Properties of ultraviolet light-induced suppressor lymphocytes within a syngeneic tumor system

It has been reported that ultraviolet light (uv) irradiation of normal C3Hf mice generates theta-positive suppressor lymphocytes which, as assayed by adoptive transfer studies, mediates susceptibility to transplanted syngeneic uv-induced tumors. We now report that the cells with suppressor activity can be recovered and enriched for using nylon wool columns. These suppressor cells are found in the nylon nonadherent fraction. Further, while uv irradiated mice are susceptible to supporting the growth of transplanted syngeneic uv tumors long after termination of the uv exposures, adoptively transferred suppression wanes and normal mice exhibit time-dependent recovery.     

Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

Relative susceptibilities of caffeine-sensitive and caffeine-resistant strains of Candida albicans to inactivation and mutation by ultraviolet radiation

Summary Stable variants having increased resistance to growth inhibition by caffeine were obtained from four different absolute, amino acid auxotrophs of Candida albicans. Differences in growth rates and expression of auxotrophy between the resistant (Caf^R) variants and their sensitive (Caf^S) progenitors suggest that caffeine resistance arises through suppressor mutations which affect the fidelity of messenger RNA translation.Both Caf^S and Caf^R strains of C. albicans are more susceptible to inactivation by ultraviolet radiation (uv) when grown at 37°C rather than 25°C following exposure. Post irradiation growth on caffeine potentiates ultraviolet inactivation of all Caf^S strains at both temperatures. Depending on its origin, a Caf^R strain (i) may show greater, lesser or the same intrinsic susceptibility to uv inactivation as its Caf^S parent at 25°C or at 37°C and (ii) may or may not be refractory to post-irradiation contact with caffeine. Caf^R variants independently isolated from a given auxotroph are alike in inactivational responses whereas those obtained from different auxotrophs are dissimilar. This implies that different suppressor mutations are unique in the way they affect expression of potentially lethal uv damage and that only one kind of suppressor is obtained by selection for caffeine resistance in a particular auxotroph.The histidine requiring Caf^R strain WB-2CR is much more resistant to uv inactivation that its Caf^S parent WB-2. Moreover, post-irradiation survival of WB-2CR is unaffected by caffeine. However, WB-2CR and WB-2 are equally susceptible to uv-induced reversion to prototrophy. In both strains, caffeine does not enhance uv-induced reversion at 25°C or 37°C and exhibits an antimutagenic activity at high uv dosage at 37°C.The findings reinforce previously reported indications that, in C. albicans, (i) caffeine-sensitive excision-repair of uv damaged DNA does not occur and (ii) caffeine potentiates uv cellular inactivation by disturbing post-irradiation synthesis of protein essential for recovery from non-genetic damage.     

Influences of cellular susceptibility to amphotericin B and of post-irradiation growth conditions on inactivation of Candida albicans by ultraviolet radiation

Earlier investigations have shown that inactivation of non-budding cells ofC. albicans by ultraviolet radiation is determined in part by (i) their post-irradiation incubation temperature, (ii) opportunity for exposure to long chain fatty acids or sterols and (iii) the nutri tional quality of their post-irraditation growth medium. These cultural conditions do not affect the viability of cells which are budding when irradiated nor the frequency of mutants among the survivors of irradiated, unbudded populations. Several lines of evidence have indicated that the post-irradiation growth conditions influence recoveries from non-genetic forms of uv-induced damage which are potentially lethal only for cells which are not in the process of division when irradiated. Studies reported here show that mutation to resistance to the polyene antibiotic, amphotericin B, changes the composite pattern of survival responses of uv treated unbudded cells to the three post-irradiation conditions. Acquisition of amphotericin B resistance does not alter the uv mutability of such cells nor the vulnerability of budding cells to uv inactivation. Differences in susceptibilities of cells to polyene antibiotics are known to be determined by differences in the composition and structural arrangement of their membranes. The present observations are consonant, therefore, with a previous proposal that post-irradiation temperature, nutrition and contact with lipid affect cellular recoveries from damages to processes which (i) are essential for initiation of cell division and (ii) are related to the organization of the cell membrane.     

