Episome-mediated transfer of drug resistance in Enterobacteriaceae. IX. Recombination of an R factor with F

Watanabe, Tsutomu (Keio University, Tokyo, Japan), and Chizuko Ogata. Episome-mediated transfer of drug resistance in Enterobacteriaceae. IX. Recombination of an R factor with F. J. Bacteriol. 91:43-50. 1966.-R factors can be transduced in Salmonella typhimurium with phage P-22, and a majority of the drug-resistant transductants are unable to transfer their drug resistance by cell-to-cell contact, as we have previously reported. Several exceptional types of transductants of S. typhimurium, with the markers of resistance to sulfonamide, streptomycin, and chloramphenicol, were recently obtained by transduction with phage P-22 of a four-drug-resistance R factor carrying the markers of resistance to sulfonamide, streptomycin, chloramphenicol, and tetracycline. They were exceptional in that they had low conjugal transferability of their drug resistance. When one of these exceptional transductants (38R) was transferred to an F(+) strain of Escherichia coli K-12, 38R acquired high transferability in its further transfer. This high transferability was found to be due to the recombination of 38R with F. Transductant 38R was of the fi(+) (fi = fertility inhibition) type, and did not show superinfection immunity against fi(+) and fi(-) R factors. The recombinant 38R.F was genetically very stable and resistant to elimination with acridines. It did not show superinfection immunity against fi(+) and fi(-) R factors, but did show superinfection immunity against F. Further, 38R.F did not restrict a female-specific phage (W-31), unlike wild-type F. F(-) and R(-) segregants were isolated from this recombinant 38R.F, and these segregants exhibited genetic characteristics different from the original R, its transductant 38R, and wild-type F.     

Studies on the cell-cycle dependency of the actions of insulin-like growth factor-I in Balb/c 3T3 cells

The present study was conducted to determine the cell-cycle dependency of various actions of IGF-I in Balb/c 3T3 cells. When autophosphorylation of the IGF-I receptor was determined in [32P]-labelled cells, IGF-I increased radioactivity in a 100 K-Da phosphoprotein, presumably beta-subunit of the IGF-I receptor, both in quiescent and in primed competent cells. Likewise, IGF-I stimulated uptake of [3H]deoxyglucose independent of the cell cycle. In contrast, IGF-I increased calcium entry, radioactivity in [3H]diacylglycerol, and [3H]thymidine incorporation in primed competent cells while these reactions were not induced by IGF-I in quiescent cells. The latter three reactions were attenuated when cells were pretreated with pertussis toxin. These results indicate that some, but not all, of the actions of IGF-I are dependent on the cell cycle in Balb/c 3T3 cells. They also suggest that a pertussis-toxin-sensitive G protein may be involved in the cell-cycle-dependent actions of IGF-I.     

Domain II mutants of Pseudomonas exotoxin deficient in translocation

Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.     

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

Effects of activin A and somatostatin on intact FSH secretion and intracellular Ca2+ concentration in human FSH-secreting pituitary adenoma cells.

Activin A stimulated synthesis and secretion of intact FSH in dispersed human FSH-secreting adenoma cells. Significant stimulation was observed after 24 hr. Activin A caused an increase in Ca2+ concentration ([Ca2+]i). This response occurred soon after the activin A action. These effects were blocked in Ca(2+)-deficient medium and by nitrendipine (5 microM). Somatostatin inhibited the activin A-induced intact FSH secretion and the [Ca2+]i response. These findings indicated that Ca2+ influx through voltage-gated Ca2+ channel was involved in the activin A induced synthesis and secretion of intact FSH.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli,on the pulmonary metastases of lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor,IL-4

In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5×10⁷ cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.