Incorporation of lysine into Y base of phenylalanine tRNA in Vero cells.

Vero cells, a line derived from African green monkey kidney, contains a hypermodified base, called Y, adjacent to the 3' end of the anticodon of tRNAPhe. Two types of evidence are presented suggesting that lysine is involved in biosynthesis of Y base in these cells. First, when Vero cells are starved for lysine, a new, early-eluting species of tRNAPhe which lacks the fully modified Y base can be detected by reversed phase chromatography (RPC-5). After addition of lysine to the medium, this new species disappears. Second, when these cells are grown in low-lysine medium and then exposed to [3H]lysine, radioactivity from the lysine comigrates with tRNAPhe. The Y base can be selectively excised from tRNAPhe by incubation at pH 2.9, and extracted into ethyl acetate. Thin-layer chromatography of acid-excised material from these cells reveals that lysine-derived radioactivity comigrates with genuine Y base from calf liver tRNAPhe and the acid-excised tRNA no longer contains radioactivity. These results are consistent with the model that lysine is a structural precursor of Y base in tRNAPhe of Vero cells.     

Multiple isoacceptor forms of several transfer ribonucleic acids in a mutant yeast strain.

By use of reverse phase 5 chromatography, a strain of Saccharomyces cerevisiae (XB 109-5B) has been shown to exhibit multiple isoaccepting forms for several of the transfer ribonucleic acids (tRNAs). This is in contrast with a standard wild-type strain where only one acceptor is found for each tRNA studied. Multiple peaks for tRNATyr, tRNAPhe, tRNASer, and tRNAVal have been detected for strain XB 109-5B. However, the observation of multiple isoacceptors cannot be extended to all tRNAs in this strain since tRNAAsp appears as a single form that is the same as in the wild type. The appearance of multiple peaks was found to depend on the growth conditions of the cells. The tRNA profiles of XB 109-5B that was grown rapidly with vigorous aeration differed the most from profiles of comparably grown wild-type yeast, whereas tRNA from this mutant, grown without shaking or supplementary aeration, appeared the same as the wild type. The minor nucleoside composition of the isoacceptors of tRNAPhe was obtained.     

Evidence for a role of specific isoacceptor species of tRNA in amino acid transport.

Soybean leghemoglobins ā and b?were compared by microscale peptide mapping after heme removal with acid-acetone. Maps generated by trypsin or the combined action of trypsin and thermolysin indicated a large amount of homology between the proteins with the only variations detected being the N-terminal peptides. The N-terminal tryptic peptide of leghemoglobin b? was found to be both blocked and to lack the first amino acid of the corresponding leghemoglobin ā peptide. Nuclear magnetic resonance and gas chromatography/mass spectroscopy studies showed that the N-terminal of leghemoglobin b? was N-acetyl-alanine. It is possible that leghemoglobin b? arises from leghemoglobin ā by a two-stage modification involving cleavage of the N-terminal valyl residue and subsequent acetylation of the exposed alanyl residue.     

Clonal variation in adrenocorticotropin-induced desensitization of adenylate cyclase in Y1 adrenocortical tumor cells. Association with a 68,000-dalton protein

Adrenocorticotropin(ACTH)-induced desensitization of adenylate cyclase was examined in subclones derived from the ACTH-responsive, Y1 mouse adrenocortical tumor cell line. This report describes clonal variation in ACTH-induced desensitization of adenylate cyclase and an associated variation in the level of a 68,000-dalton protein, p68. A subclone of Y1 cells with a low level of p68 (0.8% of total protein) exhibited a faster rate of desensitization and a slower rate of recovery from desensitization when compared with a clone containing a high level of p68 (10% of total protein). In three clones with low levels of p68, ACTH desensitized adenylate cyclase with ED50 values from 0.3 to 0.5 nM. In several clones with high levels of p68, the adenylate cyclase system was more resistant to ACTH-induced desensitization; the ED50 values for ACTH in these clones ranged from 2 to 12 nM. Among 11 ACTH-responsive subclones, the level of p68 correlated significantly (p less than 0.001, r = 0.87) with resistance to the desensitization induced by 1 nM ACTH. These results suggest that p68 may function in the maintenance of an ACTH-responsive adenylate cyclase system, or that the level of p68 and responsiveness to ACTH are coordinately regulated.     

Patterns of codon recognition by isoacceptor aminoacyl-tRNAs from wheat germ.

Isoacceptors of Ala-, Arg-, Glu-, Gln-, Ile-, Leu-, Lys-, Ser-, Thr- and Val-tRNAs from wheat germ have been resolved by reverse phast chromatography. Codon recognition properties have been determined on isolated fractions of each of these aa-tRNAs and codon assignments have been made to a number of isoacceptors. Evolutionary changes which have occurred in patterns of codon recognition by isoacceptor aa-tRNAs in wheat germ and other organisms are discussed.     

Association of a 68,000-dalton protein with adrenocorticotropin-sensitive adenylate cyclase activity in Y1 adrenocortical tumor cells

This report explores the biochemical basis for clonal variation in adrenocorticotropin (ACTH)-sensitive adenylate cyclase activity in the Y1 mouse adrenocortical tumor cell line. We demonstrate that the level of a specific protein, designated p68, is significantly correlated with the ability of adrenocorticotropin to stimulate adenylate cyclase activity among Y1 subclones (p = 0.004; r = 0.65). p68 was characterized by its molecular weight in sodium dodecyl sulfate polyacrylamide gels (Mr = 68,000) and by its isoelectric point as determined by two-dimensional gel electrophoresis (pI = 7.2). On two-dimensional gels, the protein migrated as a major spot with satellite spots 0.1 pH unit on either side. Homogenates and plasma membrane fractions from clones highly responsive to ACTH had large amounts of p68. In homogenates from highly responsive clones p68 represented 10 to 12% of the total protein. Homogenates and plasma membrane fractions from clones insensitive to ACTH were deficient in p68. In homogenates from the insensitive clones Y6 and OS3, p68 represented less than or equal 0.8% of the total protein. A somatic cell hybrid, formed by fusion of these two ACTH-insensitive clones recovered ACTH-sensitive adenylate cyclase activity and concomitantly expressed appreciable levels of p68. It is suggested that p68 may regulate the transfer of information from the occupied ACTH receptor ot the catalytic subunit of adenylate cyclase.     

