Expression of alien histocompatibility antigens on SJL/J tumors detected by cell-mediated and serological analyses

Summary Reticulum cell sarcoma (RCS) cells of SJL/J (H-2^s) mice have been shown to express antigens that are cross-reactive with allogeneic cells of the H-2^d and H-2^b haplotypes by cell-mediated cytotoxicity, antibody-mediated cytotoxicity, immunofluorescence, and quantitative absorption assays. These alien antigens have been detected on both spontaneous and in vivo- and in vitro-passaged RCS cells to varying degrees.The in vitro cell lines were able to stimulate a syngeneic cytotoxic T cell response detected in a 4-h ⁵¹Cr release assay. The cytotoxic cells reacted with in vitro RCS tumor targets but not with in vivo or spontaneous RCS tumors. Furthermore, the cytotoxic cells lysed H-2^d and to a lesser extent H-2^b target cells, but not H-2^k, H-2^p, or H-2^r cells. The cross-reactivity was also observed with SJL/J anti-BALB/c cytotoxic cells, which can lyse in vitro RCS targets effectively. The in vivo tumors were not stimulatory in cytotoxic responses and did not serve as targets.H-2^d specificities were also detected in cultured RCS tumor cells by cytotoxic antibody. Both allogeneic SJL/J anti-BALB/c, C57B1/6 anti-BALB/c sera reacted with RCS tumor cells and not normal SJL/J cells. Furthermore, monospecific D^d sera were also cytotoxic against RCS lines. The cytotoxic activity could be absorbed by BALB/c cells and RCS cells but not with normal SJL/J cells. The H-2^d specificities were also detected on the in vivo lines by indirect immunofluorescence. The majority (60%) of spontaneously arising tumors expressed either H-2^d or H-2^b allospecificities in the immunofluorescence assays. Although these antigens may not be inappropriate for the SJL/J strain, their differential expression on tumor cells may be significant in the etiology of the tumor.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Role of silicon in diatom metabolism

Levels of the cyclic nucleotides, cAMP and cGMP, were determined in four species of pennate diatoms; changes in their levels and ratios were monitored in silicon-starved and light-dark synchronized cultures of Cylindrotheca fusiformis. Content of both cAMP and cGMP changed during the cell cycles: when silicate was added to starved cultures, cAMP, cGMP and DNA levels rose rapidly; cAMP and cGMP declined before DNA synthesis was complete and continued to fall during the events leading to cell separation. In unstarved synchronies, net synthesis of DNA continued until cell separation; 1 h before cell separation cAMP levels fell while those of cGMP rose. The results support the proposal that cAMP and cGMP may play a part in the process of cell division in the diatom, possibly involving silicon.     

Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities.

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular "underlapping," and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24-48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12-14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3 H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus therefore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.     

Analysis of the oligosaccharides on androgen-binding proteins: Implications concerning their role in structure/function relationships

Rat androgen-binding protein (rABP), human testosterone-binding globulin (hTeBG) and rabbit (rb) TeBG are heterodimeric proteins. The source of the heterogeneity arises from the differential glycosylation of a common protein core. This glycosylation results in a heavy subunit (more glycosylation) and a light subunit (less glycosylation). Glycosylation is one factor responsible for multiple charged species seen when rABP, hTeBG, and rbTeBG are analyzed by two-dimensional gel electrophoresis. Enzymatic digestion with the endoglycosidase, peptide: N-glycosidase F indicated that all three proteins have asparagine (Asn)-linked oligosaccharides as their major glycan substituent. Treatment with exoglycosidases provided evidence for terminal sialic acid, galactose and mannose and N-acetylglucosamine residues. About 16–22% of the mass of the heavy subunit and about 8–14% of the mass of the light subunit is contributed by carbohydrate.

Serial lectin chromatography indicated that rABP is glycosylated differently from hTeBG and rbTeBG. About 40% of the rABP contains tri and tetraantennary complex oligosaccharides, while only about 20% of the hTeBG and TeBG from pregnant rabbits contains these types of glycans. About 9% of the TeBG from male rabbits bears these types of oligosaccharides. All of the biantennary complex oligosaccharides on rABP are fucosylated on the chitobiose core, but only 8% of those on hTeBG and none of those on rbTeBG are fucosylated in this manner. All three proteins are glycosylated at more than one site. The data indicate that the proteins may have more than one type of oligosaccharide on them. It is likely that differences in glycosylation are responsible for different physiological roles of the proteins.     

Molecular structure of mutations at an autosomal locus in human cells: evidence for interallelic homologous recombination.

A high proportion of spontaneous mutations at the heterozygous thymidine kinase (TK) locus in a human B-lymphoblast cell line involved loss of the entire active allele. Loss of heterozygosity often extended to other loci on chromosome 17q. The authors have developed a system for analysing the role of homologous recombination and gene conversion in such events. A heteroallelic (TK-/-) cell line containing single + 1 frameshifts in exons 4 and 7 was generated by repeated exposures to ICR-191. Revertant mutations to TK+/- were selected and analysed for the presence or absence or each frameshift as well as changes in linked polymorphic markers on 17q. The molecular changes associated with reversion to TK+ can thus be analysed. Preliminary results indicate that homologous recombination can be detected with this system, though it occurs at low frequency (less than 10(-7]. The authors believe this represents the first quantitative assay for measuring recombination between alleles of a specific intact gene in human cells. It should prove useful in evaluating the potency of various classes of mutagens in inducing recombinational and gene conversion events.     

A genetic locus responsible for salmonella susceptibility in BSVS mice is not responsible for the limited T-dependent immune responsiveness of BSVS mice

BSVS mice are known to be highly susceptible to salmonella infection. We have shown that the bulk of the difference in susceptibility between BSVS and salmonella-resistant A/J mice is the result of a genetic difference at a single locus not closely linked to H-2, Igh-C, or Hbb, and not X-linked. We have backcrossed the A/J allele at this locus into BSVS mice for 8 successive generations and have demonstrated that the salmonella resistance afforded by this allele is not the result of a restoration of the generalized poor T-dependent responsiveness of BSVS mice. The salmonella resistance locus we have examined with these 2 strains is probably the same as the Ity locus described by others.     

Can tumorigenesis result from in vivo “transfection” of a normal cell with DNA escaped from disintegrating nontransformed cells?

In this paper I present a hypothetical step on the route to turmorigenesis which may be common to many of the tumorogenic events taking place in vivo. According to this hypothesis portions of DNA from normal nontransformed dying cells may escape degradation, “transfect” other cells and under appropriate conditions also induce transformation. When various high risk factors for carcinogenesis are examined this hypothesis may easily fit into each of them. In addition, recent reports of experimental “transfection” indirectly support this hypothesis. It could now be argued that aging, ionizing radiation, immunosuppressive drugs, genetic errors, defects in DNA repair, immunodeficiency states, chronic infections and chronic inflammatory diseases may all increase the availability of free DNA or the readiness of cells to accommodate “transfection” DNA, or both. Thus the risk of cancer associated with these situations may be explained by increased chance for “transfection” with DNA.It is suggested that the minimal requirements for cell transformation consist of a certain sequence of DNA which does not have to be integrated into the nucleus at once, it may instead accumulate piece by piece so that the first “transfection” of a cell may occur long before transformation takes place. If the “transfecting” DNA sequence does not fulfil the minimal requirements for maintaining a state of repetitive uncontrolled mitosis, then this cell may wait silently or remain a benign tumor until “boosted” with an ultimate complementary DNA sequence to develop into a fully fledged cancer.