Interleukin-6 promotes human epidermal keratinocyte proliferation and keratin cytoskeleton reorganization in culture

We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5 ng/ml or higher concentrations promoted keratinocyte proliferation, with an ED₅₀ of about 15 ng/ml and a maximum effect at 50 ng/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) and supported keratinocyte growth for up to eight cumulative cell generations. IL-6-treated keratinocytes formed highly stratified colonies with a narrower proliferative/migratory rim than those keratinocytes stimulated with EGF or TGF-α; confluent epithelial sheets treated with IL-6 also underwent an increase in the number of cell layers. We also examined the effect of IL-6 on the keratin cytoskeleton. Immunostaining with anti-K16 monoclonal antibodies showed that the keratin network was aggregated and reorganized around cell nucleus and that this was not attributable to changes in keratin levels. This is the first report concerning the induction of the reorganization of keratin intermediate filaments by IL-6 in human epidermal keratinocytes.This work was supported in part by CONACyT grant nos. 1314P-N9507 and G28272-N.     

Lesser double‐collared sunbirds Nectarinia chalybea do not compensate for hatching asynchrony by adjusting egg mass or yolk androgens

Substantial amounts of maternal androgens are found in birds’ eggs and have been shown to benefit offspring development. Within‐clutch patterns of increasing androgen concentrations over the laying sequence are often hypothesized to compensate for the negative effects of hatching asynchrony. However, detrimental effects to offspring fitness of exposure to high yolk androgen levels have also been demonstrated. This suggests that mothers should forego these costs to their offspring when the need for compensation for hatching asynchrony is low or when alternative compensatory strategies, e.g. in terms of increasing egg mass, are available. Here we show that in the south‐temperate lesser double‐collared sunbird Nectarinia chalybea, a species with hatching asynchrony but also with high survival of last‐hatched chicks, mothers do not deposit resources differentially in terms of either yolk androgen concentration or egg mass across the laying sequence. We discuss to what extend this challenges the original explanation of within‐clutch variation in these egg parameters and offer some explanation for their between‐clutch variation which was related to female body mass.     

The chelating effect of citric acid,oxalic acid,amino acids and Pseudomonas fluorescens bacteria on phytoremediation of Cu,Zn, and Cr from soil using Suaeda vera

Phytoextraction is a green technique for the removal of soil contaminants by plants uptake with the subsequent elimination of the generated biomass. The halophytic plant Suaeda vera Forssk. ex J.F.Gmel. is an native Mediterranean species able to tolerate and accumulate salts and heavy metals in their tissues. The objective of this study was to explore the potential use of S. vera for soil metal phytoextraction and to assess the impact of different chelating agents such as natural organic acids (oxalic acid [OA], citric acid [CA]), amino acids (AA) and Pseudomonas fluorescens bacteria (PFB) on the metal uptake and translocation. After 12 months, the highest accumulation of Cu was observed in the root/stem of PFB plots (17.62/8.19 mg/kg), in the root/stem of CA plots for Zn (31.16/23.52 mg/kg) and in the root of OA plots for Cr (10.53 mg/kg). The highest accumulation of metals occurred in the roots (27.33–50.76 mg/kg). Zn was the metal that accumulated at the highest rates in most cases. The phytoextraction percentages were higher for Cu and Zn (～2%) with respect to Cr (～1%). The percentages of metal removal from soil indicate the need to monitor soil properties, to recognize the influence of each treatment and to increase the concentration of bioavailable metals by the use of agricultural management practices aimed at promoting plant growth.     

Genome Comparison of a Nonpathogenic Myxoma Virus Field Strain with Its Ancestor,the Virulent Lausanne Strain

