The plasma membrane of mammalian cells is one of the tight barriers against gene transfer by synthetic delivery systems. Various agents have been used to facilitate gene transfer by destabilizing the endosomal membrane under acidic conditions, but their utility is limited, especially for gene transfer in vivo. In this article, we report that the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) greatly facilitates gene transfer via membrane destabilization. We constructed recombinant phage particles displaying Tat peptide on their surfaces and carrying mammalian marker genes as part of their genomes (Tat-phage). We demonstrate that, when animal cells are briefly exposed to Tat-phage, significant expression of phage marker genes is induced with no harmful effects to the cells. In contrast, recombinant phage displaying other functional peptides, such as the integrin-binding domain or a nuclear localization signal, could not induce detectable marker gene expression. The expression of marker genes induced by Tatphage is not affected by endosomotropic agents but is partially impaired by inhibitors of caveolae formation. These data suggest that Tat peptide will become a useful component of synthetic delivery vehicles that promote gene transfer independently of the classical endocytic pathway.

By cosynthetic studies, nine butirosin-non-producing blocked mutants of Bacillus circulans

To investigate the emergence of rifampin resistance in Rhodococcus equi strains isolated from foals and their environment in Japan, we compared the in vitro antimicrobial susceptibilities to rifampin of 640 isolates from 64 infected foals and 98 soil isolates from their horse-breeding farms. As a control, 39 human isolates from patients with and without AIDS were also tested for susceptibility to rifampin. All of the isolates showed rifampin sensitivity, except isolates from one infected foal and two patients with AIDS that showed rifampin resistance. To investigate the emergence of rifampin-resistant R. equi in the infected foal, which had received rifampin monotherapy for a month before euthanasia, 99 isolates of R. equi from the lesions and 20 isolates from the intestinal contents of the one foal with rifampin-resistant organisms were analyzed for rifampin susceptibilities, pathogenicities, and ribotypes. Of the 99 isolates from the lesions, all of which were virulent R. equi strains containing a virulence plasmid with a size of 85 or 90 kb, 90 (91%) isolates were rifampin resistant (MIC, м12.5 g/ml). On the other hand, of the 20 isolates from the intestinal contents, 11 (55%) isolates showed rifampin resistance (MIC, м25 g/ml), and 5 of them were avirulent R. equi strains. Among these 101 rifampin-resistant R. equi isolates with and without virulence plasmids characterized by ribotyping, 58 were type I, 20 were type II, 11 were type III, and 12 were type IV. These results demonstrated that at least eight different rifampin-resistant R. equi strains emerged concurrently and respectively from the different lesions and intestinal contents of the infected foal.

