Correct positioning of neurons during embryonic development of the brain depends, among other processes, on the proper transmission of the reelin signal into the migrating cells via the interplay of its receptors with cytoplasmic signal transducers. Cellular components of this signaling pathway characterized to date are cell surface receptors for reelin like apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR), and cadherin-related neuronal receptors, and intracellular components like Disabled-1 and the nonreceptor tyrosine kinase Fyn, which bind to the intracellular domains of the ApoER2 and VLDL receptor or of cadherin-related neuronal receptors, respectively. Here we show that ApoER2, but not VLDLR, also binds the family of JNK-interacting proteins (JIPs), which act as molecular scaffolds for the JNK-signaling pathway. The ApoER2 binding domain on JIP-2 does not overlap with the binding sites for MLK3, MKK7, and JNK. These results suggest that ApoER2 is able to assemble a multiprotein complex containing Disabled-1 and JIPs, together with their binding partners, to the cell surface of neurons. This complex might participate in ApoER2-specific reelin signaling and thus would explain the different phenotype of mice lacking the ApoER2 from that of VLDLR-deficient mice.

There is now growing evidence that the oxidative modification of LDL plays a potential role in atherosclerosis. In this study, genistein, a compound derived from a soy diet with a flavonoid chemical structure (4′,5,7-trihydroxyisoflavone), which was found to inhibit angiogenesis, has been evaluated for its ability to act as an LDL antioxidant and a vascular cell protective agent against oxidized LDL. The results showed that genistein was able to inhibit the oxidation of LDL in the presence of copper ions or superoxide/nitric oxide radicals as measured by thiobarbituric acid-reactive substance formation, alteration in electrophoretic mobility, and lipid hydroperoxides. Bovine aortic endothelial cell- and human endothelial cell-mediated LDL oxidation was also inhibited in the presence of genistein. The 7-O-glucoside of genistein, genistin, was much less effective in inhibiting LDL oxidation in the cell-free and cell-mediated lipoprotein-oxidating systems. Incubating human endothelial cells in the absence or presence of genistein and challenging the cells with already oxidized lipoprotein revealed that in addition to its antioxidative potential during LDL oxidating processes, genistein effectively protected the vascular cells from damage by oxidized lipoproteins. The tyrosine kinase inhibitor genistein was found to block upregulation of two tyrosine-phosphorylated proteins of 132 and 69 kDa in endothelial cells induced by oxidized LDL. Parallel experiments with the inactive analogue daidzein, however, showed that the cytoprotective effect of the isoflavones seems not to be dependent on tyrosine phosphorylation. Our findings will support the suggested and documented beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.

Beside NO (nitric monoxide) and CO (carbon monoxide), H2S (hydrogen sulfide) has been identified recently as the third gasotransmitter. By acting directly on KATP-channels on smooth muscle cells (SMC) H2S possesses vasorelaxing properties. It has the potential to react with metal ions (i.e. Cu, Fe, Zn) in metalloproteins. Angiotensin-converting enzyme (ACE), responsible for vasoconstriction, is a zinc (Zn2+) containing enzyme. We therefore hypothesized that H2S may interact with the Zn2+ in the active center of ACE, modulating (inhibiting) enzyme activity. ACE activity was measured on the surface of human endothelial cells (HUVECs) monolayers in culture, ex-vivo in umbilical veins and in HUVEC protein extracts. Quantitative real-time polymerase chain reaction (PCR) was used to study the effect of H2S on ACE mRNA expression in HUVECs. H2S inhibited the activity of ACE in HUVEC protein extracts in a dose-dependent manner, and only Zn2+ but not Cd2+, Ca2+ or Mg2+ could counteract the inhibitory effect. Cell-surface ACE activity was inhibited by H2S on HUVEC monolayers and in ex-vivo umbilical veins. No influence of H2S on ACE mRNA expression was observed. H2S exhibits direct inhibitory action on ACE activity in HUVECs, obviously by interfering with the Zn2+ in the active center of the enzyme. Thus, beside the known influence of H2S on SMC KATP-channels, the observed direct ACE inhibitory effect may add to the vasorelaxant effect of H2S in the vasculature by reducing angiotensin II production and inhibiting bradykinin degradation.

