Glutamine is not an essential amino acid for Sf-9 insect cells

Summary Spodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of -galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 M) and higher concentrations (1.5 M and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 g/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

A rapid urease test for presumptive identification of Cryptococcus neoformans

A rapid method to evidence urease activity is described. Urea hydrolysis and consequent production ammonia are detected by a chemical reaction producing a blue phenol compound (indophenol blue). Three hundred and three yeast were tested. Out of 107 urease-positive organisms detected by Christensen's Urea Agar Test (CUAT) 102 were positive by our method. No false negatives were observed by this method when testing 87 Cryptococcus strains. Ths practical screening test for presumptive identification of Cryptococcus neoformans is simple, unaffected by pH changes and requires 15 minutes to be performed.     

Candida albicans stress mannoproteins expression in superficial and systemic candidiasis

The presence of heat shock mannoproteins (HSMPs) reactive with sIgA was demonstrated in several C. albicans strains. The subculture of the C. albicans isolated from mucosal surfaces on Sabouraud's dextrose agar at 25 °C switched off the HSMP expression. A re-expression of the HSMPs was obtained in the same medium by shifting the temperature of incubation to 37 °C. However, expression of HSMPs in two strains isolated from deep infections was maintained during several subcultures on Sabouraud's dextrose agar at 25 °C. A glycoprotein of 200 kDa seemed to be the main HSMP reacting with vaginal sIgA. The data presented in this study suggest that factors other than temperature can influence the expression of C. albicans HSMPs and therefore these antigens should be referred as stress mannoproteins.Abbreviations HSMPs heat shock mannoproteins - MAb monoclonal antibody - sIgA secretory IgA     

Overall Informativity,OI, in DNA Polymorphisms Revealed by inter-AluPCR: Detection of Genomic Rearrangements

We studied two systems of multilocus markers revealed by PCR using primers directing amplification betweenAlurepeats in a tail-to-tail orientation. Genomic polymorphisms were detected as the presence or absence of the electrophoretic bands representing DNA fragments of a given length. A total of 104 such fragments segregating as Mendelian markers in a panel of eight CEPH families were analyzed by two-point linkage analysis. Fifty-one of these fragments were localized with respect to CEPH markers; they represented 33 loci, 7 of which were multiallelic. Locus-specific oligonucleotides were developed and used as hybridization probes to identify the mapped loci within a complex pattern of inter-AluPCR products. A great proportion of inter-AluPCR polymorphisms represented length variants within amplified DNA segments, while others were presumably due to mutations within the priming sites. To describe the expected number of informative loci per typing experiment we introduced a parameter called overall informativity (OI), which provides a single measure of the multiplex ratio and the informativity of markers contributing to a multilocus system (OIof a single locus is equivalent to its heterozygosity and cannot exceed 0.5 for a biallelic codominant marker). HighOIvalues (5.8 and 11.5) of the two presented systems of inter-AluPCR markers of random chromosomal distribution render them suitable for mapping genomic rearrangements such as genomic deletions in tumoral tissues. This was illustrated by the detection of loss of heterozygosity in the 9q22–qter region in sporadic colon cancer.     

Polypores new to Japan 1. Species ofPolyporus,with a note onP. hartmanni

Polyporus admirabilis, P. dictyopus, P. guianensis, P. pseudobetulinus, P. tubaeformis andP. udus are reported for Japan for the first time.Polyporus tubaeformis had been confused withP. melanopus, but the Japanese collections were conspecific with Norwegian isolates ofP. tubaeformis. A key to allPolyporus species in Japan is provided.Polyporus hartmanni is reported for the first time outside the Australian continent. As the species has a bipolar heterothallism and produces brown rot, its taxonomic relationship withPolyporus s. str. is discussed.The Japanese Science and Technology Agency (STA) is thanked by the authors for financial support.     

Transfer of heavy metals from sediment to fish,and their biliary excretion

Uptake and biliary excretion of metals were studied in rainbow trout, Oncorhynchus mykiss, exposed through spiked sediment to a mixture of seven heavy metals. Metal concentrations and toxicity of bile and blood plasma were used as indicators of exposure. Among the seven metals (Cd, Cr, Cu, Hg, Ni, Pb, and Zn) only three (Cu, Hg, and Pb) were concentrated in the bile (bile-plasma ratio >1). Bile-plasma ratios in the rainbow trout were similar to those found in rats for Cu and Hg. Daphnia magna bioassays were used to determine toxicity of bile and blood plasma in the same trout. Toxicity of bile and blood plasma increased after treatment with acid. An analysis of variance (ANOVA) showed that toxicity of bile and blood plasma to D. magna in metal-exposed trout was significantly correlated with (1) bile and blood plasma test concentration, (2) acid treatment of bile and blood plasma (hydrolysis of metal-plasma and metal-bile complexes) and (3) sediment concentration of metals during exposure of trout. In order to significantly detect the magnitude of the exposure to a xenobiotic the biomarker must respond in a dose- or time-dependent manner. Therefore, the potential use of bile toxicity as a biomarker of heavy metal exposure in fish is probably limited by the low bioconcentration of many of these toxicants in bile.