Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Polyamine metabolism in BHK21/C13 cells : Loss of spermidine from cells following transfer to serum-depleted medium

The growth rate of BHK21/C13 cells in culture was slowed down by transferring growing cells to serum-depleted medium Following deprivation of serum, the intracellular concentration of polyamines decreased. The amount of spermidine relative to spermine decreased, and this change was the result of the spermidine content per cell decreasing more than the spermine content. The decrease in cell content of polyamines was accompanied by release of polyamines from the cells into the culture medium. The polyamines released were examined using cells whose polyamines had been labelled by prior incubation of the cells with radioactive putrescine. Almost all of the radioactivity released into the medium was found in spermidine, even though the cells contained most of their radioactivity in spermine. It is suggested that specific release of spermidine may be an important mechanism by which these cells can regulate their intracellular content of polyamines.     

Calmodulin activity in yeast and mycelial phases of Ceratocystis ulmi

Abstract The yeast and mycelial phases of Ceratocystis ulmi contained roughly equivalent levels of calmodulin activity as determined by their ability to stimulate calmodulin-deficient bovine brain cAMP phosphodiesterase. This stimulation was calcium-dependent and could be inhibited by either dibucaine or trifluoperazine. Also, the concentration of dibucaine necessary to achieve the mycelium-to-yeast morphological conversion was found to be 3-fold greater in the presence of exogenous calcium. A model is presented in which only 30% of the cellular calmodulin need be complexed with calcium ions for mycelial development.     

Investigation of organic anions in tree root exudates and rhizosphere microbial communities using in situ and destructive sampling techniques

Purpose

Understanding of the role of low molecular weight organic anions (OAs) in structuring rhizosphere microbial communities in situ is limited due to challenges associated with sampling. Improved techniques are needed for such studies.

Methods

This study used in situ and destructive sampling techniques and compared two exudate extraction methods [anion exchange membrane (AEM) capturing and water extraction] from rhizosphere and non-rhizosphere samples of genetically modified (GM) and control Pinus radiata D. Don trees grown in large-scale rhizotrons for ~10?months. Metabolically active soil microbial communities were analysed using rRNA-DGGE.

Results

Recovery of eight out of 12 anions was influenced by extraction methods, and in situ sampling using AEM was shown to be the most efficient method. Only minor differences were detected in OAs in root exudates collected from the GM and control trees. Significant differences in α-Proteobacterial and Pseudomonas communities were associated with the two tree lines in the topsoil at both sampling events. Additional differences in β-Proteobacterial and fungal communities between tree lines were detected in the rhizosphere using destructive sampling.

Conclusion

This study demonstrated that in situ sampling was superior to destructive sampling for the efficient collection of root exudates and analysis of associated rhizosphere microbial communities.     

Stable transformation of papaya via microprojectile bombardment

Summary Stable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1^–1 2,4-D, 400 mg 1^–1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1^–1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - X-gluc 5-Br-4-Cl-3-indolyl--D-glucuronic acid - CaMV cauliflower mosaic virus - NOS nopaline synthase - NPTII neomycin phosphotransferase II Journal Series no. 3448 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Effects of plant odor on oviposition by the black swallowtail butterfly,Papilio polyxenes (Lepidoptera: Papilionidae)

Black swallowtail females laid more eggs on plant models treated with contact stimulants and volatiles from carrot leaves than on models treated only with contact stimulants. The volatiles enhanced landing rates and females alighted more frequently on artificial leaves treated with host volatiles than on adjacent control leaves. Volatiles from cabbage, a nonhost, inhibited landing rates on artificial leaves treated with carrot contact stimulants. Examination of antennae revealed two major types of sensilla, believed to be olfactory in function. Electroantennogram preparations responded more strongly to carrot volatiles than to cabbage volatiles and several shared responses at particular retention times to carrot volatile components eluting from a gas chromatograph. Our results are consistent with a long-standing hypothesis that behavioral responses to essential oil components characteristic of the larval food plants have facilitated host shifts in the genus Papilio.     

Deriving phylogenetic trees from the similarity analysis of metabolic pathways

MOTIVATION: Comparative analysis of metabolic pathways in different genomes can give insights into the understanding of evolutionary and organizational relationships among species. This type of analysis allows one to measure the evolution of complete processes (with different functional roles) rather than the individual elements of a conventional analysis. We present a new technique for the phylogenetic analysis of metabolic pathways based on the topology of the underlying graphs. A distance measure between graphs is defined using the similarity between nodes of the graphs and the structural relationship between them. This distance measure is applied to the enzyme-enzyme relational graphs derived from metabolic pathways. Using this approach, pathways and group of pathways of different organisms are compared to each other and the resulting distance matrix is used to obtain a phylogenetic tree. RESULTS: We apply the method to the Citric Acid Cycle and the Glycolysis pathways of different groups of organisms, as well as to the Carbohydrate metabolic networks. Phylogenetic trees obtained from the experiments were close to existing phylogenies and revealed interesting relationships among organisms.     

High-resolution molecular epidemiology and evolutionary history of HIV-1 subtypes in Albania

Background

HIV-1 epidemic in Western Europe is largely due to subtype B. Little is known about the HIV-1 in Eastern Europe, but a few studies have shown that non-B subtypes are quite common. In Albania, where a recent study estimated a ten-fold increase of AIDS incidence during the last six years, subtype A and B account for 90% of the know infections.

Methodology/Principal Findings

We investigated the demographic history of HIV-1 subtype A and B in Albania by using a statistical framework based on coalescent theory and phylogeography. High-resolution phylogenetic and molecular clock analysis showed a limited introduction to the Balkan country of subtype A during the late 1980s followed by an epidemic outburst in the early 1990s. In contrast, subtype B was apparently introduced multiple times between the mid-1970s and mid-1980s. Both subtypes are growing exponentially, although the HIV-1A epidemic displays a faster growth rate, and a significantly higher basic reproductive number R₀. HIV-1A gene flow occurs primarily from the capital Tirane, in the center of the country, to the periphery, while HIV-1B flow is characterized by a balanced exchange between center and periphery. Finally, we calculated that the actual number of infections in Albania is at least two orders of magnitude higher than previously thought.

Conclusions/Significance

Our analysis demonstrates the power of recently developed computational tools to investigate molecular epidemiology of pathogens, and emphasize the complex factors involved in the establishment of HIV-1 epidemics. We suggest that a significant correlation exists between HIV-1 exponential spread and the socio-political changes occurred during the Balkan wars. The fast growth of a relatively new non-B epidemic in the Balkans may have significant consequences for the evolution of HIV-1 epidemiology in neighboring countries in Eastern and Western Europe.