Search

The transfer of melanin from melanocytes to keratinocytes is upregulated by UV radiation and modulated by autocrine and paracrine factors. Among them, the keratinocyte growth factor (KGF/FGF7) promotes melanosome transfer acting on the recipient keratinocytes through stimulation of the phagocytic process. To search for possible differences in the melanosome uptake of keratinocytes from different skin color, we analyzed the uptake kinetics and distribution pattern of fluorescent latex beads in primary cultures of light and dark skin-derived keratinocytes stimulated with KGF and we compared the direct effect of KGF on the melanosome transfer in co-cultures of human primary melanocytes with light and dark keratinocytes. KGF-promoted melanosome transfer was more significant in light keratinocytes compared to dark, due to an increased expression of KGF receptor in light skin keratinocytes. Colocalization studies performed by confocal microscopy using FITC-dextran as a phagocytic marker and fluorescent beads as well as inhibition of particle uptake by cytochalasin D, revealed that beads internalization induced by KGF occurs via actin-dependent phagocytosis. 3D image reconstruction by fluorescence microscopy and ultrastructural analysis through transmission electron microscopy showed differences in the distribution pattern of the beads in light and dark keratinocytes, consistent with the different melanosome distribution in human skin.

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

Background: Dermatitis herpetiformis (DH), the skin's expression of coeliac disease (CD), is induced by the presence of IgA antibodies and epidermal transglutaminase (TG3) as the main autoantigen, stored in the papillary dermis and on the vessel walls., Aims: To evaluate the presence of IgA and TG3 deposits, considered to be the first step in inducing DH, in healthy skin of coeliac patients without cutaneous manifestations., Methods: Punch biopsies were taken from 11 consecutive coeliac patients, two with DH and nine without cutaneous manifestations, three of whom were adhering to a gluten-free diet (GFD), and evaluated for the presence of deposits in the upper dermis and vessel walls by immunofluorescence and confocal microscopy., Results: In coeliac patients affected by DH we found the presence of IgA and TG3 deposits mainly on the upper dermis, but also in vessel walls. In all coeliac patients without DH and also in those patients who were following a strict GFD, we found widely variable deposits of IgA and TG3 in both the papillary dermis and the vessel walls, although a lower intensity of the fluorescence signal was detected than with coeliac patients affected by DH. Double immunostaining with anti-IgA and anti-TG3 antibodies showed a strong co-localization in the upper dermis in patients with DH and a weaker co-localization in those without DH., Conclusions: We have demonstrated the presence of IgA and TG3 deposits in the healthy skin of coeliac patients, which are considered to play a central role in the pathogenesis of DH.

The aetiopathogenic mechanism underlying clear cell acanthoma (CCA) is not completely clear and it has been postulated that CCA and psoriasis may have a similar pathogenesis because of the common features shared by the two diseases. As it has been recently demonstrated that in psoriatic lesions the paracrine epithelial growth factors [keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)-7 and FGF-10] are involved in promoting and sustaining the keratinocyte hyperproliferation, the aim of this study was to analyse the expression of KGF on CCA lesions and to search for a role of this growth factor in CCA pathogenesis. Immunohistochemical analysis showed an up-modulation of KGF in CCA, although the immunostaining was variable among the different samples collected. Positive immunoreactivity for KGF was detected mainly on dermal areas where the inflammatory infiltrate was more pronounced suggesting a relationship between lymphocyte activation and KGF up-modulation. Real-time quantitative RT-PCR assay performed on mRNA extracted from formalin-fixed paraffin-embedded CCA and normal skin (NS) samples further demonstrated the overexpression of the KGF/FGF-7 gene in all CCA samples compared with NS. Moreover, the evaluation by immunohistochemistry of KGF receptor distribution, the high-affinity tyrosine kinase receptor for KGF, showed a down-modulation of this receptor, as previously reported in the presence of increased levels of KGF. Taken together these results suggest the inflammatory nature of CCA and further support the hypothesis that this disease may represent, like psoriasis, an inflammatory dermatosis in which KGF up-modulation may be responsible for keratinocyte hyperproliferation and may represent a new common feature of both diseases.

