A mutant of Bacillus thuringiensis var. israelensis (B.t.i.) resistant to antibiotics

Summary PST, a spontaneous mutant of Bacillus thuringiensis var. israelensis (B.t.i.) resistant to penicillin, streptomycin and tetracycline was isolated by serial selections. In the absence of antibiotics it showed genetic stability for 16 generations. Mosquito larvicidal activity of PST was similar to that of B.t.i., its parental strain. It also maintained the specific antigenicity of B.t.i. although its rate of growth was somewhat lower, a generation time of 55 min for PST vs. 38 min for B.t.i. Cell concentration plays a major role in the phenomenon of PST resistance to penicillin.This antibiotic resistant mutant of b.t.i. provides us with an efficient tool to trace B.t.i. among the indigenous bacteria present in septic habitats in the field as well as inside the larval gut.     

The microheterogeneity of the crystallizable yeast cytoplasmic aspartyl-tRNA synthetase

Yeast aspartyl-tRNA synthetase is a dimeric enzyme (alpha 2, Mr 125,000) which can be crystallized either alone or complexed with tRNAAsp. When analyzed by electrophoretic methods, the pure enzyme presents structural heterogeneities even when recovered from crystals. Up to three enzyme populations could be identified by polyacrylamide gel electrophoresis and more than ten by isoelectric focusing. They have similar molecular masses and mainly differ in their charge. All are fully active. This microheterogeneity is also revealed by ion-exchange chromatography and chromatofocusing. Several levels of heterogeneity have been defined. A first type, which is reversible, is linked to redox effects and/or to conformational states of the protein. A second one, revealed by immunological methods, is generated by partial and differential proteolysis occurring during enzyme purification from yeast cells harvested in growth phase. As demonstrated by end-group analysis, the fragmentation concerns exclusively the N-terminal end of the enzyme. The main cleavage points are Gln-19, Val-20 and Gly-26. Six minor cuts are observed between positions 14 and 33. The present data are discussed in the perspective of the crystallographic studies on aspartyl-tRNA synthetase.     

Regulation of biogenesis of liver delta-aminolevulinate synthase: effects of structural modifications of heme on the enzyme's RNA

1. The aim of this study was to determine the effects of several metallo-porphyrins, derived by modifications of heme, on the concentration delta-aminolevulinate (ALA) synthase RNA in hepatocytes. 2. Primary cultures of chick embryo hepatocytes were incubated with allylisopropylacetamide (AIA) for 5 hr in the presence and absence of each metallo-porphyrin (10 microM). At the end of each incubation, total RNA was isolated from the cells and analyzed for ALA synthase-specific RNA by solution hybridization. 3. The concentration of ALA synthase RNA increased 7.3 fold in hepatocytes incubated with AIA alone. The AIA-induced elevations in the enzyme's RNA were blocked partially and equally in cells. incubated with zinc- or with iron-protoporphyrin IX. The block was greater in cells incubated with cobalt-protoporphyrin IX. 4. Modifications of the side chains of the porphyrin ring at positions 2 and 4, giving mesoporphyrin IX and deuteroporphyrin IX, changed the effectiveness of the iron- and the cobalt-porphyrins to limit the AIA-induced increase in ALA synthase RNA. The modifications did not affect the capacities of the zinc-porphyrins to inhibit the rise in RNA. 5. In conclusion, the effect of a given metallo-porphyrin on liver ALA synthase RNA following side chain modification depended on the coordinated metal.     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

Identification and characterization of E.coli ribosomal binding sites by free energy computation.

Sequences upstream from translational initiation sites of different E.coli genes show various degrees of complementarity to the Shine-Dalgarno (SD) sequence at the 3' end of the 16S rRNA. We propose a quantitative measure for the SD region on the mRNA, that reflects its degree of complementarity to the rRNA. This measure is based on the stability of the rRNA-mRNA duplex as established by free energy computations. The free energy calculations are based on the same principles that are used for folding a single RNA molecule, and are executed by similar algorithms. Bulges and internal loops in the rRNA and mRNA are allowed. The mRNA string with maximum free energy gain upon binding to the rRNA is selected as the most favorable SD sequence of a gene. The free energy value that represents the SD region provides a quantitative measure that can be used for comparing SD sequences of different genes. The distribution of this measure in more than 1000 E.coli genes is presented and discussed.     