One of the best-studied examples of host-virus coevolution is the release of myxoma virus (MV) for biological control of European rabbits in Australia and Europe. To investigate the genetic basis of MV adaptation to its new host, we sequenced the genome of 6918, an attenuated Spanish field strain, and compared it with that of Lausanne, the strain originally released in Europe in 1952. Although isolated 43 years apart, the genomes were highly conserved (99.95% identical). Only 32 of the 159 MV predicted proteins revealed amino acid changes. Four genes (M009L, M036L, M135R, and M148R) in 6918 were disrupted by frameshift mutations.Myxoma virus (MV), the causative agent of myxomatosis, belongs to the Leporipoxvirus genus of the Poxviridae family (9). Two distinct types of MV have been identified: South American MV, which circulates in Sylvilagus brasiliensis, and Californian MV, which circulates in Sylvilagus bachmani. Each virus is highly adapted to its host, causing a benign cutaneous fibroma at the site of inoculation. Both types of MV infect the European rabbit (Oryctolagus cuniculus), causing myxomatosis. The Californian strain MSW is more virulent for European rabbits than South American strains such as SLS or Lausanne (54). Another leporipoxvirus, Shope fibroma virus (SFV), is found in eastern North America in Sylvilagus floridanus. SFV protects European rabbits against myxomatosis (24), and it is routinely used as a vaccine.One of the best-studied examples of host-virus coevolution is the use of MV for biological control of European rabbits (22, 23, 29). It is particularly unusual because the precise time the virus was released is known, and the original viruses are available for comparison with current strains. MV (the SLS strain) was deliberately released in Australia in 1950 and soon after (1952) in France (the Lausanne strain), whence it rapidly spread across Europe, and it has become endemic since then. For almost 60 years, a complex coevolution of host and virus has occurred, characterized by the emergence of attenuated viral strains and rabbits selected for resistance to MV (11, 12, 30).The MV Lausanne strain and SFV have been completely sequenced (13, 61). MV encodes 171 genes, versus 165 encoded by SFV. The genetic information is highly conserved between the two viruses. Recently, preliminary sequencing of the MSW strain indicated that the major genomic differences with the Lausanne strain localize at the left terminal end of the MSW genome (31). In MSW, the terminal inverted repeats (TIRs) are extended, causing the duplication of five complete open reading frames (ORFs), which are present as a single copy near the right TIR in the Lausanne strain (9). To date, little molecular analysis concerning the adaptation of MV to its new host has been performed. Studies involving Australian field strains found small differences with reference to the SLS and Lausanne strains (49, 50), suggesting that adaptation (and the concomitant attenuation) of MV is not associated with major genetic changes such as large deletions. This finding is in contrast to what has been reported for attenuated poxviruses obtained by extensive cell culture passaging, which usually present substantial genomic deletions or rearrangements (5, 25, 36, 47, 48).Strain 6918 is a naturally attenuated MV isolated in Spain in 1995 (7). It is therefore a descendant of the Lausanne strain recovered after 43 years of continuous evolution in the field. It has been used for the development of a “transmissible vaccine” intended to protect wild-rabbit populations against both myxomatosis and rabbit hemorrhagic disease virus (RHDV) in Spain, where the European rabbit plays a key role in the Mediterranean ecosystems (18). For this purpose, a recombinant virus, 6918VP60-T2, was constructed by inserting the capsid gene of RHDV into the genome of strain 6918 (4, 6, 7, 56, 57). The genomes of 6918 and 6918VP60-T2 have been sequenced. Here we report the results of our comparison of the genomic sequences of Lausanne and 6918. To our knowledge, this is the first comparative genomic analysis involving two poxvirus field strains linked by a clearly recorded lineage, one being fully virulent and the other virtually nonpathogenic. The results provide relevant insights into the mechanisms of MV attenuation that occurred as a consequence of the adaptation of the virus to its new host.     

Postfledging habitat selection of juvenile middle spotted woodpeckers: a multi-scale approach

Despite its relevance for the persistence of populations, the ecological mechanisms underlying habitat use decisions of juvenile birds are poorly understood. We examined postfledging habitat selection of radio-tracked juvenile middle spotted woodpeckers Dendrocopos medius at multiple hierarchically-nested spatial scales in NW Spain. At the landscape and home range scales, old oak forest was the most used and selected habitat, young oak forests and pine plantations were avoided, and riverside forests were used as available. At a lower scale, birds selected larger diameter trees for foraging. Home ranges had higher densities of large deciduous trees (mainly oaks Quercus spp., but also poplars Populus spp. and willows Salix spp. >22  cm and >33  cm DBH) selected for foraging by juveniles than non-used areas. These results suggest that foraging conditions may drive, at least partly, habitat use decisions by juvenile birds. We also discuss the potential influence of intraspecific competition, the search for a future breeding territory in the early postfledging period and predation avoidance on habitat use decisions by juvenile birds. Contrary to previous studies on migrant forest birds, postfledging juvenile woodpeckers selected the same habitat as for the breeding adults (i.e. old oak forest), indicating that migrant and resident specialist avian species may require different conservation actions. Conservation strategies of woodpecker populations should consider the protection of old oak forests with high densities of large trees to provide suitable habitat to breeding adults and postfledging juveniles. The habitat improvement for this indicator and umbrella species would also favour other organisms that depend on characteristics of old-growth oak forests.     

