Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.     

Production of isolated somatic embryos from sunflower thin cell layers

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.Abbreviations NAA 1-naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-benzylamino-purine - MS Murashige and Skoog medium - B5 Gamborg medium     

Subsociality and female reproductive success in a mycophagous thrips: An observational and experimental analysis

Oviparous females of the haplodiploid, facultatively viviparous thrips Elaphrothrips tuberculatus(Thysanoptera: Phlaeothripidae) guard their eggs against female conspecifics and other egg predators. The intensity of maternal defense increases with clutch size. Field and laboratory observations indicate that cannibalism by females is an important selective pressure favoring maternal care. Experimental removals of guarding females showed that egg guarding substantially increases egg survivorship and that the survivorship of undefended eggs is higher in the absence of nonguarding female conspecifics than in their presence. The fecundity of viviparous females increases with the number of eggs cannibalized. The reproductive success of oviparous females increases with body size and local food density and decreases with local density of breeding females. Social behavior may not have advanced beyond maternal care in Elaphrothrips tuberculatusbecause, relative to Hymenoptera, capabilities for helping relatives are few or nonexistent, and the causes of variation in female reproductive success are not influenced easily by cooperation among females.     

Callus culture ofPterocladia capillacea (Rhodophyta) and analysis of cell wall polysaccharides

Calluses induced fromPterocladia capillacea have been kept in culture for more than three years. They exhibit a fast growth rate, owing to the release of single cells, which in turn develop into new callus. The effect of various media and culture conditions upon growth was investigated. In order to confirm the identity of the callus cells, a 0,45 mg incoculum was grown that yielded 15 g dried callus within six weeks. Polysaccharides from this material (5.5 g) were analysed by¹³C NMR spectroscopy. This produced a spectrum typical of agar and very similar to the one obtained for agar extracted fromP. capillacea plants. However, the callus agar displayed no gel-forming properties, even after alkali modification.author for correspondence     

On the evolutionary ecology of marking pheromones

Summary Many parasitic insects mark hosts with a pheromone after oviposition. The evolutionary ecology of such marking pheromones was studied to determine (i) under what ecological and behavioral conditions such pheromones could evolve and (ii) why so many of these marking pheromones are water-soluble and thus short-lived. We used a number of different techniques. First, the fitness values of individual normal (nonmarking) and mutant (marking) insects foraging for hosts were computed using dynamic state-variable models. Second, population level models were used to study when a population of non-marking individuals can be invaded by marking individuals. Third, behavior-rich simulations (developed originally for apple maggot,Rhagoletis pomonella) were used to test experimentally some of the hypotheses generated using the individual and population-level models. Finally, we developed a model for the benefit over time to an individual by marking. This model shows that when benefit is measured in terms of larval survival, nearly all of the benefit to a mother is obtained from short-lived marks. Genetical theories of pheromone evolution and the connection between our results and existing theories of altruistic behavior are discussed.     

Adhesion of canine and human uropathogenicEscherichia coli andProteus mirabilis strains to canine and human epithelial cells

Escherichia coli strains causing urinary tract infections in dogs produce fimbriae composed of fimbrial subunits closely related to the F12 and F13 fimbriae of human uropathogenic strains [4]. The adhesins carried by the fimbriae of human and canine isolates differ, however, as concluded from a different hemagglutination pattern and from the fact that the dog strains do not agglutinate latex beads coated with P-fimbriae receptor. This possible difference in adhesive specificity was confirmed by experiments in which the adhesion of human and dog isolates to dog kidney epithelial cells (MDCK cells) and human bladder epithelial cells (T24 cells) was compared. Dog uropathogenic strains, in contrast to human uropathogenicE. coli strains, adhere to MDCK cells but hardly to T24 cells. Adhesion to MDCK cells correlates with the presence of F12 or F13 fimbriae on the dog strains. These results suggest that homologous fimbrial subunits can carry different adhesin molecules and that these adhesin molecules can be responsible for species-specific adherence. On the contrary, adhesion of a number of dog uropathogenicProteus mirabilis strains to MDCK and T24 cells was not species specific; it depended on the mere presence of fimbriae.     

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.     

Microbial cell responses to a non-ionic surfactant

Summary The effects of a non-ionic surfactant, Pluronic F-68, on growth of microbial cell cultures have been studied. Growth ofSaccharomyces cerevisiae at 30°C or 37°C as measured by viable cell counts was unaffected by culture with pluronic. However, corresponding absorbance measurements forS. cerevisiae incubated with 5–10% pluronic were lower than controls at both temperatures. Absorbance ofE. coli cultures was also significantly reduced by incubation with 5.0–10.0% pluronic at both temperatures although viable counts again revealed no significant inhibition of growth.     

Circumnutation in Phaseolus vulgaris. I. Growth, osmotic potential and cell ultrastructure in the free-moving part of the shoot

Circumnutation of Phaseolus vulgaris L. cv. Blanc de Juillet twining shoots was examined at the cellular level. Cell growth was monitored inside the free-moving part of the shoot as a function of time and position with respect to the terminal bud. Complete elongation of epidermal cells required >7 days. A 60% increase in length of the cells in the bending zone was observed. Scanning and transmission electron microscopy studies showed the elasticity of cell walls, the extent of plasmodesmata and endoplasmic reticulum of epidermal cells. The osmotic potential in epidermal cells of the bending zone displayed significant differences only between the concave and convex sides. The pattern of cell growth in twining shoots is discussed. Circumnutation could be seen as a consequence of periodic turgor changes inducing periodic growth.