The efficacies of the new quinolones temafloxacin, ofloxacin, and ciprofloxacin were investigated against Mycoplasma pneumoniae in an experimental hamster pneumonia model. Hamsters were infected intratracheally with M. pneumoniae and sacrificed 18 h after the final medication, and their lungs were aseptically removed, homogenized, and cultured quantitatively. The efficacies of these drugs were determined by the CFU of M. pneumoniae in lungs. Temafloxacin and ofloxacin, but not ciprofloxacin, were active when the oral administration of200 mg/kg ofbody weight per day (once per day) for 5 days was initiated 24 h after infection. Although no effect on the elimination of M. pneumoniae was observed after the administration of these drugs at 200 mg/kg/day at 5 days after infection, the continuous administration for 15 days of temafloxacin, but not ofloxacin or ciprofloxacin, significantly reduced viable M. pneumoniae in the lungs. These results suggest that temafloxacin and ofloxacin are effective in the acute phase of infection and, moreover, that temafloxacin is effective in the late stage of infection during which progressive lung alterations and continuous increases in mycoplasmal growth occurred. The peak levels of temafloxacin in sera and lungs after oral administration were similar to those of ofloxacin and higher than those of ciprofloxacin. The areas under the curve of temafloxacin in the lung tissue, however, were higher than those of ofloxacin and ciprofloxacin. On the basis of these results, temafloxacin and ofloxacin might be promising antimicrobial agents for the treatment of mycoplasmal infection.Mycoplasma pneumoniae is the causative agent of upper respiratory tract infections and pneumonia in humans and the experimental pneumonia of hamsters (3,5,8,10). Macrolide and tetracycline antibiotics have been widely used in chemotherapy against M. pneumoniae infections because of the susceptibility of the infectious agent to these antibiotics (1,4,13,17,23). At the same time, the reported incidences of mutants resistant to erythromycin from patients treated with or without the antibiotics have increased (14,15).Recently, the efficacy of new quinolones against various respiratory pathogens, including M. pneumoniae, has been demonstrated (4,16,22). We have also reported that temafloxacin, ofloxacin, and ciprofloxacin, among several new quinolones, possess more mycoplasmacidal activity against M. pneumoniae than macrolide and tetracycline antibiotics. In this report, we evaluated the in vivo potency of new quinolones, administered orally against M. pneumoniae pneumonia in hamsters, which exhibited the potent mycoplasmacidal activity in vitro described previously (1). MATERIALS AND METHODSM. pneumoniae. M. pneumoniae 242, freshly isolated from the throat swab of a patient with mycoplasma pneumonia and passaged five to seven times in broth, was used as the challenge strain. Culture samples were kept at -80°C until used for inoculation. The MICs of temafloxacin, ofloxacin, and ciprofloxacin for the strain were 1.56,...

The spontaneous mutant 18a derived from Streptomyces kasugaensis MB273 exhibited pleiotropic effect such as loss of aerial mycelium formation, aureothricin (AT) production, and of citrullin biosynthesis, as well as changes in plasmid; the mutant required cystine for production of aureothricin.An improved method of protoplast regeneration was applied to S. kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained. Sixty to ninety percent of the colonies regenerated from the 18a protoplasts exhibited reversion of the pleiotropic mutation in 18a. Moreover, of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied. These types could be divided into two groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected. From these results, the following conclusions were obtained.First, plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protoplast regeneration. Second, the structural genes for biosynthesis of AT probably exist on chromosome.Third, regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency. A working hypothesis was proposed to explain these complex phenomena.Recent studies of genetic determinants for antibiotic biosynthesis are making it clear that plasmids are often involved.However, the role of plasmids in antibiotic production is still not clear, except for the plasmid SCP1 carrying the structural genes of methylenomycin in Streptomyces coelicolor A3(2) 1,2).Streptomyces kasugaensis strain M338 and the newly isolated strain MB273 have been used in our laboratory; both produce aureothricin (AT), thiolutin and kasugamycin. AT and thiolutin are N-propionylpyrrothine and N-acetylpyrrothine, respectively.The results of our early experiments showed that L-cystine was the precursor of AT biosynthesis and that pyrrothine was an intermediate in the pathway from L-cystine to AT' .4). AT-non-producing mutants were induced by acriflavine treatment. When these non-producers were incubated in synthetic liquid medium supplemented with cystine, neither AT nor pyrrothine was detected. However, when pyrrothine was added in place of cystine, all non-producers produced AT4,5) even "natural" AT-nonproducers such as S. venezuelae or S. erythreus produced AT from added pyrrothine4,5). Acylation of pyrrothine was also confirmed in cell-free systems prepared from these non-producing mutants5).Because of the facts mentioned above, we thought that plasmid-borne genes might play a role in the biosynthetic pathway from cystine to pyrrothine, and that chromosomal genes might be involved in the acylation of pyrrothine to AT. In this paper, it is shown that this conclusion is in need of revision.S. kasugaensis MB273 carries three kinds of plasmids; pSK1, pSK2 and pSK3. Both pSK1 and pSK2 are 6.8Md, and pSK3 is 14.5Md. The pSK...