Immunological probes for oestradiol receptors in human breast tumours

Monoclonal antibodies have been prepared against a soluble oestradiol receptor (REC) preparation partially purified from human myometrium by oestradiol affinity chromatography. The antibodies were detected by their ability to immunoprecipitate receptor bound [125I] oestradiol. One of the antibodies (D5) has been studied in detail. It will only precipitate REC after activation by salt, heat, low pH or KCNS and will not react with nuclear RE. It will not react with androgen, progesterone or glucocorticoid receptors nor with sex hormone binding globulin; it will only combine with REC from human sources. D5 recognizes a cytoplasmic 29 kdalton protein (p29) that can be separated from both type I and II soluble oestradiol binding proteins. p29 can react with activated REC and is qualitatively and quantitatively related to REC. IRMA and histochemical methods have been developed for quantitating p29 and relating its amount to receptors in human breast tumours. With both methods, highly significant (P less than 0.001) correlations with REC but not RP have been obtained. Both methods indicate that many REC-RP+ tumours contain p29. The histochemical method detects marked cellular heterogeneity in some tumours. The function of p29 is not known. It is an REC-related antigen that may be a previously undetected component of the oestradiol receptor machinery.     

Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.     

Cell-associated acid β-xylosidase production by Penicillium sclerotiorum

In recent decades, β-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 °C, under stationary condition for six days. The optimum activity temperature was 60 °C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 °C, with half-life of 240 and 232 min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The β-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V_max and K_m values was 0.48 μmol PNXP min^?1 mg^?1 protein and 0.75 mM, respectively. The enzyme was xylose tolerant with a K_i of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.     

Disturbance and predation in an assemblage of herbivorous Diptera and algae on rocky shores

Summary Speculation about the effects of disturbance in marine benthic communities is often based on competition theory. Disturbances are thought to provision numerically depleted or competitively inferior species with resources associated with open substrate. However, disturbances that remove entire assemblages of sessile species also alter trophic structure, and thereby, influence the outcome of predator/prey relations. Aspects of community structure may be determined by patterns of disturbance and predation.The influence of disturbance and predation on the distribution and seasonality of blooms of ephemeral algae and associated Diptera was investigated with field experiments at several rocky beaches in central California. Blooms of ephemeral algae developed on high intertidal rock faces that were subject to severe seasonal disturbances caused by shifting sediment. These were subsequently colonized by the herbivorous larvae of several Diptera species for predictable periods each year. Other areas, without blooms, were not so disturbed.Experiments were done to determine if seasonal blooms were caused by seasonal disturbances that remove predators which otherwise might prevent the establishment of the Diptera/algae assemblage. The predators were crabs and limpets which eat both algae and larvae while foraging. Blooms of algae and larvae did not develop when limpets were transplanted to disturbed areas in periods between disturbances. Adjacent control areas did support blooms. Transplanted limpets did not survive periods of burial. When both limpets and crabs were excluded from treatment plots in undisturbed areas, blooms developed where they would not otherwise have occurred; controls remaied unchanged. Crabs and limpets differed in their effects on this assemblage. Crabs recruited quickly to the site of a bloom, but did not crop algal cover as closely, nor decrease larval density as much as the slowly recruiting limpets.The results suggest that disturbances favor blooms of some species by reducing predation. Severe localized disturbances increased the variability of the upper shore community by creating a patchwork of differing predator/prey abundances.     

Isolation of plasma membrane glycoprotein from bovine thymocytes

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.