Tumor immunity and autoimmunity induced by immunization with homologous DNA

The immune system can recognize self antigens expressed by cancer cells. Differentiation antigens are prototypes of these self antigens, being expressed by cancer cells and their normal cell counterparts. The tyrosinase family proteins are well characterized differentiation antigens recognized by antibodies and T cells of patients with melanoma. However, immune tolerance may prevent immunity directed against these antigens. Immunity to the brown locus protein, gp75/ tyrosinase-related protein-1, was investigated in a syngeneic mouse model. C57BL/6 mice, which are tolerant to gp75, generated autoantibodies against gp75 after immunization with DNA encoding human gp75 but not syngeneic mouse gp75. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection. Manifestations of autoimmunity were observed, characterized by coat depigmentation. Rejection of tumor challenge required CD4(+) and NK1.1(+) cells and Fc receptor gamma-chain, but depigmentation did not require these components. Thus, immunization with homologous DNA broke tolerance against mouse gp75, possibly by providing help from CD4(+) T cells. Mechanisms required for tumor protection were not necessary for autoimmunity, demonstrating that tumor immunity can be uncoupled from autoimmune manifestations.     

SSCP analysis of long DNA fragments in low pH gel

AIM: Antigen retrieval (AR) procedures are based on the effect of heating (by either microwave or pressure cooking treatments) on routinely fixed and paraffin embedded tissues. We observed that AR procedures restore the reactivity of endogenous biotin (EB) and report on the distribution of EB following AR in a series of routinely fixed and embedded tissues. METHODS AND RESULTS: Following pressure cooking or microwave treatments, a simple streptavidin-peroxidase staining revealed retrieved endogenous biotin (REB) in normal tissues (such as liver, kidney and adrenal cortex), in oxyphylic cells and in some tumours, especially in carcinomas of the kidney and of the adrenal cortex. In formalin-fixed (but not in alcohol-fixed) tissue sections, the heating procedures caused an intense and finely granular cytoplasmic reaction, following a routine streptavidin-conjugated peroxidase treatment. The staining was prevented by blocking of EB by a sequential avidin-biotin treatment. CONCLUSIONS: Retrieval of EB reactivity can cause pitfalls in diagnostic immunohistochemistry but, alternatively, it might also constitute a useful and novel diagnostic marker.     

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.     

Structural and functional properties of linker histones and high mobility group proteins in polytene chromosomes

Variants of histone H1 and high mobility group (HMG) proteins and their genes in Dipteran insects are being studied in our laboratory and have revealed different properties of DNA binding and intrachromosomal distribution. One of the H1 variants of Chironomus is found only in a minority of polytene chromosome bands and differs from the other H1 proteins of the same organism by genomic organization and by an inserted structural motif, the KAPKAP repeat, that is present also in single H1 variants of other, evolutionarily remote organisms. NH2-terminal peptides containing the KAPKAP repeat were found in vitro to interact with DNA, whereas no DNA interaction was observed with the homologous peptide of another H1 variant that does not contain the inserted KAPKAP repeat. We assume that H1 variants containing the KAP motif may interact with a stretch of linker DNA and package chromatin more tightly than other H1 variants. A large series of antibodies directed against different sites in all regions of the H1 molecule is being applied in studying the sites of interaction of the H1 molecule with other molecules in interphase chromatin in terms of antibody epitope accessibility. A search for insect proteins that share properties of the mammalian HMG proteins resulted in isolation and sequencing of two different HMG1 proteins and an HMGI protein. The HMG1 protein of the midge, Chironomus tentans, show a differential distribution in chromosomes. The more abundant cHMG1a protein appears uniformly distributed, whereas the less abundant cHMG1b protein could be localized only in chromosomal puffs. This strongly indicates that these highly similar proteins have different functions in chromatin. The Chironomus HMGI protein and the intron/exon organization of its gene were found to be very similar to human HMGI/Y proteins that are highly abundant in rapidly proliferating cells. Common properties of HMG1 and HMGI proteins include high affinity interaction with AT-rich DNA, irregular DNA structures, and the capacity to bend DNA. These properties suggest that the HMG proteins may have an architectural role in assembling different types of chromatin.     

Messenger RNA translation in the presence of homologous and heterologous tRNA

The effect of tRNA distribution in the cell on the rate of specific protein synthesis has been investigated. A tRNA-dependent cell-free protein-synthesizing system derived from Krebs-II ascites cells has been worked out. In this system it is possible to synthesize specific proteins (egg-white proteins or globin) by the translation of oviduct or reticulocyte messenger RNA in the presence of tRNA from homologous or heterologous tissues. The rate of translation of a give messenger RNA is optimal in the presence of tRNA from the homologous tissue. The lower level of protein synthesis obtained with an excess of heterologous tRNA can be overcome by adding the homologous tRNA. Nevertheless homologous isoaccepting species partly purified by a reversed-phase chromatography or a benzoylated DEAE-cellulose column cannot fully restore the optimal synthesis, eliminating the hypothesis according to which a particular tRNA species is involved in this phenomenon. The correct distribution of tRNA is necessary for optimal translation of a messenger RNA.     

