Keratinocytes as depository of ammonium-inducible glutamine synthetase: age- and anatomy-dependent distribution in human and rat skin.

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component beta-catenin. Inhibition of, glycogen synthase kinase 3beta in cultured keratinocytes and HaCaT cells, however, did not support a direct role of beta-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8-10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.

Use of the Porcine Intestinal Epithelium (PIE)-Assay to analyze early stages of colonization by the human fungal pathogen Candida albicans.

Virulence of C. albicans strains can be tested using a mouse model of haematogenously disseminated Candida cells. Initial steps of host-pathogen contact such as adhesion and colonization are not taken into account due to the injection of Candida cells into the blood stream. Here we describe an assay, based on the ex vivo usage of porcine intestinal epithelium (PIE), that is useful to monitor the early stages of a C. albicans infection. The ability of C. albicans to undergo morphogenetic switching between yeast and hyphal stages is thought to contribute to its virulence. We found that hyphal formation was required to allow cells to colonize the PIE. The non-filamentous mutant strains efg1/cph1 which lacks two of the central transcription factors that are required to promote hyphal growth and wal1 that carries a deletion of the C. albicans homolog of the human Wiskott-Aldrich Syndrome Protein and is deficient in endocytosis showed only weak adherence. Furthermore, the wal1 mutant was found to be reduced in virulence using the mouse tail vein injection assay. We also analyzed the colonization properties of a variety of other mutant strains carrying deletions of either secreted aspartyl proteinase (SAP)-family genes or amino acid permease encoding genes (GAP1, SSY1, and PUT4). Interestingly, the nag5 strain which lacks an N-acetylglucosamine kinase showed enhanced filamentation and invasive growth as well as increased resistance against farnesol.

[Comparison of conventional and real-time RT-PCR for the quantitation of jun protooncogene mRNA and analysis of junB mRNA expression in synovial membranes and isolated synovial fibroblasts from rheumatoid arthritis patients]. / Vergleich zwischen konventioneller und Real-Time RT-PCR zur Quantifizierung der jun-Protoonkogen-mRNA und Analyse der junB-mRNA-Expression in Synovialmembranen und isolierten synovialen Fibroblasten von Patienten mit rheumatoider Arthritis.

AP-1 dependent genes, e.g., matrix-metallo-proteinases, are involved in the pathogenesis of rheumatoid arthritis (RA). Therefore, the transcription factor AP-1 and its subunits, proteins of the Jun and Fos proto-oncogene families, are interesting targets for analysis in RA. In this study, we analyzed the mRNA expression of junB in synovial membrane (SM) samples and isolated synovial fibroblasts of patients with RA, osteoarthritis (OA), and normal, non-inflammatory controls. To address the suitability of real-time RT-PCR for the quantitation of Jun proto-oncogene family members, conventional RTPCR and real-time PCR were comparatively applied for junD, a gene representing a major challenge because of its high GC-content (70%, increasing the probability of secondary structures interfering with the PCR) and its sequence homology to other Jun proto-oncogenes. In addition, a comparison was performed concerning the precision, reproducibility, costs, as well as labor and time consumption of the two PCR methods. Real-time RT-PCR proved superior to conventional PCR in terms of precision (mean deviation of measured from employed concentration 58% for real-time PCR vs 225% for conventional PCR), reproducibility, as well as labor and time consumption (4 times less for real-time RT-PCR). Experimental cDNA normalization for equivalent cDNA concentrations by sample dilution was more reliable than mathematical cDNA normalization. However, real-time PCR was 3.6-fold more expensive. Applying the more reliable real-time RT-PCR for the ex vivo analysis of junB mRNA-expression, no significantly different expression of junB was observed in SM or isolated synovial fibroblasts from RA as compared to OA. Interestingly, however, junBmRNA expression was significantly lower in RA SM and borderline significantly lower in OA SM than in normal/non-inflammatory SM, with potential effects on the functional properties of the resulting AP-1 complexes. Immunohistochemical staining of the SM with JunB-specific antibodies showed comparable JunB protein expression in SFB (collagen III mRNA-positive) of RA and OA samples. Thus, real-time RT-PCR appears suitable and time-saving for the quantitation of jun proto-oncogene mRNA-expression in tissue and cell samples with high precision and reproducibility.