Intratibial Moloney sarcoma virus-induced osteosarcoma in the rat: tumor incidence and pathologic evaluation.

Osteosarcomas were induced in approximately 80% of young New Zealand Black rats by the intratibial inoculation of Moloney murine sarcoma virus from day 1 to day 5 after birth. The neoplasms were composed of a spectrum of well-differentiated to poorly differentiated osteoblasts, osteocytes, and osteoclasts. Budding of C-type viral particles was associated with tumor induction. Compared to rats inoculated on day 1 after birth, rats inoculated at 4 days of age developed consistently more osteoproliferative bone tumors that often were associated with hypercalcemia, increased serum alkaline phosphatase, and elevated urinary hydroxyproline.

Adriamycin-induced cardiac damage in the mouse: a small-animal model of cardiotoxicity.

After one dose of adriamycin, a subacute cardiomyopathy was observed in the mouse by both light and electron microscopy. The microscopic alterations were characterized by single-cell necrosis and mitochondrial degeneration. These lesions were similar to those seen in man and shortly preceded fatal toxicity.

Virus-induced animal model of osteosarcoma in the rat: Morphologic and biochemical studies.

Osteosarcomas were produced by the intratibial inoculation of New Zealand black rats with Moloney sarcoma virus (MSV) at 1 day and 4 days of age. Radiographic evidence of osteosarcoma development was first demonstrated at 10 to 15 days postinoculation in both groups. Subsequent radiographic and light and electron microscopic evaluation of tumor-bearing rats demonstrated that osteosarcomas in rats inoculated at Day 4 of age were more osteoproliferative osteosarcomas than those in rats inoculated on Day 1. Rats inoculated at 4 days of age lived longer, had more slowly growing osteosarcomas, and developed a consistent tumor-associated cachexia compared to tumor-bearing rats inoculated at Day 1. Both groups of rats had a 93% metastasis rate involving either sublumbar lymph nodes, lungs, or both. Tumor-bearing rats inoculated at 4 days of age had consistent elevations in both urinary hydroxyproline excretion (HOP/CR) and serum alkaline phosphatase levels, and in serum calcium levels at some time points. The high tumor incidence after a short latent period and the morphologic and biochemical similarities between the MSV-induced murine osteosarcoma and the osteosarcoma in human beings makes this discrete tumor and a valuable animal model for the evaluation of new therapeutic regimens.

Ribosome structure: localization of 3' end of RNA in small subunit by immunoelectronmicroscopy.

The 3' end of the RNA in the 30S ribosomal subunit of Escherichia coli has been modified by oxidation with sodium periodate and conjugation with the (mono) dinitrophenyl derivative of ethylenediamine. Antibodies, induced with dinitrophenyl-bovine serum albumin, interact with the modified ribosomal subunits. Electron micrographs of negatively stained antibody-subunit complexes show individual ribosomal subunits to which a single antibody molecule is bound and subunit dimers cross-linked by an IgG molecule. The modified 3' terminus has been localized to a single site on the upper portion of the platform region of the 30S subunit. This location is consistent with earlier placements of proteins that react with the 3' end of the RNA.

Alterations of lactate dehydrogenase isoenzymes with subacute adriamycin toxicity.

Single doses of Adriamycin (ADR) and isoproterenol in CDF rats produce significant increases in total serum lactate dehydrogenase (LDH) activity, LDH isoenzymes, and LDH-1/LDH-2 ratio. Isoproterenol produced significant increases in LDH-1, LDH-2, and LDH-3 and the LDH-1/LDH-2 ratio; ADR produced significant increases in all isoenzymes, reflecting widespread toxicity, with peak levels of total LDH, LDH isoenzymes, and LDH-1/LDH-2 ratio at 48 hours after injection. While the increased LDH-1/LDH-2 ratio may suggest myocardial leakage with ADR administration, the results are too nonspecific to conclude that the heart is the primary source of serum LDH-1 and LDH-2.

The integration of investigative toxicology in the drug discovery process.

Integrating toxicology early in the drug discovery process adds value by providing the earliest possible identification of a compound's potential for toxicological and pathological effects relevant to intended clinical use. With this approach true 'lead' candidates, with a high probability of clinical success, are identified and advanced while reducing effort and resources expended on compounds without the requisite therapeutic index. Resources are focussed on the speed of getting a discovery 'lead' into early clinical development, defining the mechanisms of observed preclinical toxicity and their relevance to human use, and developing early safety data with in vitro test systems ahead of in vivo systems where possible, thus reducing animal use.

