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The cell membrane and the nuclear membrane are two major barriers hindering the free movement of various macromolecules through animal cells. Nevertheless, some proteins can actively bypass these barriers by dint of intrinsic peptidic signals, so incorporation of these signals might improve the efficacy of artificial gene delivery vehicles. We examined the role of the nuclear localization signal (NLS) in gene transfer, using recombinant lambda phage as a model of the polymer/DNA complexes. We prepared a lambda phage displaying a 32-mer NLS of SV40 T antigen on its surface (NLS phage), and found that this NLS phage, delivered into the cytoplasm by appropriate devices, has higher affinity for the nucleus and induces the expression of encapsulated marker genes more efficiently than does the wild-type phage. This suggests that the 32-mer NLS peptide will become a practical tool for artificial gene delivery vehicles with enhanced nuclear targeting activity.

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 lambda 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 Tat-phage 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.

The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli beta-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 x 10(9) plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.

By N-methyl-N'-nitro-N-nitrosoguanidine treatment, neamine-negative mutants which required neamine for biosynthesis of butirosins were obtained from a butirosin-producing organism Bacillus circulans. These mutants also produced butirosins from paromamine and could be divided into two types I and II. Mutants of type I could not produce butirosins from 2-deoxystreptamine, whereas those of type II could. Two typical mutants MCRL 5003 (type I) and MCRL 5004 (type II) could produce butirosin analogs, 3', 4'-dideoxybutirosins, 6'-N-methylbutirosins, 3', 4'-dideoxy-6'-N-methylbutirosins and 3', 4'-dideoxy-6'-C-methyl-butirosins from neamine analogs, gentamine Cla, 6'-N-methylneamine, 6'-N-methylgentamine Cla and gentamine C2, respectively.

To develop a host-vector system in streptomycetes for DNA cloning, we examined the technical problems encountered and the conditions required for use of Streptomyces kasugaensis MB273 as the host. Basic techniques, such as plasmid DNA isolation, regeneration of mycelia from protoplasts and elimination of plasmids from cells were investigated. These techniques were found to be useful for many streptomycetes. Strain M518, a derivative of S. kasugaensis MB273, was found to have the following useful characteristics as a host. The plasmids of MB273 were easily cured by regeneration of mycelia from protoplasts. The protoplasts prepared from M518 regenerated mycelia at high frequency using an improved method and were efficiently transformed by plasmid DNA. The extra and intra cellular DNase activities were very weak, and no restriction endonuclease activity was detected. The sensitivity to various antibiotics was determined. This strain did not show any pathogenicity in mice nor suvival in the digestive organs of rats. MB273 and its derivatives died rather quickly in natural soil. M518 still forms aerial mycelial conidia. These results indicate that S. kasugaensis M518, derived from MB273, has useful characteristics as a host for DNA cloning. The techniques thus developed were found to be useful in other streptomycetes.

Three cryptic plasmids, designated pBT1 (5.6 kb), pBT2 (9.7 kb), and pBT3 (16.6 kb), were isolated from Streptomyces griseobrunneus ISP5066 and physically characterized. pBT1 and pBT2, which differ by a 4.1-kb segment, are high copy-number plasmids (40-100 copies per chromosome) that coexist with each other. pBT3 is a low copy-number plasmid. Vectors containing amikacin (or kanamycin) and sulfomycin (or thiostrepton) resistance genes from Streptomyces litmocidini ISP5164 and Streptomyces viridochromogenes subsp. sulfomycini ATCC 29776, respectively, were constructed from pBT1. One such vector, pBT37, has unique restriction sites for cloning, including BglII, XhoI, PvuII, ClaI, and SacI, with the PvuII and ClaI sites allowing clone recognition by insertional inactivation of sulfomycin resistance. Since many Streptomyces species were very sensitive to amikacin and sulfomycin, these resistance genes serve as useful selective markers. pBT37 could transform several Streptomyces strains that produce antibiotics such as tetracyclines, macrolides, beta-lactams, and aminoglycosides. This plasmid is a potentially useful vector for cloning antibiotic biosynthetic genes.

