Early transmembrane events in tumour cell responses observed by stopped-flow fluorometry

Early transmembrane events of tumour cells (mouse myeloma X5563 and lymphoma RDM4) after binding of a monoclonal antibody against mouse MHC antigen and a mitogenic lectin, Con A, were examined by stopped-flow fluorometry with 3 different fluorescent probes. The results showed that membrane fluidities of the cells increased first after binding of anti H-2Kk monoclonal antibody (11-4.1), then calcium was released from intracellular stores into the cytoplasma, and lastly calcium influx occurred from the external medium into the cytoplasma. While Con A only induced calcium influx from the external medium into the cytoplasma.     

Nucleotide sequence of cloned cDNA for human pancreatic kallikrein

Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.     

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

Summary We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 m-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA contnet of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry of individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA contnet of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Effects of steroid hormones on the level of corticotropin messenger RNA activity in cultured mouse-pituitary-tumor cells.

Studies have been made with the mouse pituitary tumor cell line AtT-20 in culture to determine whether or not the suppression of pituitary corticotropin messenger RNA activity observed upon the administration of glucocorticoids to adrenalectomized rats is due to a direct action of these steroid hormones on the pituitary. The levels of corticotropin messenger RNA activity in AtT-20 cells treated with various steroid hormones were measured with the use of the cell-free protein-synthesizing system derived from wheat germ. The addition of dexamethasone to culture medium reduced the level of corticotropin messenger RNA activity to 30-40% of that in untreated cells. Corticosterone and cortisol exhibited a suppressive effect to a lesser extent. In contrast, nonglucocorticoids such as testosterone and 17beta-estradiol were essentially ineffective. These results indicate that at least part of the glucocorticoid action is exerted directly on the pituitary to suppress corticotropin messenger RNA activity.     

Kinetics of hydrogen-deuterium exchange in ATPase from a thermophilic bacterium PS3.

The kinetics of the hydrogen-deuterium exchange reaction in a stable ATPase (TF₁) from a thermophilic bacterium PS3 was followed by infrared absorption measurements. The rates of the hydrogen-deuterium exchange reactions decreased in following order; free form, TF₁·ADP, TF₁·ATP and TF₁·AMP-P(NH)P. TF₁ does not dissociate into subunits even in the absence of nucleotides, thus differences in exchange likely reflect differences in conformations of subunits. These results indicate that the structure is most restricted when ATP or AMP-P(NH)P is bound to the enzyme.     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

Purification and preparation of antibody to RNA polymerase II stimulatory factors from Ehrlich ascites tumor cells.

An improved method was developed for purification of the protein termed S-II that specifically stimulates RNA polymerase II of Ehrlich ascites tumor cells. The specific activity of the final preparation was 400 000 units/mg of protein, which is about 30-fold higher than that of the previous preparation [Sekimizu, K., et al. (1976) Biochemistry 15, 5064]. The final preparation gave a single band on both sodium dodecyl sulfate and nondenaturing gel electrophoresis, and the protein extracted from the band on nondenaturing gel had stimulatory activity. S-II is a basic protein with a molecular weight of 40 500. The fundamental characteristics of S-II determined with the previous preparation were confirmed with completely purified S-II. A specific antibody to S-II was prepared. This antibody inhibited only the stimulatory activity of S-II and did not affect the activity of RNA polymerase II itself. Thus, S-II is probably not a component of the multimeric proteins of RNA polymerase II.     

Distribution of the messenger RNA coding for the common precursor of corticotropin and beta-lipotropin within the bovine pituitary.

The distribution of the mRNA coding for the common precursor of corticotropin and beta-lipotropin among different parts of the bovine pituitary has been investigated by quantifying the mRNA activity with the use of a cell-free protein-synthesizing system. The results obtained have demonstrated that this mRNA activity is located both in the anterior lobe and in the intermediate lobe, while it is essentially not detectable in the neural lobe nor in the stalk. The structural identity of the translation products of corticotropin/beta-lipotropin mRNA from the anterior and from the intermediate lobe has been indicated by their molecular weight as well as by the electrophoretic patterns of the peptide fragments formed from them upon partial enzymatic proteolysis or upon cyanogen bromide cleavage. The specific activity of corticotropin/beta-lipotropin mRNA in the intermediate lobe is about 20-fold higher than that in the anterior lobe, and the total activity of this mRNA in the former is about 2-fold higher than that in the latter. In the intermediate lobe, the translation product of corticotropin/beta-lipotropin mRNA amounts to almost one-third of the products encoded by total translatable mRNA. These results indicate that corticotropin/beta-lipotropin mRNA represents a major mRNA species in intermediate lobe of the pituitary, thus suggesting that this lobe may perform a highly specialized function in producing a large amount of the common precursor of corticotropin and beta-lipotropin.