Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.     

Immunological recognition of the prolactin receptor: identification of a single binding unit of molecular weight approximately 42,000

Different polyclonal and monoclonal antibodies against the rabbit mammary prolactin (PRL) receptor were previously obtained that totally inhibited PRL binding in the rabbit mammary gland. Only polyclonal antibodies were shown to immunoprecipitate preformed PRL--receptor complexes in solubilized mammary membranes suggesting that they also recognized domains outside of the PRL binding site of the receptor. When partially purified PRL receptor preparations from both rabbit and pig mammary tissues were iodinated, immunoprecipitated and subsequently analyzed by SDS--PAGE, a single component of molecular weight approximately 42,000 was specifically recognized by all the anti-PRL receptor antibodies. This unit was the only component immunoprecipitated by the monoclonal antibody M 110. Its identification was not impaired by using reducing or non-reducing conditions. Moreover, a further purification of the [125I]-labeled receptor preparations from both species by a second PRL affinity chromatography selected a single binding unit of the same molecular weight. In contrast, polyclonal antibodies immunoprecipitated additional components apart from the 42,000 unit, especially one unit of molecular weight 70,000-80,000 in both species. We conclude that rabbit and pig mammary PRL receptors exhibit striking immunological similarities. Both contain a single binding unit of molecular weight approximately 42,000 that is not linked to other units via disulfide bridges. This binding unit could be associated with a larger component of MW 70,000-80,000 in the holo receptor.     

Migration of gall stones.

The factors influencing the migration of gall stones are ill understood. Altogether 331 patients undergoing cholecystectomy were studied prospectively. The diameters of the cystic and common bile ducts and of stones in the gall bladder and bile ducts were measured. Increasing pressure was applied to the freshly excised gall bladder in an attempt to evacuate stones through the cystic duct. Stones passed in 33 (60.0%) of patients with choledocholithiasis, 45 (67.2%) of patients with pancreatitis, and 7 (3.2%) of patients without either pancreatitis or choledocholithiasis. Stones migrated in 6 (3.0%) who had a normal cystic duct diameter (less than or equal to 4 mm) and in 46 (32.5%) with a duct over 4 mm diameter. Common bile duct stones were often larger than the diameter of the cystic duct and when reintroduced into the gall bladder would not migrate. The passage of debris (less than or equal to 1 mm) through the cystic duct bore no relation to the presence or absence of choledocholithiasis or a dilated cystic duct. Small stones (1-4 mm diameter) must migrate to initiate and facilitate further migration; some must increase in size in the common bile duct. Increased biliary pressure consequently dilates the duct system retrogradely, allowing larger stones to follow. Patients at risk of stone migration and thereby pancreatitis and jaundice have large ducts that can be detected by ultrasound assessment.     

Restriction fragment length polymorphisms distinguish ectomycorrhizal fungi

Basidiomycetous fungi, two saprophytes and three mycorrhizal, were used to assess the specificity of DNA hybridization for distinguishing genera from one another. Interspecific comparisons were done with several isolates of mycorrhizal fungi,Laccaria bicolor andL. laccata, collected from diverse geographical sites. The DNAs were digested with four restriction nucleases and separated by gel electrophoresis into patterns of DNA fragments called restriction fragment length polymorphisms (RFLPs). The RFLPs were hybridized with a radioactively-labeled DNA probe encoding Basidiomycetous ribosomal RNA genes. The five genera were discernable using both unprobed and probed RFLPs. Hybridization of probe DNA with RFLPs was isolate-specific for all nine Laccaria isolates examined. The reclassification of aL. bicolor isolate is supported, demonstrating that hybridization of RFLPs offers an additional tool for taxonomy of ectomycorrhizal fungi. The method may have field application for distinguishing known isolates if their DNA fingerprints are previously ascertained and are distinct from RFLPs of indigenous organisms.     

Calcium and proton activities in rat cardiac mitochondria. Effect of matrix environment on behaviour of fluorescent probes.