Three plasmids, pSCY 2, pSCY 3 and pSCY 4, were detected in Streptomyces noursei B3, a producer of cycloheximide and nystatin. pSCY 3 and pSCY 4 had molecular sizes of 15.1 megadaltons (Md) and 3.2 Md, respectively. When covalently closed circular (ccc) DNAs were extracted from the mycelia during the course of cultivation, the yield of ccc DNA extracted per mycelium reached the highest value at the starting point of the exponential growth phase and decreased rapidly during exponential phase; the yield increased gradually in stationary phase. The smallest plasmid, pSCY 4, could not be detected after 42 hours of cultivation. To purify and characterize DNA of the major plasmid pSCY 3, it was cloned into Escherichia coli using pACYC 184 as a vector, and the physical maps of the composite plasmids were constructed.

By using 2 neamine-negative mutants, MCRL 5003 and MCRL 5004, of Bacillus circulars, subunit assembly in the biosynthesis of butirosins was investigated. The two mutants were blocked at different steps in the biosynthetic pathway of butirosins, but could produce butirosins when the culture medium was supplemented with neamine. Mutant MCRL 5003 accumulated 2-deoxystreptamine (DOS) in the fermentation broth but could not utilize DOS for the biosynthesis of butirosins, whereas mutant MCRL 5004 could produce butirosins from DOS. Based upon the ability of these 2 mutants to incorporate a series of DOS-containing compounds considered as plausible biosynthetic intermediates of butirosins into butirosins or related antibiotics, the biosynthetic pathways for butirosins and 6'-deamino-6'-hydroxybutirosins were proposed. Feeding experiments with 3', 4'-dideoxy derivatives of neamine, ribostamycin and butirosins supported the pathway proposed for butirosins. A glucosamine auxotroph MCRL 5771 of B. circulars afforded some information as to the role of D-glucosamine in the biosynthesis of butirosins.

A new complex of phleomycin-bleomycin group antibiotics, YA-56, was isolated from the culture broth of a streptomycete designated as strain MCRL 0387 and identified as a new variety of Streptomyces humidus NAKAZAWA and SHIBATA in IMAMURA et al., 1956. Ultra-violet irradiation of strain MCRL 0387 gave a high-yielding mutant which formed blue aerial mycelium instead of the grayish aerial mycelium of the original strain. Fermentative production of the antibiotics YA-56 complex is described.

珪肺症160剖検例中肺癌合併は27例, 16.7%に達した. これは全日本肺癌剖検例の9.0%, 全日本死亡統計2.4%よりはるかに高率である. 珪肺と肺癌合併例の組織像は未分化癌, 扁平上皮癌が多い. その他, 粉塵歴, 鉱山歴, 病理所見に一般の肺癌あるいは珪症と著差はない. 一方, 肺癌例の肺門リソパ節珪症合併率は極めて高く, これを含めれぽ肺癌と珪症の合併は一般に考えられるよりもかなり多い. 組織発生的には, 比較的早期肺癌原発巣では珪症との接触をみる.

Two pairs of butirosin analogs were isolated from the fermentation broths obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulars in the medium supplemented with 6'-N-methylneamine and gentamine C2, respectively. These antibiotics were characterized as 6'-N-methylbutirosins A and B (NMB-A & NMB-B), and 3', 4'-dideoxy-6'-C-methylbutirosins A and B (DCB-A & DCB-B), respectively, by chemical and spectroscopic studies. NMB-A and NMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for butirosin A, but with greater activities against butirosin-resistant strain which contains acetylating enzyme AAC- (6')-I. The activities of NMB-A and NMB-B against Pseudomonas aeruginosa strains were relatively weak. DCB-B had broad-spectrum activity, and exhibited greatly improved activity against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. These new butirosin analogs were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin.