Mechanisms of depressed reactivity to dinitrochlorobenzene and ultraviolet-induced tumors during ultraviolet carcinogenesis in BALB/c mice

Chronic treatment of BALB/c mice with ultraviolet (uv) radiation produces two distinct immunologic deficiencies. These deficiencies are apparent long before visible skin tumors are induced by the uv irradiation. One is reflected in a transient inability to develop delayed hypersensitivity to dinitrochlorobenzene and appears to be due to a defect in antigen processing. The other is expressed by the failure of mice to reject syngeneic uv-induced tumors, which are highly antigenic. This lack of tumor rejection can be passively transferred with lymphoid cells and seems to be due to the presence of specific suppressor lymphocytes.     

Analysis of the excision repair of nondimer DNA damage induced by solar ultraviolet radiation in ICR 2A frog cells

The excision repair of solar uv-induced nondimer DNA damage was examined in ICR 2A frog cells through the use of the bromodeoxyuridine (BrdUrd) photolysis assay. A relatively pure population of nondimer DNA photoproducts was induced by irradiation of ICR 2A cells with the Mylar-filtered solar ultraviolet (uv) wavelengths produced by a fluorescent sunlamp followed by exposure to photoreactivating light (PRL) which removes most of the small yield of pyrimidine dimers induced by this treatment. Cultures of cells were also exposed to 254 nm uv, which induces primarily dimers, and 60Co gamma rays. Through use of a modification of the BrdUrd photolysis assay possessing enhanced sensitivity, it was found that the solar uv-induced nondimer DNA damage was repaired by a short patch repair mechanism in which less than approximately 20 nucleotides are inserted into a repaired region. Similar results were also obtained for gamma-irradiated cells. In contrast, excision repair of 254-nm-induced dimers was accomplished by a long-patch process in which an average of about 180 nucleotides are inserted into the repaired sites.     

Effect of caffeine on repair of UV-premutations in phr- mutants of E. coli

Summary The frequency of uv-induced S3-mutations (resistance to 3 Streptomycin/ml) in E. coli B/phr^–/MC₂ was not significantly increased by postincubation in NB with caffeine, though an increase is to be expected if caffeine would inhibit the dark repair of the S3-premutations. The frequency was even decreased by high (0,1%) caffeine concentrations (Fig. 1), which indicates an enhancement of the (caffeine-resistant) repair. This enhancement may be caused indirectly by the observed prolongation by caffeine of the lag phase which gives more time for repair. Also the strong photoprotection and (indirect) photoreversion of the S3-mutations in this (non-photoreactivable) strain were not influenced by caffeine-posttreatment (Fig. 2 and 3). Thus, the dark repair assumed to be stimulated by pre- or post-illumination would be of the caffeine-resistant type. The repair of S3-premutations occurring during post-treatment in saline was inhibited by caffeine (Fig. 4). Also the dark-reactivation of cells killed by uv was inhibited by caffeine in the NB-agarmedium (Fig. 5). It is assumed that the repair of S3-premutations going on in NB-suspended cells is due to a mechanism which is not or only weakly inhibitable by caffeine and which is different from the caffeine-sensitive mechanism working under hunger conditions (perhaps by excising uv-products). Since reactivation of killed cells is caffeine-sensitive but reversion of S3-premutations is caffeine-resistant in NB-cells the uv-induced lethal lesions must be different from the S3-premutations.     

Immunological reconstitution of T-cell-deprived mice. I. Inability of thymosin to restore spleen cell mitogen and tumor allograft response.

Ultraviolet light (uv)-induced tumors appear to possess unique and common determinants, both of which can function as rejection antigens in appropriate in vivo tumor transplantation assays. We postulate that the common tumor antigens can play an important role in anti-tumor immunity. We have previously reported on the phenomenon of ultraviolet light generated suppressor lymphocytes which mediate susceptibility to transplanted uv-induced murine skin tumors. We now report that these uv generated suppressor cells are capable of specifically inhibiting effector responses directed against the common tumor antigens. A hypothesis is presented to explain how the presence of suppressor cells might account for the observed differences between in vivo and in vitro assays of tumor immunity.     

Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos

Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.     

