An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.     

Actin stringently accumulated in the specifically positioned,differentiating female gametangia of Allomyces

Summary Female gametangia of the normal bisexual Allomyces species are richer in fluorescently probed (FITC) actin, independent of their apical or subapical positioning during differentiation on the fertile hyphae. The anti-actin, cytochalasin D, can selectively suppress male differentiation in both species.     

Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer

Of the total number of breast cancers approx. 30-50% are hormone-dependent and estradiol is one of the main factors of cancerization. Consequently, the control of this hormone inside the cancer cell is of capital importance because it is well established that the inhibition of estradiol biosynthesis can have a positive effect on the evolution of the disease. The blockage of estradiol can be obtained by the action of anti-aromatases, anti-sulfatases, the control of the 17 beta-hydroxysteroid dehydrogenase activity or by the stimulation of the sulfotransferase which converted the estrogens in their sulfates. In breast cancer tissue estrone sulfate is quantitatively the most important source of estradiol. In the intact cell, estrone sulfatase activity is very intense in the hormone-dependent cell lines (e.g. MCF-7, T-47D) but very small activity is observed in the hormone-independent (e.g. MDA-MB-231, MDA-MB-436) cell lines. However, this activity became very strong after homogenization in the hormone-independent cells, suggesting the presence of repressive factor(s) for this enzyme or its sequestering in an inactive form, in the intact cells of these cell lines. In a series of previous studies it was found that in hormone-dependent cell lines different anti-estrogens: tamoxifen and derivatives, ICI 164,384, very significantly decrease the estradiol concentration originated from estrone sulfate, and recently it was observed that Decapeptyl (D-Trp6-gonadotropin-releasing hormone) in the presence of heparin can also decrease the conversion of estrone sulfate into estradiol. No significant effect was obtained in the presence of heparin or Decapeptyl alone. The estrone sulfatase activity can be inhibited by progesterone, the progestagen R-5020, and testosterone. In another series of recent studies the presence of very strong estrogen sulfotransferase activity has been shown in one breast cancer cell line, the MDA-MB-468. We can conclude that: (1) the control of estradiol concentration can be carried out in the breast cancer tissue itself; (2) estrone sulfate can play an important role in the bioavailability of estradiol in the breast cancer cell; and (3) as is the case for the aromatase, the control of: the estrogen sulfatase, estrogen sulfotransferase, and 17 beta-hydroxysteroid dehydrogenase can be new targets for therapeutic applications in breast cancer.     

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.     

UV-induced interstrand cross-linking of d(GT)n.d(CA)n is facilitated by a structural transition

Photochemical alterations following ultraviolet irradiation of the alternating copolymer d(GT)n.d(CA)n were studied. We found that in solution conditions which produced circular dichroism spectra compatible with B-form or A-form DNA, no interstrand cross-linking or photoproduct formation could be demonstrated. Zimmer et al. (Zimmer, C., Tymen, S., Marck, C., and Guschlbaumer, W. (1982) Nucleic Acids Res. 10, 1081-1091) and Vorlickova et al. (Vorlickova, M., Kypr, J., Sotkrova, S., Sponar, J. (1982) Nucleic Acids Res. 10, 1071-1080) have reported a number of solution conditions which produce a structural transition of this polymer characterized by a negative deviation of the circular dichroism spectrum in the region of 280 nm. The nature of this transition has not yet been elucidated. Following ultraviolet irradiation of d(GT)n.d(CA)n under two conditions which produce this transition (manganese solution or ethanol plus trace salts solution) we found ultraviolet dose-dependent interstrand cross-linking as well as dose-dependent formation of thymine-containing photoproduct. Interstrand cross-linking is demonstrated by two criteria: increase in polymer size as detected by alkaline agarose gel electrophoresis, and generation of intermediate density material in alkaline cesium sulfate isopycnic gradients. The thymine-containing photo-product was demonstrated by thin layer chromatography of acid hydrolysates of the polymer. The photo-product is at least partially photoreversible. These findings suggest that the geometry of the alternative conformation is such that pyrimidines from different strands are closely approximated, allowing for photodimerization.     

Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligonucleotides covalently linked to intercalating agents. Influence of positively charged substituents on binding to complementary sequences

A pentamethylene chain was used to covalently link the 3'-phosphate of oligothymidylates to the 9-amino group of an acridine derivative. Positively charged substituents were further attached to the 3'-phosphate group to form 3'-phosphotriesters. These molecules form specific complexes with poly(rA) which involve the formation of a number of A X T base pairs equal to that of thymines in the oligonucleotide. Absorption changes induced in the acridine absorption bands are similar to those expected upon intercalation of the acridine dye between A X T base pairs. The acridine covalently linked to the 3'-phosphate strongly stabilizes the complexes formed with poly(rA) as compared with the corresponding unsubstituted oligodeoxynucleotide. The presence of a positively charged substituent on the 3'-phosphate together with the acridine dye further enhances the interaction. The effect of salt concentration on complex stability depends on the number of negatively charged phosphate groups of the oligodeoxynucleotide and on the nature of the substituents borne by the 3'-phosphate group. When the oligothymidylate is substituted by an acridine dye, the stability of the poly(rA) complexes increases when salt concentration increases. If an additional positively charged substituent is present on the 3'-phosphate group, stability decreases when salt concentration increases for the shortest oligonucleotide (trimer) and increases with longer oligonucleotides. Thermodynamic parameters have been calculated from the concentration dependence of melting temperatures.     

A stochastic model of mutant growth due to mutation in tumors,based on stem cell considerations

A stochastic model of the evolution of mutant subpopulations from stem cells in a human tumor system is derived. From the model, the growth of mutants (both stem cell mutants and overall mutants) due to mutation of tumor stem cells during growth is explored in detail. This allows one to relate the mutant stem cell and overall tumor mutant cell population sizes. The relation of these average sizes is derived for large tumor size and confirms the result of the model due to MacKillop et al. [4], which is based on three tumor cell subpopulations: stem, transitional, and end cells. Furthermore, the results of the stem cell statistics obtained are the same as those obtained from the filtered Poisson process approach [2].