By using 2 neamine-negative mutants, MCRL 5003 and MCRL 5004, of Bacillus circulars, subunit assembly in the biosynthesis of butirosins was investigated. The two mutants were blocked at different steps in the biosynthetic pathway of butirosins, but could produce butirosins when the culture medium was supplemented with neamine. Mutant MCRL 5003 accumulated 2-deoxystreptamine (DOS) in the fermentation broth but could not utilize DOS for the biosynthesis of butirosins, whereas mutant MCRL 5004 could produce butirosins from DOS. Based upon the ability of these 2 mutants to incorporate a series of DOS-containing compounds considered as plausible biosynthetic intermediates of butirosins into butirosins or related antibiotics, the biosynthetic pathways for butirosins and 6'-deamino-6'-hydroxybutirosins were proposed. Feeding experiments with 3', 4'-dideoxy derivatives of neamine, ribostamycin and butirosins supported the pathway proposed for butirosins. A glucosamine auxotroph MCRL 5771 of B. circulars afforded some information as to the role of D-glucosamine in the biosynthesis of butirosins.

Three plasmids from Micromonospora species were isolated and characterized. Micromonospora zionensis NRRL5466 (a producer of sisomicin and G-52) carried a high-copy-number plasmid pMZ1 (9.9 kb). Micromonospora rosaria NRRL3718 (a producer of rosamicin) contained a large plasmid, pMR1 (53.5 kb), and a relatively small plasmid, pMR2 (11.0 kb).

Two pairs of butirosin analogs were isolated from the fermentation broths obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulars in the medium supplemented with 6'-N-methylneamine and gentamine C2, respectively. These antibiotics were characterized as 6'-N-methylbutirosins A and B (NMB-A & NMB-B), and 3', 4'-dideoxy-6'-C-methylbutirosins A and B (DCB-A & DCB-B), respectively, by chemical and spectroscopic studies. NMB-A and NMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for butirosin A, but with greater activities against butirosin-resistant strain which contains acetylating enzyme AAC- (6')-I. The activities of NMB-A and NMB-B against Pseudomonas aeruginosa strains were relatively weak. DCB-B had broad-spectrum activity, and exhibited greatly improved activity against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. These new butirosin analogs were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin.

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.

A series of basic 16-membered macrolide antibiotics, M-4365 A1, A2, A3, G1, G2 and G3, were isolated from the culture broth of strain MCRL 0940 which is assigned to be a new species of Micromonospora and for which the name Micromonospora capillata sp. nov. is proposed. Among these antibiotics, M-4365 A2 and G2 showed strong inhibitory activity against Gram-positive and Gram-negative bacteria.

To elucidate the biosynthetic pathway of platenomycin (PLM), biosynthetic relationships of platenolides I (PL-1) and II (PL-II), 3-O-propionyl-5-O-mycaminosyl platenolides I (PPL-I-MC) and II (PPL-II-MC), 9-dehydro demycarosyl platenomycin (DDM-PLM), demycarosyl platenomycin (DM-PLM) and 4"-deacyl platenomycin (DA-PLM) were examined with growing cultures or the washed mycelium of blocked mutants of Streptomyces platensis subsp. malvinus MCRL 0388, a platenomycin-producing organism. As a result, it was revealed that PLM was biosynthesized from PL-I via DM-PLM and DAPLM along the pathways shown in Chart 1. 4"-Isovaleroyl unit of PLM-A1 and 4"-propionyl unit of PLM-B1 were respectively derived from L-leucine and L-isoleucine.

A pair of new butirosin analogs was isolated from the fermentation broth obtained by cultivating a neamine-negative mutant of the butirosin-producing organism Bacillus circulans in the medium supplemented with 6'-N-methylgentamine C1a. These antibiotics were characterized and elucidated as 3', 4'-dideoxy-6'-N-methylbutirosins A and B (DMB-A & DMB-B), by chemical and spectroscopic studies. DMB-A and DMB-B exhibited broad-spectrum antibacterial activities with in vitro potency similar to or slightly less than that for the butirosin A, with the exception of strains of Pseudomonas aeruginosa and Serratia marcescens against which they exhibited activities equal to or slightly greater than that for butirosin A. As expected, they exhibited stronger activities against butirosin-resistant organisms which contain acetylating enzymes AAC(6')-I and AAC(6')-IV, and phosphorylating enzyme APH(3')-II. They were also active against some of the clinical isolates resistant to butirosins, dibekacin and/or gentamicin. The acute intravenous toxicity in mice of the DMB complex (B:70 APPROXIMATELY 80%) was somewhat less than that of the butirosin A.