In this study, we analyzed the effects of synthetic vitreous fibers (SVFs) on a mesothelial (MeT5A) and a fibroblast cell line (NIH3T3), compared to those exerted by crocidolite asbestos fibers. SVFs (glass wool, rock wools) do not induce significant changes in cell mortality, whereas crocidolite asbestos fibers caused a dose-dependent cytotoxicity. We investigated the correlation between the fiber-induced cytotoxicity and the extent and type of interaction of the fibers with the cell surface, and we observed that SVFs, unlike crocidolite asbestos fibers, establish few and weak interactions. Moreover, after internalization, crocidolite asbestos fibers are often found free in the cytoplasm, whereas glass wool fibers are mainly localized inside cytoplasmic vacuoles. After treatments, we also detected signs of oxidative stress, revealed by an increased reactive oxygen species (ROS) production and by an induction of superoxide dismutase (SOD) activity. The lipoperoxidative damage was characterized by a decrease in polyunsaturated fatty acids (PUFA), an increase in the content of thiobarbituric reactive species (TBARS) and a consumption of vitamin E, as a lipophilic antioxidant. Furthermore, we investigated the effect of fiber exposure on cell proliferation. and it was found that, unlike crocidolite asbestos fibers, SVFs did not induce a significant increase in DNA synthesis.

Saccharomyces cerevisiae strains isolated from pregnant women were identified and characterized by molecular techniques which disclosed a wide chromosomal variability and possible segregations due to sporulation. The morphological analysis showed that very few strains were able to sporulate and generate pseudohyphae, whereas none produced proteases, raising some doubts on the importance of these characters in strain pathogenicity. The analysis of ethanol production revealed that these strains are quite similar to those found in fermentative plants, suggesting a possible derivation from the food industrial environment. Since the absence of relevant amounts of sugar does not confer selective advantage to strong fermentative metabolisms, these findings suggest that a metabolic adaptation to the vaginal environment did not occur yet.

Iprodione is a contact fungicide used to control several pathogens such as Botrytis cinerea, Monilia, and Sclerotinia. This paper reports the ability of an iprodione-resistant strain of Zygosaccharomyces rouxii to degrade iprodione at a concentration of 1 mg L(-1). The yeast Z. rouxii was chosen also for its ability to grow at high osmolarity. Also of note is that in bioremediation situations and in the food industry such resistance could be important. The kinetic and metabolic behaviors of the fungicide in the media are described. The results show a new transformation pathway of iprodione by the yeast leading to the formation of N-(3,5-dichlorophenyl)-2,4-dioxoimidazoline, 3-isopropylhydantoin, and 3,5-dichloroaniline. These compounds were identified by 1H NMR, 13C NMR, and GC-MS analyses. This study provides a basis to employ yeast strains in biodegradation studies in relation to their ability in the disappearance and degradation of xenobiotics into simpler molecules.

Recently, after the identification of ferritin light chain (L-ferritin) gene and protein over-expression in human metastatic melanoma cells, we engineered, starting from the LM metastatic melanoma cell line, clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. The present investigation started from the observation that L-ferritin down-regulated LM cells displayed a less pigmented phenotype, confirmed by a major decrease of total melanin, when compared to control LM cells. This finding was accompanied by a dramatic decrease in tyrosinase activity, which was not paralleled by a concomitant reduction of the amount of tyrosinase specific mRNA. Western blot analysis of tyrosinase in control LM cells displayed a pattern, which corresponds to the progressive glycosylation of the native protein up to the 80 kDa form, considered the functional one. Tyrosinase pattern assayed in L-ferritin down-regulated LM cells showed the remarkable absence of the 80 kDa form and a prevalence of endoglycosidase H (endo H)-sensitive immature (70 kDa) tyrosinase, accumulated in the endoplasmic reticulum (ER), as confirmed by confocal microscopy analysis. These results demonstrate that, in a human metastatic melanoma cell line, the stress condition promoted by L-ferritin down-modulation, can substantially influence proper maturation of tyrosinase., (Copyright 2005 Wiley-Liss, Inc.)

