Proteinases of Leishmania mexicana amastigotes and promastigotes: Analysis by gel electrophoresis

The proteinases of Leishmania mexicana mexicana amastigotes and promastigotes have been analysed by electrophoresis on polyacrylamide gels containing denatured haemoglobin. Eleven bands of activity were detected indicating multiple proteinases. There were significant quantitative and qualitative differences between the proteinases of the two developmental forms. Four, B–E, were present in both forms but were of much higher activity in the amastigote. There were two major activities in promastigotes, A and D. The other proteinases, F–K, were of lower activity; I and K were not detected in promastigotes. All proteinases were active optimally at pH 4.0. Most of them, including the major proteinases A–E, were thiol proteinases since they were stimulated by 1 mM dithiothreitol and were sensitive to inhibitors such as HgCl₂, leupeptin, antipain and iodoacetic acid.     

Efficient generation of cowpea mosaicvirus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants

To elucidate the mechanism of formation of cowpea mosaic virus (CPMV) particles, RNA-2-encoded precursor proteins were expressed in Spodoptera frugiperda cells. Processing of the 105K and 95K polyproteins in trans to give the mature Large (L) and Small (S) coat proteins required both the 32K proteinase cofactor and the 24K proteinase itself, while processing of VP60, consisting of the fused L-S protein, required only the 24K proteinase. Release of the L and S proteins resulted in the formation of virus-like particles (VLPs), showing that VP60 can act as a precursor of virus capsids. Processing of VP60 expressed in plants also led to efficient production of VLPs. Analysis of the VLPs produced by the action of the 24K proteinase on precursors showed that they were empty (RNA-free). This has important implications for the use of CPMV VLPs in biotechnology and nanotechnology as it will permit the use of noninfectious particles.     

Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12-O-tetradecanoylphorbol-13-acetate

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer-causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low-density asynchronous cultures of C3H/101/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, N-nitrosomethylurea, N-nitrosoethylurea, mitomycin C, 5-fluorodeoxyuridine, and 5-azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3-methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.     

A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells

This study identifies instability of MHC class I/peptide complexes and intermolecular competition for MHC class I presentation as factors responsible for the subdominance of cyto toxic T lymphocyte (CTL) epitopes. This evidence is based on the characterization of a new CTL epitope derived from the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV). This epitope, peptide GP117-125 (GP117) is presented to T cells by the mouse MHC class I molecule, H-2D^b. In short-term experiments induction of GP117-specific CTL by vaccination rendered C57BL/6 mice only partially resistant to infection with wild-type LCMV (LCMV-WE) but completely resistant to challenge with a previously described LCMV variant. The variant virus, LCMV-8.7B23, bears point mutations within both known LCMV-GP, H-2 D^b-restricted epitopes GP33-41 (GP33) and GP276-286 (GP276) resulting in a valine to leucine change at position 35 in peptide GP33 (V35L) and an asparagine to serine change at position 280 in peptide GP276 (N280S). Although variant peptide GP33/V35L stimulates a weak CTL response, GP276/N280S does not. Elution of peptide GP117 from both LCMV-WE- and LCMV-8.7B23-infected cells revealed that the difference in the capacity of GP117-specific CTL to protect against LCMV-WE and the virus variant LCMV-8.7B23 was due to differences in the level of GP117 presentation on the surface of both types of cells. Thus, it appears that the protective capacity of CTL specific for the subdominant epitope GP117 is influenced by the extent of presentation of other immunodominant peptide epitopes present within infected cells.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

Measurement of deoxyribonuclease I (DNase) in the serum and urine of systemic lupus erythematosus (SLE)-prone NZB/NZW mice by a new radial enzyme diffusion assay

A new radial enzyme diffusion (RED) method for the measurement of DNase activity in serum and urine is described. The sensitivity of the assay is in the range of 15.6–500 ng/ml. The assay is based on the hydrolysis of double-stranded (ds) DNA (or nucleosomes) in agarose. The specificity of the reaction for DNase I was established by showing that either EDTA in the reaction buffer or G-actin abolished DNase activity. Being a functional assay, RED has advantages over radioimmunoassay (RIA) or ELISA, since antigenic assays may also measure complexes of DNase with actin. This method was used to measure DNase activity in the sera and urine of lupus-prone mice (NZB/NZW F₁ hybrids, aged 4–6 weeks). Serum DNase activity in these mice was significantly lower (mean 9 ng/ml) than in control, normal mice of the same age and sex (mean 37 ng/ml). Concentration of DNase in the urine of 4–6-week-old female NZB/NZW F₁ hybrids (24 ng/ml) was significantly lower then in control mice (521 ng/ml). The RED method was used to measure the concentration of actin as the DNase inhibitor in serum. G-actin in the presence of ATP binds DNase and inhibits its nucleolytic activity. Since ATP is necessary for the actin inhibition of DNase I, this shows that there is actin as well as DNase I in the serum. Actin is not only ATP-dependent, but also heat-labile. Heating the sera for 10 min at 50°C increases DNase activity. This is an alternative method for measuring the concentration of actin in the serum. An almost identical estimate of actin concentration in sera of normal mice was found from the difference of DNase activity in the presence or absence of ATP (mean actin concentration = 21 ng/ml) or from the difference of DNase activity in heated and non-heated serum (mean actin concentration 18 ng/ml). We were not able to demonstrate DNase inhibitors in the urine of either control or NZB/W F₁ hybrid mice.