Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.     

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.     

Dual effects of estradiol on normal and tumor pituitary cell multiplication

We have compared the effects of estradiol on the [3H]thymidine (TdR) incorporation into the DNA of 2 rat tissues whose growth is controlled by estradiol in vivo in 2 opposite directions: the normal anterior pituitary and the MtF4 pituitary tumor transplanted under the kidney capsule. Small pieces of pituitary or tumor from Fischer rats, treated or not by estradiol in silastic tubing, were incubated in vitro with [3H]TdR. The [3H]TdR incorporated per microgram DNA was decreased in tumor after 2 to 8 day-estradiol treatment while simultaneously, in the same rats, it was increased in the pituitary. In addition, we studied the effect of estradiol in vitro on the F4C1 cell line obtained from the MtF4 tumor. A dose-dependent decrease of both the [3H]TdR incorporated into DNA and the DNA amount was observed between 10(-6) and 10(-5) M estradiol. These results suggest that the control of the pituitary or MtF4 tumor growth by estradiol in vivo is in part due to an inhibition of cell multiplication. Although estradiol inhibits the growth of a clone of MtF4 tumor cells in vitro we cannot decide whether or not the in vivo effect of estradiol is direct.     

Telomeres and P-element of Drosophila melanogaster contain sequences that replicate autonomously in Saccharomyces cerevisiae

Summary We have isolated restriction fragments from a shotgun collection of Drosophila DNA which function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae and hybridize with telomeric regions of the 2L, 2R, 4, and X chromosomes. In an independently obtained set of D. melanogaster clones five fragments hybridize in situ with telomeres and a number of internal sites. Two of them also contain ARSs. A Drosophila mobile P-element also possesses ARS activity in yeast.     

Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli

Summary The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion GT, leading to amino acid substitution Val¹⁴⁶Phe. This mutational change marks the second domain of the subunit involved in rifampicin binding.     

Uptake of U-14C-glucose by Streptococcus mutans in the presence of saccharin

The uptake of U-14C-glucose by resting cells of Streptococcus mutans OMZ-176 was studied in the presence of the artificial sweetener saccharin as well as sodium chloride. Glucose grown cells were resuspended in phosphate buffer (0.05 M, pH 7.8), and the uptake of U-14C-glucose was observed for 150 min in time intervals of 30 min, in the presence of 0.02 and 2.00 mg/ml of sodium saccharin as well as sodium chloride. As compared to the control and the sodium chloride treatments, sodium saccharin at the highest concentration range more than doubled the accumulation of radioactive labelled carbon within the cells.     

Production of wine from mango (Magnifera indica L.) using Saccharomyces and Schizosaccharomyces species isolated from palm wine

Juice extracted from pulp of the mature ripe tropical fruit, mango ( Magnifera indica L.), containing 15.9% soluble solids, was fermented with four strains of yeast isolated from palm wine. Two of the strains belonged to the genus Schizosaccharomyces (T1 and T2) while the other two were Saccharomyces (B2 and M1). The two strains of Schizosaccharomyces were found to be suitable for the production of sweet, table mango wine with alcohol contents of 8.0 and 9.0% for T1 and T2, respectively. The two strains of Saccharomyces were found suitable for the production of dry mango fruit wines containing 10.0% alcohol.     

Bilateral convergence of pulmonary stretch receptor inputs on I beta-neurons in the cat

Extracellular recordings were made from inspiratory beta- (I beta) neurons in the nucleus of the tractus solitarius in decerebrate cats. A reversible direct current block of myelinated fibers in the ipsilateral vagus nerve was used to evaluate the input from pulmonary stretch receptor afferents (PSR) of the contralateral vagus to individual I beta-neurons. This block served to remove all ipsilateral (which includes all monosynaptic) inputs from PSR to I beta-cells. The effect of withholding inflation on the firing rate and the time of onset of firing of I beta-neurons was determined before, during and after application of the direct current block. There was considerable variation in the strengths of the inputs from the ipsilateral and contralateral nerves; some cells received PSR inputs from only the ipsilateral vagus, but the majority were excited with varying magnitude from both vagi. Several neurons had powerful excitatory inputs from PSR of the contralateral vagus, with the ipsilateral (monosynaptic) contribution being of minor importance.