UVB exposure of epidermal cells is known to trigger early and late molecular pathways dependent on receptor tyrosine kinases and reactive oxygen species (ROS). We have recently reported that UVB irradiation induces tyrosine phosphorylation, kinase activation, and internalization of the receptor for the keratinocyte growth factor (KGFR), a paracrine mediator of epithelial growth, differentiation, and survival. Here we analyzed in more detail the UVB-induced endocytic pathway of KGFR and the role of KGFR activation and internalization in regulating UVB-promoted apoptosis and cell cycle arrest. Immunogold electron microscopy and confocal analysis revealed that the UVB-induced endocytosis of KGFR occurs through clathrin-coated pits and that the internalized receptors are sorted to the degradative route and reach the lysosomal compartment with a timing similar to that induced by their ligand KGF. Treatment with the anti-oxidant N-acetylcysteine inhibited KGFR endocytosis, suggesting that the receptor internalization is mediated by the intracellular production of ROS. The ligand-independent KGFR endocytic pathway induced by UVB requires receptor kinase activity and tyrosine phosphorylation and involves transient receptor ubiquitination. Inhibition of KGFR activity reduces both the KGF-mediated proliferative response and the UVB-promoted apoptotic cell death, indicating a different effect of ligand-induced and UVB-induced KGFR triggering. In addition, receptor internalization leads to protection from apoptosis caused by UVB exposure. Finally, we compared directly the behavior of KGFR with that of the epidermal growth factor receptor (EGFR) upon UVB exposure. Surprisingly, biochemical and immunofluorescence analysis showed that EGFR, differently from KGFR, does not undergo UVB-induced tyrosine phosphorylation and internalization. Taken together, our results suggest a differential role of KGFR and EGFR in the response of epidermal cells to UVB possibly because KGFR endocytosis could be crucial for attenuation of survival signals in the suprabasal layers of human skin.

Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

Different phenotypical or molecular techniques can be used to describe and classify microorganisms for taxonomic, phylogenetic or genetic purposes. In yeast taxonomy the official hierarchic classification, based on morphological and physiological characters, is used together with more convenient molecular techniques such as the DNA sequencing. The question on whether these procedures produce coherent classifications is critical both to interpret taxonomic data consistently and to outline species correctly. In this paper, a set of type strains from the major genera of the budding hemiascomycetes yeast is examined with a series of physiological and molecular techniques, widely employed in taxonomy, in order to compare the among-strains correlations obtained with different methods. Results showed that the level of correlation among different techniques is relatively low, showing that different classifications and species organization could be obtained with diverse approaches. This is particularly interesting, considering that the official description of the yeast species is based on characters different from those becoming increasingly popular in the routine identification.

Over a period of 18 months 3 clusters of central venous catheter-related Ralstonia pickettii bacteremia occurred in 3 different units of the same hospital. In order to investigate the relatedness of the clinical isolates we studied 15 strains using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques. The combined analysis of the results obtained by these two methods led us to conclude that all the patients except one were infected by a single clone comprising two variants circulating in the units. Only one case was due to a different strain, probably originating outside the ward. PFGE and RAPD appear to be discriminatory techniques to study the clonal relationship among the isolates and can represent a good tool to perform the epidemiological investigation of an outbreak.

