2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computer aided analysis for biosensing and screening

Biosensors with animal and microbial cells immobilized close to the tip of a membrane electrode have been developed for chemical and drug testing. Our experimental results show that biosensors can be used for drug screening and to provide useful information about various cell-chemical interactions. A computer aided analysis (CAA) software package is being developed here using the biosensor for various screening purposes. This software package enables us to use a computer to analyze the biosensor dynamic responses. Computer simulation and parameter estimation techniques are used to select the best model and to describe the biochemical and pharmacologic effects of various chemicals and drugs on different cell lines.     

Pharmacologic characterization of cirazoline-activated inositol phospholipid hydrolysis in rat brain cortical slices

Interaction of cirazoline, an imidazoline derivative, with alpha 1-adrenoceptor coupled inositol phospholipid hydrolysis was characterized in rat brain cortical slices. Norepinephrine, a full alpha 1-agonist, and phenylephrine, a partial alpha 1-agonist, on inositol phospholipid hydrolysis were included for comparison. Norepinephrine produced a fourfold stimulation of inositol phospholipid hydrolysis, whereas cirazoline and phenylephrine caused only submaximal responses (40-60%) when compared with norepinephrine. The stimulation of inositol phospholipid hydrolysis by cirazoline was completely blocked by the alpha 1-adrenoceptor antagonist prazosin, but not by selective alpha 2- or beta-adrenoceptor antagonists. Furthermore, the norepinephrine dose-response curve was shifted to the right in the presence of cirazoline, without affecting the maximal response. These results suggest that cirazoline behaves as a partial agonist at brain alpha 1-adrenoceptors linked to inositol phospholipid hydrolysis.     

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.     

Proliferative response of seminal vesicle cells to androgen in mice castrated neonatally and pretreated with estrogen or androgen at adulthood

Seminal vesicle cells of neonatally castrated adult mice show poor response to androgen, compared to those of mice castrated at adulthood; effects of pretreatment with androgen or estrogen at adulthood on androgen-induced proliferation of the seminal vesicle cells were examined in neonatally castrated mice. Male mice castrated at day 0 after birth were pretreated with daily injections of testosterone propionate (TP, 100 micrograms/mouse), 17 beta-estradiol (E2, 5 micrograms/mouse) or vehicle for 20 days starting from day 60; daily TP injections (100 micrograms/mouse) for 30 days were started again from day 110 in all the pretreated mice to examine androgen-induced proliferation by incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles. Both TP and E2 pretreatments significantly increased the seminal vesicle weight found before TP treatment. However, androgen-induced proliferation of the seminal vesicle found in neonatally castrated mice (poor response; long duration with a low peak on day 3) was changed at least in part to that found in mice castrated at adulthood (good response; short duration with a high peak on day 3) only following the TP pretreatment but not at all following the E2 pretreatment. The E2 pretreatment induced poor androgen-induced proliferation with a low peak on day 7.     

Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.     

Monoclonal antibody detection of transformation associated protein (TAP) in ts110 Moloney murine sarcoma virus transformed 6M2 cells that are different from the mos gene product

By the use of a highly specific monoclonal antibody (designated MC), we were able to detect three radiolabeled bands with molecular weights of 60,000, 63,000, and 66,000 daltons in the ts-110 Moloney murine sarcoma virus mutant-transformed rat kidney cells known as 6M2. Expression of transformation properties as well as these three bands in 6M2 cells was found to be temperature sensitive. Therefore, MC detected factors that are apparently associated with the transformation of 6M2 cells. These factors are tentatively referred to as transformation associated proteins. These transformation proteins were found in two other Moloney murine sarcoma virus-transformed rat cell lines. These proteins were found to differ from known gene products of the ts-110 Moloney murine sarcoma virus mutant and do not have kinase activity. The transformation associated proteins may represent rat cellular factors activated during the transformation of rat cells by Moloney murine sarcoma virus.     

Heat shock proteins within the mammalian cell cycle: relationship to thermal sensitivity, thermal tolerance, and cell cycle progression

We have measured endogenous and induced rates of 70-kD, 89-kD, and 110-kD heat shock proteins in highly pure G1-, S-, or G2-M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence-activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two-dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S-phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70-kD polypeptide were measured in S and G2-M as compared to G1 following heat shock. Little increase in either the 89-kD or 110k-kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat-induced cell cycle delay or to the resumption of phase-specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.     

Unique sequence, ski, in Sloan-Kettering avian retroviruses with properties of a new cell-derived oncogene.

The Sloan-Kettering viruses (SKVs) are a group of transforming retroviruses that were isolated from chicken embryo cells which had been infected with the avian leukosis virus transformation-defective Bratislava 77 (tdB77). Each of the SKV isolates was shown to contain multiple genomes of different sizes indicating the presence of several viruses in addition to tdB77. To identify and characterize the putative transforming gene(s) of the SKVs, we used hybridization selection to isolate the fraction of a representative cDNA which was SKV specific. Both solution and blot hybridization studies with viral RNAs showed that the specific probe contained a sequence, ski, that was at least partially held in common by the multiple SKV genomes. This conclusion was confirmed by the observation that a molecularly cloned ski probe also hybridized to each of the multiple SKV genomes. Southern blots of chicken DNA revealed homologs of ski (c-ski) which were not associated with endogenous viral loci. Results showing that c-ski was expressed in polyadenylated cytoplasmic RNA of uninfected chicken cells indicated that it is a functional gene. Other data showed that c-ski was conserved in avian and mammalian evolution, suggesting a functional role for the gene in species other than chickens. Using ski cDNA in solution hybridizations with viral RNAs and in Southern blot hybridization with cloned retroviral oncogenes, we did not detect any relationship between ski and any of 15 previously identified oncogenes.     

Folic acid and chromosome breakage. III. Types and frequencies of spontaneous chromosome aberrations in proliferating lymphocytes

The types and frequencies of spontaneous chromosome aberrations were studied in human lymphocytes cultured for 96 h in minimal essential medium (MEM) or MEM without folic acid (MEM-FA). In both media, the most frequent aberrations were chromatid gaps, isochromatid gaps and chromatid breaks. Chromosome (isochromatid) breaks and dicentrics were seen less frequently. Neither of these less frequent aberrations was seen in 4000 cells from MEM, but both were seen in 4000 cells from MEM-FA.