Growth of sebaceous cells in monolayer culture

Summary Rat preputial cells were grown in an epithelial cell primary monolayer culture system identical to that used for culturing epidermal cells, which were studied for comparison. Despite similar appearance when observed by phase contrast microscopy, other features identified the preputial cells as a unique epithelial cell population. Preputial cells grew as a relatively small number of large colonies, formed domes before confluence, and expressed a specific acinar keratin, K4, which had previously been found in human sebaceous glands. In addition, preputial cells formed fewer cornified envelopes than epidermal cells, too few to discern the reduction of envelope formation by retinoic acid treatment in vitro which was found in epidermal cells. Rat preputial cells in monolayer culture, therefore, are a promising model for studying the effects of hormones on sebaceous cell growth and differentiation.     

Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS

Background

Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine.

Methodology/Principal Findings

Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M’sila where P. (Phlebotomus) papatasi was the only sand fly species detected.

Conclusion

The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases.     

Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea <Emphasis Type="Italic">Photobacterium phosphoreum</Emphasis> ANT-2200

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.     

Sebaceous epithelial cell differentiation requires cyclic adenosine monophosphate generation

The cyclic adenosine monophosphate (cAMP) generator choleratoxin is known to promote the growth of sebaceous epithelial cells (sebocytes) in monolayer culture in classical serum-containing media. Now that sebocytes can be grown in serum-free medium, we have examined whether choleratoxin or other cAMP generators are required for differentiation of rat preputial sebocytes in response to specific ligand activators of peroxisome proliferator-activated receptors (PPARs). Unexpectedly, choleratoxin reduced sebocyte proliferation. However, sebocyte differentiation in response to specific PPARalpha and PPARgamma agonists required a cAMP generator such as choleratoxin, and this response was suppressed by a protein kinase A inhibitor. In contrast, the stable prostacyclin analog, carbaprostacyclin (cPGI2), a PPARalpha,delta agonist that also generates cAMP, stimulated differentiation independently of choleratoxin. Furthermore, unlike the selective PPARalpha and PPARgamma agonists, cPGI2 stimulated both sebocyte DNA synthesis and proliferation. These data are compatible with the evidence that prostacyclin has the additional effect of generating cAMP. In addition, we addressed the possibility that choleratoxin may act as a surrogate for beta-adrenergic catecholamines in generating cAMP. In contrast with choleratoxin, both alpha- and beta-adrenergic catecholamines stimulated sebocyte growth and interfered with the choleratoxin effect on differentiation. These data suggest ligand-dependent, complex interactions between cAMP and the other signal transduction pathways involved in sebocyte growth and development.     

Sexual hormones in human skin.

The skin locally synthesizes significant amounts of sexual hormones with intracrine or paracrine actions. The local level of each sexual steroid depends upon the expression of each of the androgen- and estrogen-synthesizing enzymes in each cell type, with sebaceous glands and sweat glands being the major contributors. Sebocytes express very little of the key enzyme, cytochrome P450c17, necessary for synthesis of the androgenic prohormones dehydroepiandrosterone and androstenedione, however, these prohormones can be converted by sebocytes and sweat glands, and probably also by dermal papilla cells, into more potent androgens like testosterone and dihydrotestosterone. Five major enzymes are involved in the activation and deactivation of androgens in skin. Androgens affect several functions of human skin, such as sebaceous gland growth and differentiation, hair growth, epidermal barrier homeostasis and wound healing. Their effects are mediated by binding to the nuclear androgen receptor. Changes of isoenzyme and/or androgen receptor levels may have important implications in the development of hyperandrogenism and the associated skin diseases such as acne, seborrhoea, hirsutism and androgenetic alopecia. On the other hand, estrogens have been implicated in skin aging, pigmentation, hair growth, sebum production and skin cancer. Estrogens exert their actions through intracellular receptors or via cell surface receptors, which activate specific second messenger signaling pathways. Recent studies suggest specific site-related distribution of ERalpha and ERbeta in human skin. In contrast, progestins play no role in the pathogenesis of skin disorders. However, they play a major role in the treatment of hirsutism and acne vulgaris, where they are prescribed as components of estrogen-progestin combination pills and as anti-androgens. These combinations enhance gonadotropin suppression of ovarian androgen production. Estrogen-progestin treatment can reduce the need for shaving by half and arrest progression of hirsutism of various etiologies, but do not necessarily reverse it. However, they reliably reduce acne. Cyproterone acetate and spironolactone are similarly effective as anti-androgens in reducing hirsutism, although there is wide variability in individual responses.     

Expression level of heterologous tat genes is crucial for in vivo reconstitution of a functional Tat translocase in Escherichia coli

The Tat system has the remarkable capacity of exporting proteins in folded conformation across the cytoplasmic membrane. The functional Tat translocase from Gram-negative bacteria consists of TatA, TatB and TatC proteins. To gain information about the species specificity of the Tat translocase, we cloned tat genes from Gram-negative pathogens Shigella flexneri 2a str. 301, Vibrio cholerae El Tor N16961, Pseudomonas aeruginosa PAO1, thermophilic Sulfolobus solfataricus P2, Thermus thermophilus HB8 and from three Magnetospirillum species (AMB-1, MS-1 and MSR-1), and assessed the capacity of these Tat systems to restore the Tat-dependent growth defect of Escherichia coli tat mutants. We found that whereas the tat genes from the thermophilic bacterial and archaeal species were not functional in E. coli, other tat genes could all complement the phenotype of the E. coli tat mutants. In addition, a chimera composed of the N-terminus of V. cholerae TatE and C-terminus of M. magneticum TatA was functional. Whereas the expression of the tatABC genes from P. aeruginosa and Magnetospirillum strains must be induced to obtain a functional Tat system, overproduction of the V. cholerae TatABC proteins abolished the complementation. The complementation impairment seemed to be correlated with increasing level of slow-migrating TatC isoforms. In vitro studies showed that slow-migrating TatC isoforms in the purified V. cholerae TatABC complex increased with storage time. Together these results showed that the Tat translocases from the Gram-negative bacteria are generally functional in E. coli and the expression level is crucial for in vivo reconstitution of a functional Tat translocase.     

Polar positional information in Escherichia coli spherical cells

Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.