The pathogenic mechanism underlying the hyperproliferation of keratinocytes in psoriasis is still not completely clarified. The production of cytokines released by activated T lymphocytes infiltrating the upper dermis probably has a crucial role. Even dermal fibroblasts can participate in the process through the secretion of growth factors, and some studies have reported an increased expression of the insulin-like growth factor 1. Few studies, however, have focused on the possible involvement of the keratinocyte growth factor (KGF/FGF-7) and the fibroblast growth factor 10 (FGF-10/KGF-2), which are secreted by fibroblasts and stimulate keratinocyte proliferation acting through a receptor specifically expressed by epithelial cells. The aim of this study was to investigate the expression of KGF and FGF-10 on the skin of patients with psoriasis by immunohistochemical analysis and to evaluate the correlation with the lymphocyte infiltrate and the epidermal proliferation. Immunostaining for KGF and FGF-10 showed that both the growth factors are upregulated in the upper dermis of psoriatic skin, and that the expression is correlated with the presence of T-cell infiltrate and with keratinocyte proliferation. Our data suggest that in psoriatic lesions activated lymphocytes can stimulate fibroblasts to produce KGF and FGF-10, which in turn contribute to sustain the hyperproliferative status of the keratinocytes.

Aims: Two different strain characterization techniques, random amplified polymorphic DNA (RAPD) and killer toxin sensitivity (KTS), were compared to assess their typing performance using a set of 30 certified Saccharomyces cerevisiae strains., Methods and Results: A sequential random resampling procedure was employed to subdivide the 32 descriptors in eight sets, in order to compare the differential performances of the two techniques with diverse number of characters. Results showed that RAPD performs better than killer, although the complete differentiation of the strains under study could be obtained only by combining profiles from the two techniques., Conclusions: The combination of different typing techniques was useful when discriminating similar organisms. In such cases, the introduction of a second typing technique can be more advantageous than increasing the number of characters obtained with a single method., Significance and Impact of the Study: The distribution of among-strains pairwise distances and the relative performance of the two techniques has implications for the study of biodiversity, taxonomy and microbial ecology.

ERGIC-53 is a lectin-like transport receptor protein, which recirculates between the ER and the Golgi complex and is required for the intracellular transport of a restricted number of glycoproteins. We show in this article that ERGIC-53 accumulates during the heat shock response. However, at variance with the unfolded protein response, which results in enhanced transcription of ERGIC-53 mRNA, heat shock leads only to enhanced translation of ERGIC-53 mRNA. In addition, the half-life of the protein does not change during heat shock. Therefore, distinct signal pathways of the cell stress response modulate the ERGIC-53 protein level. Heat shock also affects the recycling pathway of ERGIC-53. The protein rapidly redistributes in a more peripheral area of the cell, in a vesicular compartment that has a lighter sedimentation density on sucrose gradient in comparison to the compartment that contains the majority of ERGIC-53 at 37 degrees C. This effect is specific, as no apparent reorganization of the endoplasmic reticulum, intermediate compartment and Golgi complex is morphologically detectable in the cells exposed to heat shock. Moreover, the anterograde transport of two unrelated reporter proteins is not affected. Interestingly, MCFD2, which interacts with ERGIC-53 to form a complex required for the ER-to-Golgi transport of specific proteins, is regulated similarly to ERGIC-53 in response to cell stress. These results support the view that ERGIC-53 alone, or in association with MCFD2, plays important functions during cellular response to stress conditions.

The introduction of man-made vitreous fibers (MMVFs) as a substitute for asbestos in industrial and residential applications raises concerns about their potential health hazards. The aim of our study was to assess cytotoxic and oxidative effects induced on a human mesothelial cell line (MeT-5A) by exposure to glass wool (GW), rock wool (RW) and refractory ceramic fibers (RCF) in comparison with crocidolite asbestos (CR). MeT-5A cells were exposed for 24 h to 2, 5 and 10 microg/cm2 of MMVF and crocidolite fibers and analysed by scanning electron microscope (SEM) for cell surface alterations. Cells were exposed for 2 h to 1, 2, 5 and 10 microg/cm2 of the same fibers and analysed by enzyme Fpg-modified comet test for direct and oxidative DNA damage. SEM revealed loss of microvilli in cells exposed to RCF and numerous blebs in cells exposed to higher doses of RW. Comet test showed significant direct DNA damage in cells exposed to RCF even at the lowest dose. Comet test with Fpg, that permits the detection of oxided DNA bases, showed significant oxidative DNA damage in cells exposed to higher doses of RW. The presence of DNA damage and alterations of cell surface induced by low doses of RCF and the presence of oxidative DNA damage and blebs on cell surface in cells exposed to higher dose of RW suggest possible cytotoxic, oxidative and genotoxic effects for these MMVFs.

