titration with Cat-Floc was not affected by pH between 2 and 12. The polymer combined with heparin, forming a flocculant, and the binding was shown to be stoichiometric by gravimetric analysis of heparin-sulfur. Results showed that heparin could be measured quantitatively by colloid titration with Cat-Floc. The applicability of this method for measurements of other biochemical materials was discussed.

Alteration in phospholipid composition of Staphylococcus aureus during formation of autoplast

The phospholipid composition of autoplasts (protoplasts made by autolysis of Staphylococcus aureus 209P was examined. The autoplasts were prepared by incubation of 209P cells in 1.2 M sucrose--0.33 M acetate buffer (pH 5.8). Cardiolipin comprised nearly half the total phospholipid in these autoplasts. Autoplasts had a lower phosphatidylglycerol content than intact cells but similar lysylphosphatidylglycerol content. The increase in cardiolipin content during release of autoplasts was not affected by pH or temperature. The result indicates that removal of the cell wall caused the increase in cardiolipin content. The total amount of phospholipids increased slightly during autoplast formation, but there was no significant increase in fatty acids or diglycerides. The changes of phospholipid composition during formation of the autoplast was due to de novo synthesis of cardiolipin from phosphatidylglycerol.

Lipid Composition of Staphylococcus aureus and Its Derived L-forms

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L‐forms were cultured in serum‐containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L‐forms, but for less than 25% in parent bacteria. The cardiolipin content of L‐forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L‐forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L‐forms but not in the parent strains when they were cultured in serum‐free broth. To examine the ability of L‐forms to synthesize cholesterol, the cholesterol content of L‐forms cultured in serum‐free broth was compared with that of the medium. The results indicated that staphylococcal L‐forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptation‐al change in response to the disappearance of the cell wall.

Adaptational Changes of Fatty Acid Composition and the Physical State of Membrane Lipids Following the Change of Growth Temperature in Yersinia enterocolitica

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0, Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.

Studies on Osmotic Stability of Liposomes Prepared with Bacterial Membrane Lipids by Carboxyfluorescein Release

The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic‐shock release of entrapped carboxyfluorescein as an indicator. The sub‐second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L‐form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.

Etiological Agents Responsible for Acute Diarrhea in Children in an Urban Community in Burma

Le pourcentage des bacteries pathogenes isolees varie de 46% en hiver a 79% pendant la mousson. Les agents isoles sont Shigella, Salmonella, Vibrio cholerae, Yersinia et Campylobacter

A Latex Agglutination Test for the Detection of Mycoplasma pneumoniae in Respiratory Exudates: A Comparative Study with a Commercially Available DNA‐Probe Test

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA‐probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 × 105 CFU/ml and that of DP was 5 × 104 CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half‐life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.

Establishment of a Mouse Model of Cystitis and Roles of Type 1 Fimbriated Escherichia coli in Its Pathogenesis

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non‐fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate‐buffered saline or non‐fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.

The Presence of Cis‐9,10‐Methylene Hexadecanoic Acid in Yersinia enterocolitica

The fatty acids of Yersinia enterocolitica were investigated by Abbas and Card. In their report, they stated that the major fatty acids were C16:0, C16:1, C17:0 and C18:1 and branched or cyclopropane side‐chain fatty acids could not be detected. We, however, found a moderate amount of cyclopropane side‐chain fatty acid. We could determine that this fatty acid was cis‐9, 10‐methylene hexadecanoic acid by gas‐liquid chromatography, gas chromatography‐mass spectrometry, silver‐nitrate‐treated TLC and PtO2 catalyzing hydrogenation method.

An indirect immunofluorescence method for detection of Mycoplasma hominis in vaginal smears

We developed a novel method for the detection of Mycoplasma hominis from vaginal swabs using an indirect immunofluorescence technique. It is a rapid and simple method that can be finished in only 5 hr and is more sensitive than the usual culture isolation method. The indirect immunofluorescence method was applied to vaginal smears from 193 healthy women and 33.7% gave a positive test. This value was much higher than that (11.4%) obtained from the same specimens by the culture method. When vaginal smears were subjected to Papanicolaou staining after the indirect immunofluorescence method, the specific immunofluorescence of the epithelial cells was located exactly at the sites of granular aggregates stained with Papanicolaou stain. A histological examination by Papanicolaou staining showed that the incidence of inflammation seems to be slightly higher in M. hominis‐carriers than in non‐carriers.