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.     

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.     

Disturbances in toxicological experiments caused by Microsporum canis

Cutaneous lesions which can lead to false positive results have been observed in several rabbits used for the determination of the cutaneous irritation capacity of a product (ITA, PII). The responsible agent was Microsporum canis. A preventive treatment by an antifungal agent did not modify toxicological experimental results.     

Evidence for a fucose-binding protein in boar spermatozoa

A fucose binding protein was detected in boar spermatozoa by means of a specifically developed modified enzyme-linked-lectin-assay using glycosylated peroxidase derivatives. The distribution of the fucose binding protein was assessed by means of fluorescence microscopy with fluoresceinyl-glycosylated peroxidase. Fucose binding was particularly prominent at the apical region of the sperm head. In order to gain more insight into the precise localization of the carbohydrate binding protein electron microscopical studies were performed using fucosyl peroxidase coupled to colloidal gold. In ultrathin sections as well as in specimens prepared in toto for TEM an intensive binding of fucosylperoxidase-colloidal gold was predominantly found at the apical part of the acrosome appearing as a crescent-like area. In some cases this binding pattern was replaced by a triangle-like intensive labelling at the equatorial segment as revealed clearly by specimens prepared in toto. By SDS-PAGE of the SDS-extractable sperm-proteins, followed by transblotting to nitrocellulose and visualization with the fucosylperoxidase by enzymatic amplification with 4-chloro-1-naphthol mainly one protein with the reduced molecular weight of approximately 53 kdal and some small proteins with apparent molecular weights less than 20 kdal was found to be responsible for the fucose-binding ability of porcine spermatozoa.     

Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.     

Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.     

Recombinant outer-surface protein A (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi: high production levels in Saccharomyces cerevisiae yeast cultures

The recombinant outer-surface protein A with an N-terminally truncated form (des-Cys¹-OspA) from the Lyme disease spirochete Borrelia burgdorferi was expressed in Saccharomyces cerevisiae at high production levels. Since the recombinant vaccine candidate expressed in Escherichia coli exhibits low production yields and the purification of lipoproteins appears to be difficult, we have investigated the secretion of a soluble recombinant OspA in the yeast S. cerevisiae. In this way, a Leu⁺ derivative of S. cerevisiae cI3ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding des-Cys¹-OspA and driven by the MF1 promoter. The fed-batch culture results revealed that an efficient secretion of des-Cys¹-OspA is obtained with a high production level of about 2.1 g 1^–1 at a cell density of 101 g 1^–1 cell dry weight. The accumulation of recombinant protein in the supernatant exceeds 6% of the total yeast proteins when estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Moreover, des-Cys¹-OspA showed lower solubilities at high cell densities and, as a consequence, a fraction of the recombinant protein precipitated. An internal cleavage of the MF1 pro::des-Cys¹-OspA precursor was also detected. However, in this case the cleavage occurred at a frequency such that the large amounts of the secreted des-Cys¹-OspA could be employed for the evaluation of an immunogenic effect on animal immunization. These studies will extend the knowledge of the usefulness of OspA as a vaccine for Lyme borreliosis.     

Increased shikonin production by hairy roots of Lithospermum erythrorhizon in two phase bubble column reactor

Summary Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with n-hexadecane in a shake flask and a bubble column bioreactor. Over 95 % shikonin produced was recovered in the n-hexadecane layer. In flask cultures the maximum concentration of shikonin with n-hexadecane extraction was 3 times higher than that obtained without extraction. In the two phase bubble column reactor, 572.6 mg/L of shikonin and 15.6 g/L of dry cell mass were obtained after 54 days. Shikonin was produced at a constant level of 10.6 mg/L day during this period.