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco-2 cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands., (Copyright 2004 Wiley-Liss, Inc.)

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly., (Copyright 2004 Wiley-Liss, Inc.)

Many descriptions of yeast species are based on a limited number of strains collected at one time from a single locale. Often, little is known of phenotypic or genotypic variation and covariation within species. We compare 36 strains of an asexual cactophilic yeast, Candida sonorensis, collected from Opuntia cacti. Comparisons were based on geographical distances between collection locales, responses to physiological assimilation and stress tests, random amplified polymorphic DNA (RAPD) profiles, partial Lage Subunit (LSU) rDNA sequences, relative DNA-DNA hybridization values, and electrokaryotypes. There was significant variation among strains in all types of data collected. Comparisons among the different data types found significant positive associations between RAPD profiles, geographical distances, physiologies, reassociation values, and electrokaryotypes. No significant associations were found between rDNA sequences and any other type of variation measured. Based on RAPD, reassociation, electrokaryotype, and physiological data, the 36 strains could be divided into two groups: those collected in West Texas (nine strains) and all others. RAPD data indicated that 10 (of 12) Australian strains also formed a distinct clade. The taxonomic and phylogenetic status of these clades is discussed. Evidence that new genotypes can sweep through large geographic areas is also discussed.

Variability of HXT2 at the protein and gene level was investigated among Saccharomyces sensu stricto and other yeast species. Results showed that the HXT2 gene is probably present in yeast genera other than Saccharomyces, suggesting that this gene is widely distributed in the yeast world. Chromosomal analyses indicated the stable location of HXT2 on the same chromosome and with the same copy number throughout the entire sensu stricto group. Results of the immunoblotting assay demonstrated that all strains tested (with the exception of S. cerevisiae DBVPG 6042) exhibited a lower level of Hxt2p expression than that shown by laboratory wild-type. Moreover, Hxt2p expression seems to reinforce the taxonomical differences between the two pairs of species (S. cerevisiae and S. paradoxus vs. S. pastorianus and S. bayanus) within the sensu stricto group of the genus of Saccharomyces that also reflect their different ecological niche.

Summary: This paper presents ClassMaker, a macro of MS Excel able to classify continuous data of molecular weight data as binary (1/0) values. The output is represented by a binary matrix, which can be introduced in every software application for phylogenetics or multivariate statistics. This application is designed in order to be a link between image analysis programs and statistical or phylogenetic applications, in order to produce a complete series of free programs able to carry out the complete analysis from the gel to the dendrogram., Availability: ClassMaker is freely available from http://www.agr.unipg.it/cardinali/index.html, where a list of the URLs from which programs of image analysis, statistics and phylogenetics can be freely downloaded.

Keratinocytes play a key role in the pathogenesis of allergic contact dermatitis (ADC) induced by the sensitizing agent nickel. We analyzed here the effects of treatment with nickel and of the pretreatment with zinc on HaCaT cells and primary human keratinocytes. Cell counting, 5-bromo-2'-deoxyuridine incorporation assay and adenosine triphosphate (ATP) bioluminescence detection showed that treatment with NiSO4 induced DNA synthesis and cell proliferation and that pretreatment with ZnSO4 was able to abrogate this proliferative effect. This nickel-induced cell growth appeared enhanced when primary human keratinocytes were co-cultured with fibroblasts. Western blot analysis demonstrated that nickel ions induced up-modulation of the expression of the keratinocyte growth factor receptors (KGFR) without affecting the keratinocyte differentiation, whereas the protein levels of the epidermal growth factor receptor (EGFR) and of its ligand transforming growth factor-alpha (TGF-alpha) appeared unmodified by the treatment. Double immunofluorescence showed that the effect of nickel on DNA synthesis was mainly exerted on KGFR expressing cells, suggesting that KGFR up-modulation could be required for the nickel-induced cell proliferation. These results indicate that KGFR and its ligands may play a role in the mechanism of action of nickel ions and in the protective effect of zinc pretreatment.