In situ cytotoxic T cells in a methylcholanthrene-induced tumor.

The in situ localization of cytotoxic T-cells was examined in a methylcholanthrene-induced fibrosarcoma. The MCA 2 tumor was initiated in this laboratory and utilized before extensive in vivo passage. Tumor cell suspensions were separated by unit velocity sedimentation. The T cell-enriched tumor-derived fractions and spleen cells from MCA 2-bearing mice were cytotoxic for in vivo isolated MCA 2 and in vitro cultured MCA 2 tumor cells, but not for SAD2 tumor cells. The cytotoxic activity of effector cells isolated from MCA 2 tumors was abrogated by treatment with anti-theta serum plus complement. The significance of cytotoxic T cells with in a progressing tumor is discussed.     

Mapping of two isoleucine tRNA isoacceptor genes in bacteriophage T5 DNA.

T5 bacteriophage codes for the synthesis of more than 14 different tRNA species, which map in four separate clusters in the C segment of the T5 chromosome. In this study, two tRNAile isoacceptor species have been identified by reverse-phase chromatography and shown to be transcribed from two different tRNA loci along the T5 chromosome. The map positions of the tRNA isoacceptors were aided by the use of several T5 deletion mutants in which the position and size of the deleted DNA segments had been previously determined by heteroduplex mapping. Hybridization analysis suggests the presence of some sequence homology between the two tRNAile species.     

Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells.

The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase's inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Coerulospinal cells containing neuropeptide Y in the cat.

Spinally projecting neuropeptide Y (NPY)-immunoreactive cells were sought in the feline locus coeruleus (LC) nuclear complex after horseradish peroxidase (HRP) injection into the lumbar cord; HRP injection was followed by intracerebroventricular colchicine administration. Our results revealed that a significant number (approximately 20% of all descending cells from the LC complex) of spinally projecting NPY-immunoreactive neurons arise from the LC alpha, the subcoeruleus and the K?lliker-Fuse nuclei. Other nonspinally projecting NPY-containing cells were also evident in the laterodorsal tegmental nucleus and the LCd, in addition to those occurring in the aforementioned LC nuclear complex.     

Y. enterocolitica inhibits antigen degradation in dendritic cells

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.     

Differences in nucleotide and base DNA excision repair observed during mitogenic stimulation of bovine lymphocytes.

The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.     

Heterobivalent dual-target probe for targeting GRP and Y1 receptors on tumor cells

Receptor targeting ligands for imaging and/or therapy of cancer are limited by heterogeneity of receptor expression by tumor cells, both inter-patient and intra-patient. It is often more important for imaging agents to identify local and distant spread of disease than it is to identify a specific receptor presence. Two natural hormone peptide receptors, GRPR and Y1, are specifically interesting because expression of GRPR, Y1 or both is up-regulated in most breast cancers. We describe here the design and development of a new heterobivalent peptide ligand, truncated bombesin (t-BBN)/BVD15-DO3A, for dual-targeting of GRPR and Y1, and validation of its dual binding capability. Such a probe should be useful in imaging cells, tissues and tumors that are GRPR and/or Y1 positive and should target radioisotopes, for example, ⁶⁸Ga and/or ¹⁷⁷Lu, to more tumors cells than single GRPR or Y1 targeted probes. A GRP targeting ligand, J-G-Abz4-QWAVGHLM-NH₂ (J-G-Abz4-t-BBN), and an Y1 targeting ligand, INP-K[ε-J-(α-DO3A-ε-DGa)-K]-YRLRY-NH₂([ε-J-(α-DO3A-ε-DGa)-K]-BVD-15), were synthesized and coupled to produce the heterobivalent ligand, t-BBN/BVD15-DO3A. Competitive displacement binding assays using t-BBN/BVD15-DO3A against ¹²⁵I-Tyr⁴-BBN yielded an IC₅₀ value of 18 ± 0.7 nM for GRPR in T-47D cells, a human breast cancer cell line. A similar assay using t-BBN/BVD15-DO3A against porcine ¹²⁵I-NPY showed IC₅₀ values of 80 ± 11 nM for Y1 receptor in MCF7 cells, another human breast cancer cell line. In conclusion, it is possible to construct a single DO3A chelate containing probe that can target both GRPR and Y1 on human tumor cells.     

Annulate lamellae in human tumor cells.

Annulate lamellae have been found in a primitive neuroectodermal tumor, a metastatic cerebellar tumor, a testicular seminoma, a retinoblastoma and three melanomas. These annulate lamellae are arranged in stacked parallel arrays in the cytoplasm of tumor cells. The number of annulate lamellae observed to comprise a single stack varies from 2--4 in the seminoma tumor to 5--18 in the cerebellar tumor. Although the functional significance of annulate lamallae is still unknown, in many instances they have been found to be continuous with rough-surfaced cisternae of the endoplasmic reticulum and ribosomes have been demonstrated on the surface of annulate lamellae. This may suggest that annulate lamellae participate in protein synthesis.