Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture     

Expression of Dictyostelium early gene, dutA, is independent of cAMP pulses but dependent on protein kinase A

Abstract The untranslatable, RNA polymerase II-dependent gene ( dutA ) of Dictyostelium discoideum is induced early in development. However, unlike other early genes, dutA induction was not affected by cAMP pulses and occurred normally in various cAMP-related mutant cells, the results indicating that this induction depended solely on factors other than cAMP. In the knockout strain of the catalytic subunit of protein kinase A, dutA expression was severely blocked and not recovered by cAMP pulses. This demonstrates that even the cAMP-independent gene, dutA , requires protein kinase A for its expression.     

Phencyclidine-induced expression of c-Fos-like immunoreactivity in mouse brain regions

For the purpose of studying a role of immediate early genes in psychotomimetic-induced behavioral excitation, we experimentally enhanced the locomotor activity of mice by acute administration of phencyclidine and examined the expression and localization of the c-Fos-like and c-Jun-like immunoreactivities in brain regions. A single injection of phencyclidine (5.0 mg/kg, i.p.) significantly increased not only the locomotor activity but also the expression of c-Fos-like immunoreactivity in several brain regions, particularly in the parietal cortex, hippocampal dentate gyrus, piriform cortex and hypothalamus. Interestingly, the c-Fos-like immunoreactivity in the parietal cortex continued to increase for 1 week after the phencyclidine injection. These results indicate that phencyclidine, even injected only once, can induce the persistent expression of c-Fos or c-Fos-related protein(s) in the mouse brain, and also suggest the possibility that such a c-Fos expression may underlie the behavioral and/or psychotomimetic effects of phencyclidine.     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Cloning of the macrolide antibiotic biosynthesis gene acyA, which encodes 3-O-acyltransferase, from Streptomyces thermotolerans and its use for direct fermentative production of a hybrid macrolide antibiotic.

A gene encoding the macrolide modification enzyme 3-O-acyltransferase (acyA) was cloned by chromosome walking onto the carbomycin biosynthetic region in Streptomyces thermotolerans TH475, with the 3' region of the gene encoding the macrolide modification enzyme 4"-O-acyltransferase (acyB1) as a probe. A shortened fragment (1.8 kb) containing acyA was subcloned with pIJ350. A high-level tylosin producer, Streptomyces fradiae MBBF, transformed with the plasmid could produce a hybrid macrolide, 3-O-acetyltylosin, most efficiently.     

Microheterogeneity of PSI-E Subunit of Photosystem I in Nicotiana sylvestris

Microheterogeneity of a photosystem I (PSI) subunit encodedby a nuclear gene psaE was examined in Nicotiana sylvestris,with the aid of cDNA cloning, peptide mapping analysis and proteinsequencing. The psaE product of this plant has four isoformswhose mobilities in PAGE are slightly different from each other.We isolated two types of psaE cDNAs from a N. sylvestris cDNAlibrary, and designated the corresponding genes as psaEa andpsaEb, respectively. The psaEa and psaEb genes are 77% homologousat DNA level, and their translation products share 80.4% homologyfor the precursor proteins and 89.1% for the mature forms. Comparativeanalysis of the four isoproteins and the putative products ofthe two psaE genes revealed that two isoproteins out of fourare derived from psaEa gene, and the difference between thesetwo isoproteins lies in the respective presence or absence ofN-terminal alanine. Likewise, the other two proteins are derivedfrom psaEb with similar N-terminal heterogeneity. These resultsindicate that multi-gene organization and heterogeneous N-terminalformation at post-translational level are two possible causesfor PSI subunit polymorphism in isogenic plant lines. (Received October 8, 1993; Accepted November 30, 1993)     

Production of poly(3-hydroxyalkanoates-co-3-hydroxy-ω-fluoroalkanoates) byPseudomonas oleovorans from 1-fluorononane and gluconate

Summary Pseudomonas oleovorans grew well and synthesized copolyesters of 3-hydroxyalkanoates and 3-hydroxy--fluoroalkanoates in the mineral medium containing 1-fluorononane and sodium gluconate. The content of fluorinated units in copolyesters could be controlled from 0 to 40 mol%. The copolyesters were shown to have a random sequence distribution of different monomeric units by analysis of the¹³C NMR spectra. The melting temperatures of copolyesters were 52–58°C, and the enthalpy of fusion decreased with the content of fluorinated units.     

Enhanced expression of group II phospholipase A2 in human hepatocellular carcinoma

Enzyme activity, protein contents, and mRNA contents of group II phospholipase A₂ (PLA₂) in hepatocellular carcinoma (HCC) surgically obtained from 8 patients were compared with those in either its neighboring liver tissues or control liver tissues. The PLA₂ specific activity towards the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was significantly greater in the tumor tissues (6.62 ± 1.46 nmol/min/mg) than those in the surrounding liver tissues (1.33 ± 0.22 nmol/min/mg) and controls (0.43 ± 0.04 nmol/min/mg). The results of immunoblot analysis using a specific anti-human group II PLA₂ antibody and of Northern blot analysis using a human group II PLA₂ cDNA as a probe demonstrated that group II PLA₂ was responsible for the increased enzyme activity. The contents of immunoreactive group II PLA₂ in the tumor tissues (8.81 ± 1.24 ng/mg) were significantly higher than those in the surrounding liver tissues (1.77 ± 0.27 ng/mg); those in the control tissues were below the analytical range of the method used. The group II PLA₂ mRNA was also significantly increased in the tumor tissues, compared with that in the surrounding liver tissues, whereas it was not detectable in th controls. This indicates that group II PLA₂ in HCC is induced at the pretranslational level.