The concept of species currently in use in microbial taxonomy is based on sex because derives from that developed in zoology and botany. The absence of sex as the only system to reproduce does not allow to use the hybridization as the test to assess the conspecificity of microbial strains, forcing microbial taxonomists to use relative resemblance among strains as the only tool to define microbial species and to classify new microorganisms. Relative resemblance can be intuitively defined as the situation in which two strains of the same species must be more similar than each of them with a strain of any other species. Unfortunately, there are several algorithms to define the similarity between two strains, but none can be used to estimate the average distance between several members of the same species. This paper describes the problems inherent with the definition of species without hybridization tests and proposes two algorithms for a standard estimation of the overall variability of a species with the data obtained from a sample of strains. These measures will allow the non-subjective determination of the overall similarity among members of the same species using results from both phenotypic and molecular analyses. Both algorithms are based on a bootsrapping procedure implemented by RHO and SMA, two software applications freely available from the internet. These applications allow an easy evaluation of parameters such as the overall variability and levels of confidence associated with the sample of strains and the panel of characters under study.

Ultraviolet irradiation of mammalian cells induces several events that include activation of growth factor receptors and triggering of signal transduction pathway. Most of the UV responses are mediated by the production of reactive oxygen species (ROS) and can be blocked by antioxidants. In this study, we analysed the effect of UVB irradiation at physiologic doses and that of the pro-oxidant agent cumene hydroperoxide (CUH) on the activation of the receptor for keratinocyte growth factor (KGF), a key mediator of epithelial growth and differentiation. Exposure to both UVB (30-150 mJ/cm(2)) and CUH (200 microM of NIH3T3 KGFR (KGF receptors) transfectants caused a rapid tyrosine phosphorylation and activation of KGFR similar to that induced by KGF, and internalization of the activated receptor. The KGFR expression appeared unmodified by the treatments. Ultrastructural observations of both UVB- and CUH-treated cells showed a normal morphology of the plasma membranes and intracellular organelles. The antioxidant N-acetylcysteine inhibited UVB-induced receptor phosphorylation. The generation of an intracellular oxidative stress was detected as a decrease of catalase activity and of vitamin E, and reduced glutathione levels, whereas superoxide dismutase activity was not significantly modified. A peroxidation of polyunsaturated fatty acids of cell membranes was observed after both treatments, associated with the intracellular oxidative stress. Similar biochemical events were observed on NIH3T3 untransfected control cells, suggesting that KGFR activation follows intracellular generation of ROS and is not associated with a scavenging effect. Taken together our results demonstrate that exposure to UVB and to oxidant stimuli induces a rapid intracellular production of ROS, which in turn are capable of triggering KGFR activation and internalization, similar to those induced by KGF.

The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA)(4), and contour-clamped homogeneous electric field patterns from strains of Cryptococcus neoformans var. neoformans isolated in different clinical and geographical situations and a standard Saccharomyces cerevisiae strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place S. cerevisiae out of the C. neoformans cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system.

For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.

The aim of this study was to examine three serial isolates of Cryptococcus neoformans from a patient with AIDS for genotypical and phenotypical characteristics. The isolates were obtained during a first episode of cryptococcosis (simultaneous sampling of blood and cerebrospinal fluid) and after a relapse 3 years later (sampling of cerebrospinal fluid). Pulsed-field gel electrophoresis and random amplification of polymorphic DNA revealed that the blood isolate 1525 (first episode) was different from the two cerebrospinal fluid isolates (1526, first episode; 1782, relapse), yet the cerebrospinal fluid isolates were indistinguishable from each other regardless of the analysis performed. Phenotypical studies showed that isolate 1782 had significantly improved resistance to phagocytosis and killing by monocytes and polymorphonuclear cells and an altered efficacy in evoking cytokine response (augmentation of tumour necrosis factor-alpha, interleukin [IL]-1beta, IL-10, and interferon-gamma, decrease of IL-12). Interestingly, capsule size and antifungal drug resistance remained unchanged, while production of phospholipase and protease was consistently enhanced in the 1782 isolate with respect to the 1525 and 1526 isolates. In conclusion, in serial Cryptococcus neoformans isolates from a patient with AIDS, phenotypical changes but not molecular changes were documented, thus supporting the role of microevolution as a pathogenetic mechanism(s) for persistence/reactivation of fungal organisms.