Conserved domains in DNA repair proteins and evolution of repair systems

A detailed analysis of protein domains involved in DNA repair was performed by comparing the sequences of the repair proteins from two well-studied model organisms, the bacterium Escherichia coli and yeast Saccharomyces cerevisiae, to the entire sets of protein sequences encoded in completely sequenced genomes of bacteria, archaea and eukaryotes. Previously uncharacterized conserved domains involved in repair were identified, namely four families of nucleases and a family of eukaryotic repair proteins related to the proliferating cell nuclear antigen. In addition, a number of previously undetected occurrences of known conserved domains were detected; for example, a modified helix-hairpin-helix nucleic acid-binding domain in archaeal and eukaryotic RecA homologs. There is a limited repertoire of conserved domains, primarily ATPases and nucleases, nucleic acid-binding domains and adaptor (protein-protein interaction) domains that comprise the repair machinery in all cells, but very few of the repair proteins are represented by orthologs with conserved domain architecture across the three superkingdoms of life. Both the external environment of an organism and the internal environment of the cell, such as the chromatin superstructure in eukaryotes, seem to have a profound effect on the layout of the repair systems. Another factor that apparently has made a major contribution to the composition of the repair machinery is horizontal gene transfer, particularly the invasion of eukaryotic genomes by organellar genes, but also a number of likely transfer events between bacteria and archaea. Several additional general trends in the evolution of repair proteins were noticed; in particular, multiple, independent fusions of helicase and nuclease domains, and independent inactivation of enzymatic domains that apparently retain adaptor or regulatory functions.     

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding

To examine a role for the medullary nucleus paragigantocellularis (PGi) in mediation of the symptomatology of opioid withdrawal, bilateral electrical stimulation of the PGi was performed in conscious, unrestrained, opioid naive (nondependent) rats. A characteristic series of behaviors was elicited during each 30-min session of PGi stimulation. The profile of these behaviors resembled qualitatively, but was not quantitatively identical with those seen during precipitated withdrawal from opioid dependence. This behavioral syndrome has been termed, opioid withdrawal-like behavior. The opioid withdrawal-like behaviors were voltage-, but not frequency-, dependent. Tolerance to repeated stimulation of the PGi did not develop following a series of 30-min runs of stimulation over 3.5 h. Intracerebroventricular (i.c.v.) injections of the nonselective opioid antagonist, naloxone, significantly decreased (by 40-50%) the intensity of stimulation-induced behavioral responses, as did injections of either the mu-selective (beta-funaltrexamine, beta-FNA) or the delta-selective (naltrindole, NTI) opioid antagonists. In contrast, similar i.c.v. injections of the kappa-selective antagonist, nor-binaltorphimine (nor-BNI), did not block behavioral responses to PGi stimulation. The results indicate that activation of the PGi by electrical stimulation can elicit behaviors similar to those observed during opioid withdrawal. Endogenous opioids, acting through mu- and delta-, but not kappa-opioid receptors, participate in mediating opioid withdrawal-like behaviors induced by PGi stimulation.     

Analysis of the low molecular weight nuclear polypeptide by isoelectrofocusing of nuclear proteins first separated by gel electrophoresis in SDS

Some of the nuclear proteins, especially those tightly bound with nucleic acids, show a tendency to aggregate and some of them are also hydrophobic. Thus, SDS-containing buffers are useful for their analysis. The method of isoelectrofocusing of proteins separated by polyacrylamide gel electrophoresis in the presence of SDS was used for the analysis of the nuclear proteins. First, SDS-polyacrylamide gel electrophoresis of total chromatin proteins was conducted. The sample of the examined nuclear proteins after SDS-gel electrophoresis was isolated as a gel slice (thus corresponding to the particular molecular weight range) and used for isoelectrofocusing. This approach has been used for the analysis of about 19 kD nuclear polypeptide, tightly bound with DNA. The isoelectric point of this polypeptide has been estimated as 5.3.     

Duration of protective immunity in experimental canine babesiosis after homologous and heterologous challenge

Three Beagle dogs were monitored clinically and serologically for 55 weeks following an experimental primo-infection and two challenge infections with a heterologous strain of Babesia canis. There was no cross-protection when dogs were challenged with the heterologous strain after 7 months, but there was complete protection when challenged a second time with this heterologous strain 5 months later. Although the serological profile using indirect immunofluorescence showed the same trend whether homologous or heterologus antigen was used, antibody titres generally reached higher values for homologous antigen. Seropositivity in itself was no guarantee for protective immunity against heterologous challenge.     

Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsPro and tRNAsLys by homologous and heterologous enzymes

Gamekeeper's thumb is due to complete rupture of the ulnar collateral ligament; it should be clinically differentiated from incomplete rupture. Methods of diagnosis and treatment of gamekeeper's thumb are described. The results in 10 cases of gamekeeper's thumb treated by surgery, with more than a one-year follow-up are evaluated.     