Lysozyme deficiency-an inherited disorder of rabbits.

A genetic disorder of rabbits consisting of a deficiency of the enzyme lysozyme is characterized. The condition appears to be inherited as an autosomal recessive trait. Most of the tissues of lysozyme-deficient rabbits including bone marrow, liver, lung. spleen and bone had levels of lysozyme which were 1% or less of the levels in the corresponding tissues of normal rabbits when measured with the lysoplate method. Levels of lysozyme in the kidney and serum were 6% of controls, but the thymus of the lysozyme-deficient rabbits had normal levels of the enzyme. All leukocytes of the lysozyme-deficient rabbits were negative for lysozyme when examined by a histobacterial technic. No morphologic lesions could be detected in any of the tissues of the lysozyme-deficient rabbits. Although several species of animals have been reported to be lysozyme deficient, this appears to be the first report of lysozyme deficiency occurring as a mutant condition. It is suggested that these mutant rabbits may be useful as a resource for experiments designed to delineate the biologic role of lysozyme.

Partial oculocutaneous albinism in Mystromys albicaudatus: nonhomology with the Chediak-Higashi syndrome.

Partially albinic Mystromys albicaudatus were examined to determine if the condition in these animals was homologous with the Chediak-Higashi syndrome. Tissues and cells from partially albinic and normal Mystromys albicaudatus were studied by light and electron microscopy. No evidence of cytoplasmic granule enlargement, which is characteristic of the Chediak-Higashi syndrome, was detected in the cells of the partially albinic rats when compared to controls. It was concluded that the inherited condition of partial albinism of Mystromys albicaudatus was not homologous with the inherited partially albinic disease known as the Chediak-Higashi syndrome.

Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.

Ribosomes from Escherichia coli strain TPR201 (which lack N6,N6-dimethyladenosine) have been photoaffinity labeled with [3H]puromycin in the presence of chloramphenicol. Puromycin-modified 30S ribosomal subunits appear to be identical to untreated subunits in electron micrographs and are efficiently precipitated by antibodies to the puromycin analog N6,N6-dimethyladenosine. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of 30S subunits which appear to be crosslinked by a single IgG molecule. A predominant site of puromycin photoaffinity labeling has been identified from the apparent point of contact of antibody and ribosomal subunit. The puromycin site is localized to the small upper portion of the particle on the side opposite to the subunit platform. This location is close to that reported for ribosomal protein S14, the major puromycin-labeled protein in the small ribosomal subunit.

Differential localization of two epitopes of Escherichia coli ribosomal protein L2 on the large ribosomal subunit by immune electron microscopy using monoclonal antibodies.

Two monoclonal antibodies (mAb), directed toward different epitopes of Escherichia coli ribosomal protein L2, have been used as probes in immune electron microscopy. mAb 5-186 recognizes an epitope within residues 5-186 of protein L2; it is seen to bind to 50 S subunits at or near the peptidyl transferase center, beside the subunit head on the L1 shoulder. mAb 187-272 recognizes an epitope within residues 187-272. This antibody binds to the face of the 50 S subunit, below the head and slightly toward the side with the stalk; this site is near the translocation domain. Both antibodies can bind simultaneously to single subunits. This indicates that protein L2 is elongated, reaching from the peptidyl transferase center to below the subunit head and approaching the translocational domain. The different locations of the two epitopes are consistent with previous biochemical results with the two antibodies (Nag, B., Tewari, D. S., Etchison, J. R., Sommer, A., and Traut, R. R. (1986) J. Biol. Chem. 261, 13892-13897).

Incorporation of single dinitrophenyl-modified proteins into the 30 S subunit of Escherichia coli ribosomes by total reconstitution.

In this first of two consecutive papers, the main objective of which is to present a new approach to the systematic localization of individual proteins located in the Escherichia coli ribosome by immunoelectron microscopy, we describe the derivatization of several purified 30 S proteins (S12, S21, S14, S19, S18, S17) with 2,4-[3,5-3H]dinitrofluorobenzene at pH 7.4 and 8.4 and the uptake of each dinitrophenylated protein in place of the corresponding unmodified protein into totally reconstituted 30 S subunits. Reverse-phase high performance liquid chromatography is used to purify the proteins, to separate and characterize the products of 2,4-[3,5-3H]dinitrofluorobenzene modification, and to analyze the protein composition of the reconstituted subunits. The extent of dinitrophenyl (DNP) modification is estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214 and 360 nm. DNP derivatives of each of the six proteins are efficiently incorporated into reconstituting 30 S subunits. Incorporation of any of the six DNP-modified proteins does not interfere with binding of Phe-tRNA(Phe) in a poly(U)-dependent manner. This result, as well as data showing that unmodified protein competes with DNP-protein for uptake during reconstitution, provide evidence that each DNP-protein occupies the same position in 30 S subunit as does unmodified protein. In general, for a given protein, unmodified and/or less modified forms are incorporated in preference to more modified forms. Modification of protein S19 at pH 7.4 proceeds with selective formation of one derivative in high yield. Reverse-phase high performance liquid chromatography analysis of acid hydrolysates of a purified sample of this derivative, as well as of peptides derived from it by digestion with Staphylococcus aureus protease, show the N-terminal proline to be the predominant site of DNP-derivatization.

Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol.

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.

Messenger RNA orientation on the ribosome. Placement by electron microscopy of antibody-complementary oligodeoxynucleotide complexes.

Messenger RNA orients on the small ribosomal subunit by base pairing with a complementary sequence in ribosomal RNA. We have positioned this ribosomal RNA segment and thus oriented the mRNA using a new technique--localization of an antibody-recognizable modified complementary oligodeoxynucleotide by electron microscopy. A synthetic oligodeoxynucleotide complementary to the message-positioning ribosomal RNA sequence was modified at either or both ends with different antigenic markers. Electron microscopy of subunit-oligodeoxynucleotide-antibody complexes allowed separate placement of each terminal marker of the oligodeoxynucleotide probe. The 5'-end of the complementary sequence contacts the subunit at the platform tip (rRNA nucleotide 1542). The message then extends along the interior side of the platform to the level of the fork of the cleft separating the platform from the subunit body, and displaced slightly to the convex side of the platform (rRNA nucleotide 1531). Based on our results and data from other laboratories, we propose a model for the positioning of messenger RNA on the 30 S subunit.

Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin.

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.

Localization of two epitopes of protein L7/L12 to both the body and stalk of the large ribosomal subunit. Immune electron microscopy using monoclonal antibodies.

Four molecules of ribosomal protein L7/L12 are found as two dimers on the Escherichia coli 50 S ribosomal subunit. Immune electron microscopy using monoclonal antibodies directed against two epitopes of protein L7/L12 has allowed placement of elements of each dimer. One monoclonal antibody, directed against a determinant in the COOH-terminal domain, allows localization of two identical determinants at or near the end of the subunit stalk. The same antibody was used to place two additional determinants at the periphery of stalkless subunits, in an area from which a stalk might be expected to project. A second antibody, directed against an epitope in the amino-terminal portion of L7/L12, caused loss of stalks from the 50 S subunits. The micrographs showed symmetrical oligometric complexes of the dissociated dimeric protein with bivalent antibody. Antibodies were also seen to bind to the body of stalkless subunits, in a region near the COOH-terminal sites. The results are explained by a model in which one dimer of protein L7/L12 exists in a folded conformation on the subunit body and the second dimer occurs in an extended conformation in the subunit stalk.

Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit.

The large RNA molecule within each ribosomal subunit is folded in a specific and compact form. The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides. The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay. The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state [as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message]. Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state. Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation. Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency. Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state. With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography). We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions.

Chemical and morphologic alterations of rabbit bone induced by adriamycin.

Long term, low dose administration of adriamycin (ADR) to young growing rabbits resulted in significant alterations in bone structure and chemistry. Morphologic changes were most pronounced at epiphyseal and metaphyseal areas of long bones. Epiphyseal cartilage plates were thin and there was derangement of growth zones. Areas of primary and secondary spongiosa were deficient in trabeculae, osteoblasts and osteoclasts. Analysis of femora, humeri and lumbar vertebrae from ADR-treated rabbits revealed increased water and fat content and significant decreases in bone density compared to age-matched controls. Cortices of long bones were roentgenographically thin and contained large irregular spaces evident microscopically. Evaluation of bone ash from ADR treated rabbits revealed significant increases in the percentage of calcium and phosphorus, although Ca/P ratios were not different from controls. Results of in vitro studies indicate that ADR binds readily to nondemineralized, but not demineralized, fresh cortical bone powder. The findings of decreased bone density, histopathologic alterations, and a paucity of osteogenic cells in ADR treated rabbits are interpreted as retardation of bone maturation. It is suggested that ADR affects adversely both the organic and inorganic fractions of bone. Due to its unique characteristics of cytostatic action, binding to metal cations and orange-red fluorescence, ADR is a novel chemical agent that may be useful in experimental bone studies.

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components.

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.