Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.     

Laser scanning cytometry in human brain slices.

BACKGROUND: The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. METHODS: We adapted this technique to immunohistochemical labelled human brain slices. RESULTS: We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. CONCLUSIONS: In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections.     

Proteomic identification and characterization of secreted N‐glycosylated NPC2 following cross‐linking of the high‐affinity receptor for IgE on mast cells

Allergen‐mediated cross‐linking of the high‐affinity receptor for IgE on mast cells triggers the release of diverse preformed and de novo synthesized immunoregulatory mediators that further the allergic response. A proteomic screen applied to the detection of proteins secreted by the model rat mast cell line, RBL‐2H3 (rat basophilic leukaemia, subline 2H3.1), led to the identification of the cholesterol‐binding glycoprotein, NPC2/RE1 (Niemann–Pick Type C2/epididymal secretory protein 1). Glycosylated NPC2 is secreted early in response to an IgE‐mediated stimulus and co‐localizes with the lysosomal membrane marker, CD63. NPC2 belongs to the ML (MD‐2‐related lipid‐recognition) protein family (155 members), which includes the Toll‐like receptor co‐factors, MD‐1 and MD‐2, and perhaps most interestingly, seven major house dust mite allergens of unknown function (including Der p 2 and Der f 2). Possible role(s) for the protein in the allergic response and future applications of this approach are discussed.     

The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (Pm^R) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable Pm^R transformants ranged about 15 per 10⁶ target cells. Due to rapid and sharp selection, Pm^R transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of Pm^R transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

Interaction of CheY2 and CheY2-P with the cognate CheA kinase in the chemosensory-signalling chain of Sinorhizobium meliloti

An unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti . Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheA–CheY2 and CheA–CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal β4-α4-β5-α5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting β5 and α5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.