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

Research has proposed that when faced with a stressor, individuals test novel coping strategies when preexisting strategies fail to reduce perceived threat. However, the utilization of novel coping strategies has received scant empirical attention. This study presents data in the form of spontaneous comments or responses to open-ended questions from three previous quantitative studies of adaptation to age-related vision loss (N = 155, 95, and 343 participants). Self-reported coping strategies were identified using a "Grounded Theory" approach, and then examined for evidence of whether the strategy was recently utilized (novel) or whether it had been used prior to vision loss (preexisting). Results supported the utilization of novel coping strategies in the process of adaptation to a chronic impairment among older adults. Overall, the use of novel coping strategies was found to be associated with better adaptational outcomes, emphasizing the importance of novel coping in response to stressful life circumstances.

A peculiar characteristic of cells infected with human herpesvirus 6 (HHV6) is the absence of viral glycoproteins on the plasma membrane, which may reflect an atypical intracellular transport of the virions and/or the viral glycoproteins, different from that of the other members of the herpesvirus family. To investigate the maturation pathway of HHV-6 in the human T lymphoid cell line HSB-2, we used lectin cytochemistry and immunogold labeling combined with several electron microscopical techniques, such as ultrathin frozen sections, postembedding, and fracture-label. Immunolabeling with anti-gp116 and anti-gp82-gp105 monoclonal antibodies revealed that the viral glycoproteins are undetectable on nuclear membranes and that at the inner nuclear membrane nucleocapsids acquire a primary envelope lacking viral glycoproteins. After de-envelopment, cytoplasmic nucleocapsids acquire a thick tegument and a secondary envelope with viral glycoproteins at the level of neo-formed annulate lamellae or at the cis-side of the Golgi complex. Cytochemical labeling using helix pomatia lectin revealed that the newly acquired secondary viral envelopes contain intermediate forms of glycocomponents, suggesting a sequential glycosylation of the virions during their transit through the Golgi area before their final release into the extracellular space. Immunogold labeling also showed that the viral glycoproteins, which are not involved in the budding process, reach and accumulate in the endosomal/lysosomal compartment. Pulse-chase analysis indicated degradation of the gp116, consistent with its endosomal localization and with the absence of viral glycoproteins on the cell surface of the infected cells., (Copyright 1999 Academic Press.)

Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p--a function that can also served by Gal3p--and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gall-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p.

Ultrastructural analysis of HSB-2 T-lymphoid cells and human cord blood mononuclear cells infected with human herpesvirus 6 revealed the presence, in the cell cytoplasm, of annulate lamellae (AL), which were absent in uninfected cells. Time course analysis of the appearance of AL following viral infection showed that no AL were visible within the first 72 h postinfection and that their formation correlated with the expression of the late viral glycoprotein gp116. The requirement of active viral replication for AL neoformation was further confirmed by experiments using inactivated virus or performed in presence of the viral DNA polymerase inhibitor phosphonoacetic acid. Both conventional electron microscopic examination and immunogold fracture labeling with anti-endoplasmic reticulum antibodies indicated a close relationship of AL with the endoplasmic reticulum and nuclear membranes. However, when the freeze-fractured cells were immunogold labeled with an anti-gp116 monoclonal antibody, AL membranes were densely labeled, whereas nuclear membranes and endoplasmic reticulum cisternae appeared virtually unlabeled, showing that viral envelope glycoproteins selectively accumulate in AL. In addition, gold labeling with Helix pomatia lectin and wheat germ agglutinin indicated that AL cisternae, similar to cis-Golgi membranes, contain intermediate, but not terminal, forms of glycoconjugates. Taken together, these results suggest that in this cell-virus system, AL function as a viral glycoprotein storage compartment and as a putative site of O-glycosylation.