Protection of mice against challenge with homologous and heterologous serovars of Actinobacillus pleuropneumoniae after live vaccination

BACKGROUND: Massive neurofilament conglomeration in motor neurons has been described to occur in the early stages of both familial and sporadic amyotrophic lateral sclerosis (ALS). Previously, neurofilament conglomerates were immunolabeled for both superoxide dismutase (SOD1) and nitrotyrosine, suggesting the potential for oxidative nitration damage to neurofilament protein by peroxynitrite. Long-lived neurofilaments may also undergo modification by advanced glycation endproducts (AGEs) with concomitant generation of free radicals, including superoxide. This radical species may then react with nitric oxide to form the potent oxidant, peroxynitrite, which in turn can nitrate neurofilament protein. Such a glycated and nitrated neurofilament protein may become resistant to proteolytic systems, forming high-molecular-weight protein complexes and cytotoxic, neuronal inclusions. MATERIALS AND METHODS: Paraffin sections containing both neurofilament conglomerates and neuronal inclusions were obtained from patients with sporadic (n = 5) and familial (n = 2) ALS and were probed with specific antibodies directed against the AGEs cypentodine/piperidine-enolone, arginine-lysine imidazole, pentosidine, and pyrraline. RESULTS: Neurofilament conglomerates, but not neuronal inclusions, were intensely immunolabeled with each of the anti-AGE antibodies tested. The immunoreactivity was selective for neurofilament conglomerates and suggested that AGEs may form inter- or intramolecular cross-links in neurofilament proteins. CONCLUSIONS: These data support the hypothesis that AGE formation affects neurofilament proteins in vivo and is associated with the concomitant induction of SODI and protein nitration in neurofilament conglomerates. AGE formation in neurofilament protein may not only cause covalent cross-linking but also generate superoxide and block nitric oxide-mediated responses, thereby perpetuating neuronal toxicity in patients with ALS.     

Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross-linking of long and short single-stranded DNA molecules

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.     

Disruption of PMR1, encoding a Ca2+-ATPase homolog in Yarrowia lipolytica, affects secretion and processing of homologous and heterologous proteins

The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca2+-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the YlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca2+-ATPase genes on the secretion of rice alpha-amylase was also observed in the Y. lipolytica and S. cerevisiae PMR1-disrupted mutants. Unlike rice alpha-amylase, the secretion of Trichoderma reesei endoglucanase I (EGI) was not influenced by the YlPMR1 disruption. However, the secreted EGI from the YlPMR1-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant. Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.     

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.     

Neutralization of myonecrosis, hemorrhage, and edema induced by Bothrops asper snake venom by homologous and heterologous pre-existing antibodies in mice

The ability of pre-existing antibodies to neutralize locally-acting toxins of Bothrops asper snake venom was investigated. Hemorrhage, myonecrosis, and edema were markedly reduced in actively immunized mice, although none of these effects was completely abolished. In mice passively immunized with equine antivenom, hemorrhage was prevented completely, while myonecrosis and edema were partially reduced. Pre-existing antibodies did not modify the early stage (< 3 hr) of venom-induced edema, but significantly accelerated the normalization of this effect within 24 hr. Passive administration of antivenom either 5 or 120 min before venom injection gave similar results, suggesting that the presence of antibodies in the intravascular compartment may fully neutralize locally acting toxins, in this experimental animal model. Overall, the homologous or heterologous origin of antibodies was not a significant factor influencing their in vivo neutralizing efficiency against local venom effects. Antibody titrations by enzyme-immunoassay using purified toxins and whole venom indicated that serum from actively-immunized mice had a higher proportion of anti-myotoxin antibodies than equine antivenom.     

Control of expression, glycosylation, and secretion of HIV-1 gp120 by homologous and heterologous signal sequences

The HIV-1 gp120 signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids. The HIV-1 gp120 gene with its natural signal sequence expressed in any prokaryotic or eukaryotic expression systems showed extremely low levels of synthesis and secretion. However, deletion of the HIV-1 gp120 signal sequence results in production of large quantities of a nonglycosylated form of gp120 in Spodoptera frugiperda cells. Substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids and led us to investigate the role of the positively charged amino acids in the signal sequence of HIV-1 gp120. Four charge-altered forms of the gp120 signal sequence of HIV-1 were constructed by site-directed mutagenesis in which the positively charged amino acids were sequentially substituted with neutral amino acids. The results of these experiments showed that the expression and secretion of gp120 was progressively increased by eliminating the positively charged amino acids in a stepwise fashion. However, the substitution of all positively charged amino acids resulted in the accumulation of nonglycosylated gp120 within the cells with decreased amounts of the glycosylated form of gp120. These results demonstrate that the positively charged amino acids in the signal sequence of HIV-1 gp120 are key factors in determining its poor expression and secretion. Analyses of intracellular transport and folding of gp120 further indicate that the presence of a highly charged, uncleaved signal sequence is an important factor limiting transport of gp120 from the rough ER to the Golgi apparatus.