Photoreversible inhibition by ultraviolet light of germ line development in Smittia sp. (Chironomidae, Diptera)

Pole cell formation in embryos of the parthenogenetic midge, Smittia sp., can be delayed or inhibited by irradiation of the posterior egg pole with ultraviolet light (uv). This leaves the schedule of nuclear divisions and chromosome eliminations virtually unaffected. However, uv irradition delays the precocious migration to the posterior pole of one nucleus, which normally becomes included in the first pole cell. This effect is photoreversible, i.e., mitigated by application of blue light after uv. Photoreversibility indicates that a nucleic acid component is involved as an effective target. During normal development of Smittia a number of chromosomes are eliminated during mitosis V, not only from somatic nuclei but also in the germ line. In the latter, this mitosis takes place during the first gonial division in the larva. After uv irradiation, the first pole cell nucleus has undergone supernumerary mitoses before pole cell formation and, as a result, is driven into mitosis V precociously as the pole cell divides. This is frequently associated with chromosome elimination from pole cells, which in turn is correlated with subsequent disappearance of already formed pole cells. Adults derived from embryos without pole cells do not form ovaries. Pole cell formation, pole cell preservation, and ovary development are separately inhibited by uv, and inhibition of each step is photoreversible. The results are discussed in the context of germ cell determination, protection against chromosome elimination, and the role of chromosomes limited to the germ line.     

Ultraviolet Rays Induced Expression of Lectins on the Surface of a Squamous Carcinoma Keratinocyte Cell Line

Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either α-l-rhamnosyl or α-d-glucosyl residues. A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sun-like doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells. The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation. Such a specific modulation of receptor expression upon uv irradiation might be biologically significant, considering the numerous intercellular recognition phenomena in skin biology. α-l-Rhamnose-specific receptor on SCL-1 could not be distinguished from α-d-glucose-specific receptor on the basis of neoglycoproteins binding, uptake, and related inhibitions. Lectin expression was mainly detected on the cell surface, and its overexpression due to uv rays required ade novoprotein synthesis process.     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures. II: Validation of the chemostat model using yeast culture data from literature

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi:, 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268–1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate.     

Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light.

Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: (1) a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; (2) a Sepharose 4B column assay in which protein eluted cincident with DNA; and (3) a CsCl density gradient equilibrium assay which showed both protein and DNA banding at densities other than their respective native densities. Treatment of the irradiated chromatin with DNase provided further evidence that protein-DNA and not protein-protein adducts were being observed in the column assay. There is a fluence-dependent response of protein-DNA adduct formation when the chromatin is irradiated at low ionic strength and is linear for protein over the range studied. When the chromatin is exposed to differing conditions of pH, ionic strength, or divalent metal ion concentration, the quantity of adduct formed upon uv irradiation varies. Susceptibility to adduct formation can be partially explained in terms of the condensation state of the chromatin and other factors such as rearrangement, denaturation, and dissociation of the chromatin components. Besides providing information on the biological significance of these types of uv-induced lesions, this technique may be useful as a probe of chromatin structure.     

Differential effects of growth temperatures on inactivation and mutation of Candida albicans by ultraviolet radiation

Summary Candida albicans exhibits greater susceptibility to inactivation by ultraviolet (uv) radiation if grown before or after irradiation at 37° C rather than 25° C. Caffeine, acriflavin or amino acid analogues potentiate inactivation during postirradiation growth at 37° C but have little effect at 25° C. In contrast to inactivation, mutation induction by uv is unaffected by pre- or postirradiation growth temperatures or by metabolic antagonists. These findings are not explicable in terms of possible effects of growth temperatures on known mechanisms for repair of uv damaged DNA. They are consistent, however, with a previous proposal that a temperature dependent mechanism for dark recovery exists in C. albicans which involves synthesis of protein essential for repair of lethal, non-genetic uv damage.     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with [35S]methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.     

Isolation of Cell Membranes from Saccharomyces cerevisiae for Evaluation of Their Protein Composition

We describe a simple method for the isolation of membrane fractions from Saccharomyces cerevisiae yeasts containing a complex of plasma membranes and cell walls. The method is based on cell disruption on an INBI flow disintegrator. This device spares subcellular structures, which simplifies the isolation of cell membranes. The membrane fraction obtained by this method was suitable for studies of the protein composition of these structures by means of two-dimensional gel electrophoresis.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.     

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.