Feulgen Cytophotometry of Pine Nuclei. II. Effect of Pectinase Used in Cell Sfparation

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that hut denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.     

Establishment of functional human pituitary tumor cell cultures

Summary Five primary human pituitary tumor cell cultures were initiated from adenoma fragments obtained from patients with prolactin-secreting adenomas and acromegaly. Functional cell cultures were maintained and propagated in monolayer or suspension culture for up to 9 months. Optimal cell viability and growth were achieved using Ham’s F10 medium enriched with 20% fetal bovine serum, although cells from a patient with acromegaly also grew in serum-free, defined, hormone-containing medium. Bromocriptine (100 ng/ml) did not alter the growth curve of replicating cells derived from a patient with acromegaly. These cells initially secreted 5.5 μg human growth hormone/10⁶ cells, and hormone production diminished after 6 wk. Prolactin secretion by cells derived from prolactinomas (0.5 to 1.3 μg/10⁶ cells/24 h) was stimulated by thyrotropin-releasing hormone (10 ng/ml) in two of the cultures. Both dopamine (10 ng/ml) and nickel chloride (1 mM) suppressed PRL secretion. These studies demonstrate that responsive human pituitary tumor cell cultures can be initiated and maintained. This research was supported by VA Medical Research Funds and NIH Grant HD 7181.     

Acute steroidogenic stimulation of cultured adrenocortical tumor cells: An electron microscopic analysis

The addition of ACTH to primary cultures of functional mouse adrenocortical tumor cells results in a marked increase in the production and release of steroids by these cells. The steroidogenic response to ACTH is detectable within minutes and is associated with a 'rounding-up' of the cells. The effects of ACTH were analyzed by scanning, conventional transmission and stereoscopic high voltage electron microscopy. Cell morphology was studied in unstimulated cells, in cells stimulated for 10-15 min in which only partial rounding had occurred, and after 2 hr of continuous stimulation when most of the cells had fully rounded. In unstimulated cultures, bundles of microfilaments (stress fibers) were prominent in cell extensions, subjacent to coated regions of the plasma membrane and frequently in proximity to microtubules and clusters of lysosome-like organelles. As the rounding process commenced, stress fibers disappeared from the cell edges. In 2 hr stimulated cells, Golgi complexes were hypertrophied. There were more microprojections from the plasma membrane than in control cells, as well as evidence of increased pinocytotic activity. There was an apparent polymerization of microtubules in elongated processes extending outward from the bases of the rounded cells and a concentration of lysosome-like organelles in these formations. The possible significance of these changes with respect to the differentiated function of adrenal cells is under continuing investigation in our laboratory.     

Synthesis and Biological Activity of Various Selenenyl and Tellurenyl-Substituted Deoxyuridines and Deoxyuridylates

Abstract

5-Methylselenenyl and 5-phenyltellurenyldeoxyuridines were constructed via the transmetallation of the corresponding 5-bromo-3′,5′-bis(dimethyl-tert-butyl)silyl deoxyuridine with n-butyllithium followed by electrophilic trapping of the anion with the appropriate reagent. These substituted deoxyuridylates have been evaluated as potenital inhibitors of Lactobacillus casei thymidylate synthase.     

Differential Declines in DNA of Aging Leaf Tissues

Leaves from tobacco and peanut were sampled for cytophotometricstudy from young to senescent stages. Differences in nucleibetween tissues developed during aging. Tobacco epidermal cellsshowed an average loss of approximately one-third of their DNAand peanut epidermis lost more than one-fourth. Mesophyll nuclearDNA declined more slowly than epidermal. These results implythat leaf aging may begin in the epidermis. (Received March 6, 1982; Accepted August 23, 1982)     

Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5''-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2''F and the 4''ClCH₂ groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.     

Contraceptive steroids from pharmaceutical waste perturbate junctional communication in Sertoli cells

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERα and only express the cytoplasmic β isoform. ERβ localization was not modified by either steroid. The threshold level was surprisingly low, around 10^?16 M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.