The ionic composition of the mitochondrial matrix, under both physiological and pathophysiological conditions, remains controversial. Although fura-2 and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), fluorescent probes for [Ca2+] and [H+] respectively, have successfully been loaded into mitochondria [Lukács & Kapus (1987) Biochem. J. 248, 609-613; Davis, Altschuld, Jung & Brierley (1987) Biochem. Biophys. Res. Commun. 49, 40-45], the adaptation of fluorescence-ratio spectroscopy to the study of the matrix ion content poses unique problems. In this report, we describe a method for successfully attaching viable rat cardiac mitochondria to glass coverslips, allowing continuous superfusion of isolated organelles during fluorescence microscopy. This technique obviated the need to correct for the accumulation of ion-sensitive and -insensitive fluorescent species of dye both within the matrix and outside of mitochondria in suspension in a cuvette, a particular problem with fura-2. By using this technique for superfusion of immobilized mitochondria, we found the pKa of BCECF for H+ at 25 degrees C shifted from 6.8 in buffer to 7.2 in rat cardiac mitochondria, with a marked hysteresis effect noted for intramitochondrial BCECF calibration curves. At higher pH, photobleaching of BCECF was enhanced. The dissociation constant (Kd) of fura-2 for Ca2+ was found to be 315 nM at 25 degrees C, pH 8.0, but only at [Ca2+] below 1 microM. At matrix [Ca2+] greater than 1 microM, the Kd shifted into the micromolar range, an effect that appeared to be pH-dependent. Importantly, the matrix [Ca2+] was determined to be between 10 and 100 nM at perfusion buffer [Ca2+] below 500 nM, but rose rapidly at the higher extramitochondrial [Ca2+] reported to occur in ischaemic cardiac myocytes. Importantly, mitochondrial transmembrane H+ and Ca2+ gradients both appeared to be maximal at perfusion buffer [H+] and [Ca2+] that approximate those of the cytosol of many resting cells.     

Bimodal effects of luteinizing hormone and role of androgens in modifying superovulatory responses of rats to infusion with purified porcine follicle-stimulating hormone

Prepubertal (28-30 days old) female rats were infused s.c. over a 60-h period with a purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH specific activity 8.4 times that of NIH-FSH-S1 and luteinizing hormone (LH) specific activity less than 0.005 times that of NIH-LH-S1, based on radioreceptor assays. When the FSH infusion rate of this preparation was increased over the range of 0.5-2 units/day (mg NIH-FSH-S1 equivalent), an all-or-none response was observed, with the threshold dose for superovulation being between 1 and 2 units/day. Eleven of twelve rats receiving the 2 units/day dose ovulated a mean +/- SEM of 67 +/- 8 oocytes on the morning of the third day after the beginning of FSH infusion. Addition of human chorionic gonadotrophin (hCG), as a source of LH activity, to a subthreshold (1 U/day) FSH infusion rate resulted in 20% of rats ovulating at an hCG dosage of 50 mIU/day; increasing the hCG infusion to 200 mIU/day concomitant with the subthreshold FSH infusion rate increased ovulation rate to a mean of 69 +/- 8/rat, with 100% of rats ovulating. To determine the effect of varying both FSH infusion rates and LH:FSH ratios, FSH was infused at several rates, with hCG added to give varying hCG:FSH ratios for each FSH infusion rate. Administration of hCG alone was ineffective in causing ovulation except at the highest infusion rates. Adding hCG to FSH to reach a ratio of 0.2 IU hCG/U FSH significantly increased the superovulatory response to an intermediate, 1 U/day FSH dose, but not to the low, 0.5 U/day dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethnobotany of purple coneflower (Echinacea angustifolia,Asteraceae) and OtherEchinacea Species

The purple coneflower, Echinacea angustifolia, was the most widely used medicinal plant of the Plains Indians. It was used for a variety of ailments, including toothache, coughs, colds, sore throats, snakebite, and as a painkiller. H. C. F. Meyer used it as a patent medicine in the 1870s and introduced it to the medical profession. Recent scientific research (mostly German) onEchinacea species has shown that they possess immunostimulatory activity. Increased cultivation ofE. purpurea andE. angustifolia may be needed to meet the increased demand for its roots and to alleviate the effects of overharvesting of wild stands.