Eisaku Yoshihara

Cytolytic activity of liposomes containing stearylamine

In order to develop the cytotoxic liposome, the cytolytic effect of polycationic liposome was examined. Upon incubation of the stearylamine-containing liposome (stearylamine-liposome) with rabbit erythrocyte, a significant extent of hemolysis was observed. Hemolytic activity of the liposome depends on the amount of stearylamine in the liposome membrane. The plots of the initial rate of hemolysis versus the concentration of stearylamine-liposome showed a sigmoidal curve, suggesting that stearylamine-liposomes act cooperatively on the erythrocyte membrane. Hemolytic activity of stearylamine-liposome was markedly influenced by the composition of hydrocarbon chains of the phospholipids in the liposome membrane, suggesting that the membrane fluidity of stearylamine-liposome is important to evoke the hemolysis. Since the liposomes containing acidic phospholipids inhibited markedly the stearylamine-liposome-caused hemolysis, it is likely that the primary target of stearylamine-liposome is the negatively charged component(s) such as acidic phospholipids on the erythrocyte membrane. Furthermore, stearylamine-liposome induced the release of the intravesicular contents from the liposome made of acidic phospholipids but not from the liposome made of phosphatidylcholine only. These results suggest that stearylamine-liposome interacted with the negative charges of the erythrocyte membrane and eventually damaged the cell. Erythrocytes from rabbit, horse and guinea pig are highly susceptible to stearylamine-liposome but those from man, sheep, cow and chicken are less so.

Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB–OprM efflux pump of Pseudomonas aeruginosa

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).

Mutations Affecting DNA-Binding Activity of the MexR Repressor of mexR-mexA-mexB-oprM Operon Expression

We have isolated 25 MexR mutants that retained their dimerizing ability but were unable to bind mexOP DNA. Surprisingly, 20 mutations were located in the hydrophobic core region at alpha4, W1, alpha2, alpha3, and beta2, and only 3 were in positively charged residues. These results verified that DNA binding is mediated by distinct regions of MexR and showed the importance of the hydrophobic core region of the DNA-binding domain.

In vitro lysis of the bloodstream forms of Trypanosoma brucei gambiense by stearylamine-bearing liposomes.

Cytolytic activity of liposomes consisting of stearylamine and phosphatidylcholine (SA/PC-liposomes) was examined in vitro against the bloodstream forms of Trypanosoma brucei gambiense. More than 99% of the cells (2 X 10(6)/ml) were killed within 30 min by treatment with 15 mol% SA/PC-liposomes (100 microM total lipids). As few as 1.2 X 10(12) liposomes per ml (equivalent to 2 nM liposome) showed trypanocidal activity. Fluorescence microscopy of cells treated with the dansylated SA/PC-liposomes suggested that the liposomes bound to and accumulated on the cell surface, eventually damaging the plasma membrane. SA/PC-liposomes showed no significant hemolysis when incubated with human and mouse erythrocytes under conditions that killed greater than 99.9% of the T. b. gambiense trypomastigotes. Human leukocytes were also shown to be less susceptible to SA/PC-liposomes than T. b. gambiense. These results may point to a new direction in strategy for therapy of African trypanosomiasis. Images

Multiantibiotic resistance caused by active drug extrusion in hospital pathogens

All living organisms from bacteria to mammals extrude noxious compounds to the external medium. When exposed to antibiotics, bacteria actively extrude intracellular antibiotic and develop resistance to the drug. NosocomialStaphylococcus aureaus, Pseudomonas aeruginosa and other bacteria are resistant to a broad range of antibiotics and to structurally and functionally diverse chemotherapeutic agents and disinfectants. For this reason nosocomial infections are especially hard to treat in immunocompromised patients who may be infected by low-virulence bacteria.Extrusion-related antibiotic resistance inP. aeruginosa arises by the expression of Mex-extrusion pumps, including genetically distinctmexA-mexB-oprM, mexC-mexD-oprj, andmexE-mexF-oprN systems, each encoding two inner membrane proteins and one outer membrane protein.S. aureus becomes resistant to fluoroquinolone by expressing NorA extrusion proteins and to disinfectants by expressing Qac extrusion proteins. The drug extrusion machinery may be classified into several categories according to the number of transmembrane segments it exhibits. The proteins that belong to a major facilitator super family have 12 or 14 transmembrane segments. The extrusion proteins with molecular weight of 12,000 to 15,000 span the membrane 4 times and are collectively called small multidrug resistance proteins. The extrusion proteins that transport substrate across the inner and outer membrane of gram-negative bacteria are in the resistance nodulation cell division family.

Trypanocidal activity of the stearylamine-bearing liposome in vitro.

Liposome made of stearylamine and phosphatidylcholine showed the trypanocidal activity in vitro. Cytotoxicity of the liposome against Trypanosoma cruzi appeared to be the strongest in trypomastigotes followed by amastigotes and epimastigotes. Lysis of the human erythrocyte was undetectably low under the conditions that the liposome kills more than 95% of trypomastigotes. The liposome seems to damage the plasma membrane.

Protein D2 porin of the Pseudomonas aeruginosa outer membrane bears the protease activity.

We report here our discovery that protein D2 of the outer membrane of Pseudomonas aeruginosa is a novel porin bearing protease activity. Homogeneously purified protein D2 hydrolyzed several synthetic peptides according to the Michaelis‐Menten kinetics. A specific serine protease inhibitor, diisopropyl fluorophosphate (DFP), inactivated the protease activity and [3H]DFP covalently labeled protein D2. We tested the effect of two monoclonal antibodies raised against protein D2 on the protease activity. One antibody lowered the protease activity to about 20%, while the other enhanced it to about 300% of that without antibody. In addition, the fractions derived from the outer membrane of the protein D2‐deficient mutants showed negligible protease activity, whereas similarly fractionated outer membrane proteins of the protein D2‐positive parent strain showed strong protease activity.

Protection of Toxoplasma gondii-infected mice by stearylamine-bearing liposomes.

The cytotoxic activity of stearylamine-bearing liposomes against Toxoplasma gondii (RH strain) was examined. When tachyzoites were treated in vitro with liposomes consisting of 20 mol% stearylamine and 80 mol% phosphatidylcholine (130 micrograms/ml total lipids), more than 95% of the parasites were killed within 90 min. Intraperitoneal injection of 10 mg of 30 mol% stearylamine/70 mol% phosphatidylcholine-liposomes in mice shortly before or after T. gondii challenge afforded protection from death for more than 30 days to 70-80% of the treated mice, whereas all untreated mice succumbed within 9 days. The liposome-injected mice that survived remained symptom-free and behaved normally.

Nucleotide sequence of the protein D2 gene of Pseudomonas aeruginosa.

Protein D2 of the outer membrane of Pseudomonas aeruginosa was shown to form the imipenem-permeable pore. We cloned and sequenced the protein D2 gene. The protein D2 gene encodes a polypeptide with 443 amino acids consisting of 23 and 420 amino acid residues for the signal peptide and mature polypeptide (M(r), 46,010), respectively. Protein D2 contains the highest molar ratio of glycine and no cysteine. The polar amino acids are scattered throughout the sequence. Images

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.