Proteases of the caspase family play central roles in apoptosis and inflammation. Recently, we have described a new gene encoding caspase-15 that has been inactivated independently in different mammalian lineages. To determine the dynamics of gene duplication and loss in the entire caspase gene family, we performed a comprehensive evolutionary analysis of mammalian caspases. By comparative genomics and reverse transcriptase–polymerase chain reaction analyses, we identified 3 novel mammalian caspase genes, which we tentatively named caspases-16 through -18. Caspase-16, which is most similar in sequence to caspase-14, has been conserved in marsupials and placental mammals, including humans. Caspase-17, which is most similar to caspase-3, has been conserved among fish, frog, chicken, lizard, and the platypus but is absent from marsupials and placental mammals. Caspase-18, which is most similar to caspase-8, has been conserved among chicken, platypus, and opossum but is absent from placental mammals. These gene distribution patterns suggest that, in the evolutionary lineage leading to humans, caspase-17 was lost after the split of protherian and therian mammals and caspase-18 was lost after the split of marsupials and placental mammals. In the canine genome, the number of caspases has been reduced by the fusion of the neighboring genes caspases-1 and -4, resulting in a single coding region. Further lineage-specific gene inactivations were found for caspase-10 in murine rodents and caspase-12 in humans, rabbit, and cow. Lineage-specific gene duplications were found for caspases-1, -3, and -12 in opossum and caspase-4 in primates. Other caspases were generally conserved in all mammalian species investigated. Using the positions of introns as stable characters during recent vertebrate evolution, we define 3 phylogenetic clades of caspase genes: caspases-1/-2/-4/-5/-9/-12/-14/-15/-16 (clade I), caspases-3/-6/-7/-17 (clade II), and caspases-8/-10/-18/CFLAR (clade III). We conclude that gene inactivations have occurred in each of the 3 caspase clades and that gene loss has been as critical as gene duplication in the evolution of the human repertoire of caspases.

The evolution of amniotes has involved major molecular innovations in the epidermis. In particular, distinct structural proteins that undergo covalent cross-linking during cornification of keratinocytes facilitate the formation of mechanically resilient superficial cell layers and help to limit water loss to the environment. Special modes of cornification generate amniote-specific skin appendages such as claws, feathers, and hair. In mammals, many protein substrates of cornification are encoded by a cluster of genes, termed the epidermal differentiation complex (EDC). To provide a basis for hypotheses about the evolution of cornification proteins, we screened for homologs of the EDC in non-mammalian vertebrates. By comparative genomics, de novo gene prediction and gene expression analyses, we show that, in contrast to fish and amphibians, the chicken and the green anole lizard have EDC homologs comprising genes that are specifically expressed in the epidermis and in skin appendages. Our data suggest that an important component of the cornified protein envelope of mammalian keratinocytes, that is, loricrin, has originated in a common ancestor of modern amniotes, perhaps during the acquisition of a fully terrestrial lifestyle. Moreover, we provide evidence that the sauropsid-specific beta-keratins have evolved as a subclass of EDC genes. Based on the comprehensive characterization of the arrangement, exon-intron structures and conserved sequence elements of EDC genes, we propose new scenarios for the evolutionary origin of epidermal barrier proteins via fusion of neighboring S100A and peptidoglycan recognition protein genes, subsequent loss of exons and highly divergent sequence evolution.

The appearance of hair is one of the main evolutionary innovations in the amniote lineage leading to mammals. The main components of mammalian hair are cysteine-rich type I and type II keratins, also known as hard α-keratins or “hair keratins.” To determine the evolutionary history of these important structural proteins, we compared the genomic loci of the human hair keratin genes with the homologous loci of the chicken and of the green anole lizard Anolis carolinenis . The genome of the chicken contained one type II hair keratin-like gene, and the lizard genome contained two type I and four type II hair keratin-like genes. Orthology of the latter genes and mammalian hair keratins was supported by gene locus synteny, conserved exon–intron organization, and amino acid sequence similarity of the encoded proteins. The lizard hair keratin-like genes were expressed most strongly in the digits, indicating a role in claw formation. In addition, we identified a novel group of reptilian cysteine-rich type I keratins that lack homologues in mammals. Our data show that cysteine-rich α-keratins are not restricted to mammals and suggest that the evolution of mammalian hair involved the co-option of pre-existing structural proteins.