The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.     

Quorum sensing by <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones is not required for <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during growth with organic particles in lake water microcosms

It was investigated whether quorum sensing (QS) mediated by N-acylhomoserine lactones (AHLs) was important for heterotrophic bacteria from the littoral zone of the oligotrophic Lake Constance for growth with organic particles. More than 900 colonies from lake water microcosms with artificial organic aggregates consisting of autoclaved unicellular algae embedded in agarose beads were screened for AHL-production. AHL-producing bacteria of the genus Aeromonas enriched in the microcosms but AHLs could not be detected in any microcosm. To test for a potential function of AHL-mediated QS, growth experiments with the wild type and an AHL-deficient mutant of Aeromonas hydrophila in lake water microcosms were performed. Growth of both strains did not differ in single cultures and showed no mutual influence in co-cultures. In co-cultures with a competitor bacterium belonging to the Cytophaga–Flavobacterium group, growth of both A. hydrophila strains was reduced while growth of the competitor bacterium was not affected. Exogenous AHL-addition did not influence growth of the Aeromonas strains in any microcosm experiment. These results showed that AHL-mediated QS was not required for A. hydrophila during colonization and degradation of organic particles in lake water microcosms, suggesting that cell–cell signalling of heterotrophic bacteria in oligotrophic waters relies on novel signal molecules.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

BMPs induce dermal markers and ectopic feather tracts

Bone morphogenetic protein (BMP) signaling is known to be involved in multiple inductive events during embryogenesis including the development of amniote skin. Here, we demonstrate that early application of BMP-2 to the lateral trunk of chick embryos induces the formation of dense dermis, which is competent to participate in feather development. We show that BMPs induce the dermis markers Msx-1 and cDermo-1 and lead to dermal proliferation, to expression of β-catenin, and eventually to the formation of ectopic feather tracts in originally featherless regions of chick skin. Moreover, we present a detailed analysis of cDermo-1 expression during early feather development. The data implicate that cDermo-1 is located downstream of BMP in a signaling pathway that leads to condensation of dermal cells. The roles of BMP and cDermo-1 during development of dermis and feather primordia are discussed.     

Species-crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP-/- knock-out mouse.

BACKGROUND: Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity. MATERIAL AND METHODS: An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP. RESULTS: High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model. CONCLUSIONS: MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.     

Impact of the long chain ω-acylceramides on the stratum corneum lipid nanostructure. Part 1: Thermotropic phase behaviour of CER[EOS] and CER[EOP] studied using X-ray powder diffraction and FT-Raman spectroscopy

The stratum corneum (SC), the outermost layer of the mammalian skin, is the main skin barrier. Ceramides (CERs) as the major constituent of the SC lipid matrix are of particular interest. At the moment, 11 classes of CERs are identified, but the effect of each single ceramide species is still not known.Therefore in this article, the thermotropic behaviour of the long chain ω-acylceramides CER[EOS] and CER[EOP] was studied using X-ray powder diffraction and FT-Raman spectroscopy.It was found that the ω-acylceramides CER[EOS] and CER[EOP] do not show a pronounced polymorphism which is observed for shorter chain ceramides as a significant feature. The phase behaviour of both ceramides is strongly influenced by the extremely long acyl-chain residue. The latter has a much stronger influence compared with the structure of the polar head group, which is discussed as extremely important for the appearance of a rich polymorphism. Despite the strong influence of the long chain, the additional OH-group of the phyto-sphingosine type CER[EOP] influences the lamellar repeat distance and the chain packing. The less polar sphingosine type CER[EOS] is stronger influenced by the long acyl-chain residue. Hydration is necessary for the formation of an extended hydrogen-bonding network between the polar head groups leading to the appearance of a long-periodicity phase (LPP). In contrast, the more polar CER[EOP] forms the LPP with densely packed alkyl chains already in the dry state.     

Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.     

Clearance of picoplankton-sized partides and formation of rapidly sinking aggregates by the pteropod, Limacina reiroversa

Findings from experiments showed that the web-feeding euthecosomatouspteropod, Limacina retroversa, can produce rapidly sinking,mucous aggregates. It is suggested that, by adhesion, theseaggregates scavenged picoplankton-sized particles, which werethus effectively cleared from the medium. In contrast, Calanusfinmarchicus was not able to clear these particles in our experiments.Sedimentation velocities of 10 aggregates measured in vivo wereup to 1000 m day^–1, with an average of {small tilde}300m day^–1 (not including two aggegates with neutral buoyancy).Mean velocities measured for feces of C.finmarchicus, Calanushyperboreus and Thyssnoessa sp. were consider ably lower. Wesuggest that the sedimentation of L retroversa aggregates wasthe source of mucous flocs collected in sediment traps (Bathmannet al., Deep-Sea Res., 38,1341–1360,1991) and at the seafloor at 1200 m depth in the southern Norwegian Sea. This processmay be an important mediator of sedimentation to the deep sea,when these pteropods are present in surface waters in largeabundance.     

Two unprecedented natural Aib-peptides with the (Xaa-Yaa-Aib-Pro) motif and an unusual C-terminus: structures, membrane-modifying and antibacterial properties of pseudokonins KL III and KL VI from the fungus Trichoderma pseudokoningii.

Pseudokonins KL III and KL VI are two natural ten-residue peptides, which both contain the (Xaa-Yaa-Aib-Pro) motif and exhibit an unusual C-terminus. They have been isolated from the fungus Trichoderma pseudokoningii by intensive reversed-phase HPLC, beside peptaibols classically C-ended by a beta-amino alcohol. The amino acid sequences and the chemical structures of the C-ends have been determined by the combined use of positive ion LSI-MS and two-dimensional homo- and heteronuclear NMR, including COSY, TOCSY, ROESY, 13C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC). Instead of one of the amino alcohols usually found as C-terminal residue in peptaibols, pseudokonins KL III and KL VI are characterized by -Pro-NH2 and cyclo-(Aib-L-Proal) (Proal, prolinal), respectively. Such backbone modifications are described here for the first time for peptaibol antibiotics. The unusual cyclo-(Aib-L-Proal) C-terminus is probably the result of an intramolecular cyclization of the two last Aib and Pro residues of a ten-amino acid precursor, via a Proal intermediate. A secondary structure stabilized by -C=O...H-N-hydrogen bonds of the 1<--4 type has been deduced for both peptides from ROESY data, 3JNHCalphaH couplings and amide proton temperature coefficient values. The (Xaa-Yaa-Aib-Pro) beta-bend ribbon spiral, which has been described for the first time in the case of a 14-residue peptaibol containing three repetitive (Xaa-Yaa-Aib-Pro) motifs (Segalas G et al. Biopolymers 1999; 50: 71-85) appears to be maintained in the two shortened modified peptides. The beta-bend ribbon structure thus appears to be initiated by a single (Xaa-Yaa-Aib-Pro) motif and unaffected by the C-terminal modifications. However, the membrane and antibiotic properties of pseudokonins KL III and KL VI, point to the unfavourable effect of both shortening and cyclization of the peptide chain.     

The mitogen-activated protein kinase pathway contributes to vanadate toxicity in vascular smooth muscle cells

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44^MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC₅₀: 30 M). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44^MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44^MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.