Neonatally induced tolerance to HGG: duration in B cells and absence of specific suppressor cells.

A specific, long lasting, tolerant state to human gamma-globulin (HCG) was established in neonatal A/J mice. These suckling mice received the tolerogen in the colostrum of their mother who had been injected with DHGG. The tolerant state could not be accounted for by "factors" other than HGG in the colostrum. The duration of this tolerance in the intact animal and in the B cell population was 16 to 18 weeks. Naturally occuring nonspecific suppressor cells were evident but specific suppressor cells could not be demonstrated. These results are discussed in relation to possible mechanisms of the induction of tolerance to self.     

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.     

Ly-m19: The Lyb-2 region of mouse chromosome 4 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with 70'/3 cells, an established pre-B cell line, were fused with cells of the nonsecretor myeloma line NS.1. One established hybridoma cell line (clone K10.6) continuously secreted antibody that recognized a new antigenic specificity tentatively named Ly-m19. This newly found antigen is detectable on both T and B cells. Cytotoxicity assays reveal that 75 percent of the spleen and lymph-node cells, 35 percent of bone-marrow cells, and 15 percent of thymus cells reacted with antibody of clone K10.6. Strains expressing the specificity Ly-m19.1 are characterized by negative reactions and include the strains AKR, CE/J, RF/J, GR/A, SJL, P/J, BDP/J, and LG/J. All other strains so far tested are Ly-m19.2. This strain distribution pattern distinguishes Ly-m19 from any known murine lymphocyte alloantigen, but it parallels the Lyb-2 ^c haplotype. Linkage test of a set of AKXL recombinant inbred strains revealed close linkage of Ly-m19 and Lyb-2 loci on mouse chromosome 4.Abbreviations used in this paper LPS lipopolysaccharide - B6 C57BL/6 - Con-A concanavalin A - MLC mixed-lymphocyte culture The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

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.     

Location ofMls locus on mouse chromosome 1

Linkage between theMls locus and the chromosome 1 markersDip-1 andald was detected using two sets of recombinant inbred strains. Linkage betweenMls andDip-1 was confirmed in the fifth and sixth backcross generations of an incipient congenic strain. The AKXL data indicate that the gene order isDip-1-ald-Mls. The recombination frequency betweenald andMls is estimated to be 0.07 ±0.05, based on the AKXL data. The recombination frequency betweenDip-1 andMls is estimated to be 0.18 ±0.04, based on all the available data.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

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.     

Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7.

Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids. The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus. A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus. The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.     

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.