The prevalence of some sexually transmitted viruses, possibly involved in cervical carcinogenesis, was studied in the cervix of women with normal cytology. The presence of human papillomaviruses (HPV) type 16 and 18, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) genomes in cervical cells taken from 143 healthy Italian women was investigated using the polymerase chain reaction (PCR). The study population was divided into four groups with respect to age as follows: group I, 17 to 25 years, n = 48 women; group II, 26 to 35 years, n = 30; group III, 36 to 50 years, n = 32; and group IV, 51 to 70 years, n = 33. In the first age group prevalence rates of HPV 16, CMV and EBV infection of 23%, 21% and 19% were found respectively. The infection rates of HPV 16 and CMV were shown to decrease with age, with prevalences of HPV 16 at 10% in the second group, 6% in the third and 3% in the fourth and of CMV at 13% in the second and third and 6% in the fourth groups. The prevalence of EBV infection did not decrease with increasing age (19% in the first and third groups, 20% in the second and 18% in the fourth). The occurrence of HPV 18 genome was very low (0-3%) and independent of age. In the first age group a higher percentage of double infections (16.6%) was found than in the three other age groups (6% in the second and third and 3% in the fourth). The finding of multiple infections in younger women requires further study in order to clarify the implications of such viral infections in healthy women and their contribution to the development of genital tract malignancies.

The extreme variability of the killer phenomenon in nature, expressed differently in different strains of the same yeast species, embodies an exceptional potential for the discrimination of yeasts at the strain level. Killer-sensitive relationships between a killer reference panel of 24 yeasts belonging to 13 species of six genera, and different industrial wine-starters of Saccharomyces cerevisiae can be used profitably for a rapid and simple fingerprinting procedure.

A modified procedure is proposed for chromosomal DNA extraction based on a cell-wall lytic enzyme never applied before in pulsed field gel electrophoresis. Protoplasting efficiency is retained under very challenging conditions for enzyme activity, such as those required for non-Saccharomyces yeasts often characterized by cell walls highly resistant to lysis.

The electrophoretic karyotypes of strains of the ten species of the yeast genus Saccharomyces (sensu Vaughan-Martini & Martini 1992) were determined by the CHEF (contour-clamped homogeneous electric field) system of pulsed field gel electrophoresis. The number of bands was found to vary from 6 to 17 and the calculated molecular weights of haploid genomes ranged from 7.9 to 14.6 Mbp. The type strains of S. exiguus and the four species of the Saccharomyces sensu stricto complex (S. bayanus, S. cerevisiae, S. paradoxus and S. pastorianus) have genomes comprised of chromosomes of all three size classes: light (< 500 kb), medium (500-1000 kb) and heavy (> 1,000 kb). Saccharomyces kluyveri DNA has only heavy bands, while the remaining species exhibit medium and heavy chromosomes. When more than one strain of each species was examined, it was seen that while the species S. bayanus, S. castellii, S. cerevisiae, S. kluyveri, S. paradoxus and S. pastorianus showed uniform karyotypes, S. dairensis, S. exiguus, S. servazzii and S. unisporus comprise heterogeneous taxa.

The bacterial flora of the skin from different anatomical sites on cancer patients and a control group of medical personnel was examined. This was done to ascertain if antineoplastic therapy was able to change the pattern of microbial flora of patients and to provide a control for possible infectious complications. The results show that in the control group bacterial flora was normal and the antineoplastic treatment did not succeed in changing the bacterial pattern in the skin of patients deeply. Gram negative bacteria were isolated more frequently from the skin of leukemia patients than from either patients with malignant melanoma or other neoplastic diseases.