Two tumor models of curative adoptive chemoimmunotherapy using tumor-infiltrated spleen cells with potent antitumor cytotoxicity stimulated by antigen-sharing tumors

Previously we have established curative protocols for adoptive chemoimmunotherapy (ACIT) of mice bearing different plasmacytomas that are known to bear cross-reacting antigens: (a) the cure of mice bearing an early-stage, nonpalpable MOPC-315 tumor by a very low dose of cyclophosphamide (10 mg/kg) and cultured MOPC-315-tumor-infiltrated (TI) spleen cells (25×10⁶) and (b) the cure of mice bearing a late-stage, relatively drug-resistant, highly metastatic RPC-5 tumor with cyclophosphamide (100 mg/kg) and cultured RPC-5 TI spleen cells (25×10⁶–50×10⁶). In both models, the spleen cells were obtained from mice bearing a late-stage tumor and were cultured for 5 days in the presence of polyethyleneglycol 6000 and autochthonous tumor cells as a source of tumor antigen. Here we show that RPC-5 tumor cells could substitute for MOPC-315 tumor cells in the 5-day culture of MOPC-315 TI spleen cells so that they became curative in ACIT for mice bearing an early-stage MOPC-315 tumor. Similarly, MOPC-315 tumor cells could substitute for RPC-5 tumor cells in the 5-day culture of RPC-5 TI spleen cells so that they became curative in ACIT of mice bearing a late-stage RPC-5 tumor. In addition, RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were effective in curing all mice bearing an early-stage MOPC-315 tumor by ACIT. However, MOPC-315 TI spleen cells whether cultured with MOPC-315 or RPC-5 tumor cells, were much less effective than cultured RPC-5 TI spleen cells in curing mice bearing a late-stage RPC-5 tumor by ACIT (although the survival of these mice was extended significantly). Interestingly, whereas RPC-5 TI spleen cells cultured with either MOPC-315 or RPC-5 tumor cells were as effective as MOPC-315 TI spleen cells cultured under the same conditions in lysing MOPC-315 tumor cells in vitro, MOPC-315 TI spleen cells that had been cultured with either MOPC-315 or RPC-5 tumor cells exerted a much weaker in vitro cytotoxic T lymphocyte activity against RPC-5 tumor cells than did RPC-5 TI spleen cells that had been cultured under the same conditions.Work was supported by research grant CA-30088 from the National Cancer Institute and IM-435 from the American Cancer Society. M. B. M. was supported by Career Development Award CA-01350 from the National Cancer InstituteThis work is in partial fulfillment of the requirements for the Ph. D. degree     

Yeast tRNAAsp: codon and wobble codon-anticodon interactions. A transferred nuclear Overhauser enhancement study

The conformations of the ribotrinucleoside bisphosphates GpApC and GpApU, the codon and wobble codon for aspartic acid respectively, bound to yeast tRNAAsp in solution, have been examined by means of time-dependent transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The conformations of the two bound ribotrinucleoside bisphosphates are shown to be very similar with an overall root-mean-square difference in interproton distances of 0.03 nm. The ribose conformations of all the residues are 3'-endo; the glycosidic bond torsion angles of the A and C residues of GpApC and of the A and U residues of GpApU are in the low anti range. These features are typical of an A-RNA type structure. In contrast, the G residue of both GpApC and GpApU exists as a mixture of syn and anti conformations. The overall conformation of the two bound ribotrinucleoside bisphosphates is also similar to A-RNA and the stability of the complexes is enhanced by extensive base-base stacking interactions. In addition, it is shown that the binding of the codon GpApC to tRNAAsp induces self-association into a multicomplex system consisting of four GpApC-tRNAAsp complexes, whereas the wobble codon GpApU fails to induce any observable self-association.