Immunogenicity and tumor protective activity of B16 melanoma vaccines

The immunogenicity and tumor-protective activity of different vaccines were examined and compared with murine B16 melanoma. All vaccines were prepared from material shed into culture medium by B16 melanoma cells. Vaccine I was generated by concentrating the shed material. Vaccine II was partially purified by precipitating the shed material with 50% ammonium sulfate followed by sephadex G-200 column chromatography. Vaccine III was concentrated shed material that was treated with 0.5% NP-40 and then ultracentrifuged to remove transplantation antigens. Mice were immunized to equal protein concentrations of vaccines weekly for 5 weeks or to control buffer. Antibody, cellular, and tumor-protective immunity to melanoma was measured in all mice 2 weeks following the last immunization. All three vaccine preparations were immunogenic. Vaccine preparation I appeared to be the most immunogenic and the one that most consistently augmented tumor-protective immunity. Augmentation in tumor-protective immunity correlated better with increase in cellular than in humoral immunity to melanoma.     

A new chondrodystrophy mutation in Japanese quail (Coturnix japonica)

A second form of hereditary chondrodystrophy (ch-2) has been discovered in a selected line of Japanese quail, Coturnix japonica. This form of chondrodystrophy is autosomal and recessive, characterized by an overall shortening and bending of the long bones of the wings and legs, slight dwarfing of the trunk, bulging of the eyes, flattening of the head, and a parrot beak. The shortened long bones vary in regard to the amount of bending from nearly straight to bends of up to 90 degrees in the midshaft region. In severe cases, the bend is evident as a protuberance of the skin. Affected embryos usually survive the 18-day incubation period. Several have hatched, but most survived no longer than 4 days after hatching. Only one female has survived long enough to lay eggs. Testcrosses indicated that this mutation is not allelic to micromelia.     

Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.

nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.     

Attenuating mutations in glycoproteins E1 and E2 of Sindbis virus produce a highly attenuated strain when combined in vitro.

Alterations in either the E1 or the E2 glycoprotein of Sindbis virus can affect pathogenesis in animals. Previously, we identified two distinct E1 glycoprotein gene sequences which differed in their effect on pathogenesis. One had an attenuation phenotype following subcutaneous inoculation of neonatal mice (E1 Ala-72, Gly-75, and Ser-237), while the other was virulent (E1 Val-72, Asp-75, and Ala-237). In this study, we examined the basis for this difference in pathogenesis by using a full-length cDNA clone of Sindbis virus from which infectious RNA could be transcribed in vitro. The relative contribution of each E1 residue to the pathogenesis phenotype was determined by using site-directed mutagenesis to alter each codon individually and in combination. Residues 75 and 237, in combination, appeared to be the major E1 determinants affecting pathogenesis. In addition, the effect of directly combining independently attenuating E1 and E2 mutations in the same virus was examined. The attenuating E1 sequences characterized in this study were coupled to a previously characterized attenuating mutation at E2 residue 114. The resulting recombinant virus, constructed in vitro, exhibited an increased attenuation of neurovirulence as compared with recombinant viruses containing either of the attenuating elements alone.     

Prostaglandin E2 enhances,and leukotriene C4 inhibits,interleukin-1 inhibition of WEHI-3B cell growth

Summary Human recombinant interleukin-1 (HrIL-1) inhibited WEHI-3B cell growth in a dose-related manner (10–10000 U/ml). Prostaglandin E₂ at high concentrations (1000 ng/ml) also inhibited cell growth. When added together, HrIL-1 and prostaglandin E₂ inhibited WEHI-3B growth in a synergistic manner (HrIL-1 concentrations of 10–10000 U/ml and prostaglandin E₂ concentrations of 10–1000 ng/ml). In contrast to the effects of the cyclooxygenase metabolite of arachidonic acid leukotriene C₄, a lipoxygenase metabolite, reversed the cytostatic action of HrIL-1.     

Carbon metabolism in Bradyrhizobium japonicum bacteroids

Abstract Carbon metabolism in Bradyrhizobium japonicum bacteroids is reviewed. Additionally, the bacteroid tricarboxylic acid (TCA) cycle and its regulation under oxygen-limited conditions is considered, with emphasis on possible sites of TCA cycle rate-limiting reactions. Furthermore, we consider other adaptive pathways that may be employed by these organisms while in symbiosis. These pathways include: (1) a poly-β-hydroxy-butyrate shunt, (2) a malate-aspartate shuttle, (3) an α-ketoglutarate-glutamate shunt, (4) a modified dicarboxylic acid cycle, and (5) fermentation pathways leading to lactate, acetaldehyde and ethanol. The effects of oxygen limitation on host carbon metabolism are also considered briefly.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.