By IL-1 signaling,monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide

Epithelia react to microbial pathogens by mounting a defensive response that includes the production of antimicrobial peptides. In this study, we show that, in human epidermal cultures, Escherichia coli LPS was a very weak direct inducer of human beta-defensin (HBD)-2 mRNA and peptide, but the induction was greatly amplified when monocyte-derived cells (MoDeC) acted as intermediaries between LPS and the epidermis. IL-1R antagonist largely reversed the effect of MoDeC on epidermal HBD-2, indicating that, from among the many products of MoDeC, IL-1 was the dominant inducer of HBD-2 synthesis. In normal fresh human skin, which contains Langerhans cells and other myeloid cell types, in addition to keratinocytes, LPS also induced HBD-2 in an IL-1-dependent manner. In DNA microarray expression studies, HBD-2 was one of the most abundant mRNAs induced in epidermis by LPS-treated MoDeC, and its induction was reversed by IL-1Ra. Thus, epidermal response to LPS is potently amplified by MoDeC through IL-1-mediated signaling, leading to a selective increase in the synthesis of the antimicrobial peptide HBD-2. This pattern of responses establishes a key role for both IL-1 and HBD-2 in the host defense reaction of the epidermis.     

Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.     

Intradermal or oral delivery of GAD-encoding genetic vaccines suppresses type 1 diabetes

Genetic vaccines are promising candidates for prevention of type 1 diabetes, an autoimmune disease resulting from cell-mediated destruction of pancreatic beta cells. It is known that the prophylactic effect and immune responses induced by administration of a genetic vaccine can depend on site of delivery. In the work presented here, we used the NOD mouse model for type 1 diabetes to evaluate different routes of delivery for DNA vaccines coding for the beta-cell antigen glutamic acid decarboxylase (GAD). Plasmid DNA coding for intracellular or secreted GAD was given via either the intramuscular (i.m.), intradermal (i.d.), or oral route, using, respectively, 300, 100, or 300 micro g DNA per mouse. Results indicated that both i.d. and oral delivery of GAD-encoding DNA were more effective than i.m. delivery for disease suppression. In addition, cytokine-specific ELISpot analysis indicated that immune responses induced by the different immunization protocols were more dependent on the cellular localization of GAD antigen than on the delivery route, while ELISA of anti-GAD serum antibody isotypes indicated that i.d. delivery of DNA was most likely to induce a Th2-like response. Our results suggest that i.d. or oral delivery of a genetic vaccine for type 1 diabetes might be preferable over the i.m. route in a future clinical setting.     

Functionally-distinct proton-binding in HERG suggests the presence of two binding sites

HERG (Human ether-à-go-go-related gene) potassium channels are crucial for cardiac action potential repolarization. HERG channels are also found in neuronal and tumor cells. The effect of pH₀ on HERG is of clinical significance because of changes in pH during myocardial ischemia, inflammation, and respiratory alkalosis. We present evidence for the presence of multiple proton binding sites in HERG. Extracellular protons bind rapidly and reversibly to affect both activation and deactivation. However, these effects occur in two distinct pH₀ ranges. The deactivation rate has a pK_a of 6.76±0.02 compared to pK_a of 5.50±0.02 for changes in current suppression, which suggests the presence of at least two proton binding sites on HERG with functionally distinct properties.     

The significance of key regulators of apoptosis in the development and prognosis of prostate carcinoma. II. Products of suppressor genes Rb and PTEN, CDKI, Fas

The molecular basis for the transition of carcinoma of the prostate from androgen-dependent to androgen-independent growth is largely unknown. Currently for example, it is not clear whether the androgen-independent phenotype is a result of selection of a subgroup of genetically distinct prostate tumour cells which are already hormone-resistant or a genetic adaptation of prostate tumour cells to the hormone therapy itself. It has also been established that prostate tumour transformation is a result of homeostatic control defects, a line of thinking directed toward elucidating the apoptotic profile of prostate tumour cells that may be important in determining prognosis, response to therapy and illness progression. Main consideration in this part of rewiev is given to the role of tumour suppressor genes pRb and PTEN and also the natural inhibitors of cyclin dependent kinases - proteins p21(Waf1/Cip1) and p27(Kip1). Attention is also given to the role of FAS-mediated pathways in apoptosis induction.     

Multiple response elements and differential p53 binding control Perp expression during apoptosis

The p53 tumor suppressor gene responds to cellular stress by activating either cell cycle arrest or apoptosis. A growing number of target genes involved in each of these pathways have been identified. However, the mechanism by which the apoptosis versus arrest decision is made remains to be elucidated. Perp is a proapoptotic target gene of p53 expressed to high levels in apoptotic cells compared with those undergoing cell cycle arrest. This pattern of expression is unusual among p53 target genes, many of which are induced to similar levels during arrest and apoptosis. Here, we describe the regulation of the Perp gene by p53 through at least three response elements in the Perp promoter and first intron. These sites are occupied in vivo in E1A-expressing mouse embryo fibroblasts undergoing apoptosis but not cell cycle arrest, in contrast to the p21 5' response element, which is occupied during both. The apoptosis-deficient p53 point mutant, p53V143A, displays a selective deficit in binding to the Perp elements, demonstrating that p53 can distinguish between Perp and p21 at the level of DNA binding. These results provide mechanistic insight into the selective expression of Perp during apoptosis and may provide a useful model for studying the p53-dependent cell cycle arrest versus apoptosis decision.