Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales.

The growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.

Development and characterization of specific anti-Usutu virus chicken-derived single chain variable fragment antibodies.

Usutu virus belongs to the Japanese encephalitis serogroup within the Flaviviridae family. Mammals may become incidental hosts after the bite of an infected mosquito while birds act as the main reservoir. Human cases have become more common recently and elicit various outcomes ranging from asymptomatic to severe illness including encephalitis. Problematically, antisera against Usutu virus cross-react with other flaviviruses such as the co-circulating West Nile virus. As an approach to generate Usutu virus-specific antibodies, we immunized chickens with purified Usutu virus envelope protein domain III, isolated the spleen mRNA and generated an scFv phage display library. The most potent binders for Usutu virus domain III were selected via biopanning and their affinity to domain III was examined using SPR. Four scFvs bound the domain III of Usutu virus in the nanomolar region; two bound the protein over 40 times more strongly than West Nile virus domain III. We further characterized these scFv antibodies for suitability in standard laboratory tests such as western blots, ELISA, and neutralization tests. Four specific and one cross-reactive antibody performed well in western blots with domain III and the full-length envelope protein of Usutu virus and West Nile virus. All antibodies bound in virus ELISA assays to Usutu virus strain Vienna-2001. However, none of the antibodies neutralized either Usutu virus or West Nile virus. These antibody candidates could be crucial in future diagnostic tests to distinguish Usutu virus from other flaviviruses and might even offer virus neutralization after a conversion to Fab or IgG.

Convergent Evolution of Cysteine-Rich Keratins in Hard Skin Appendages of Terrestrial Vertebrates.

Terrestrial vertebrates have evolved hard skin appendages, such as scales, claws, feathers, and hair that play crucial roles in defense, predation, locomotion, and thermal insulation. The mechanical properties of these skin appendages are largely determined by cornified epithelial components. So-called "hair keratins," cysteine-rich intermediate filament proteins that undergo covalent cross-linking via disulfide bonds, are the crucial structural proteins of hair and claws in mammals and hair keratin orthologs are also present in lizard claws, indicating an evolutionary origin in a hairless common ancestor of amniotes. Here, we show that reptiles and birds have also other cysteine-rich keratins which lack cysteine-rich orthologs in mammals. In addition to hard acidic (type I) sauropsid-specific (HAS) keratins, we identified hard basic (type II) sauropsid-specific (HBS) keratins which are conserved in lepidosaurs, turtles, crocodilians, and birds. Immunohistochemical analysis with a newly made antibody revealed expression of chicken HBS1 keratin in the cornifying epithelial cells of feathers. Molecular phylogenetics suggested that the high cysteine contents of HAS and HBS keratins evolved independently from the cysteine-rich sequences of hair keratin orthologs, thus representing products of convergent evolution. In conclusion, we propose an evolutionary model in which HAS and HBS keratins evolved as structural proteins in epithelial cornification of reptiles and at least one HBS keratin was co-opted as a component of feathers after the evolutionary divergence of birds from reptiles. Thus, cytoskeletal proteins of hair and feathers are products of convergent evolution and evolutionary co-option to similar biomechanical functions in clade-specific hard skin appendages.

Immunolocalization and phylogenetic profiling of the feather protein with the highest cysteine content.

Feathers are the most complex skin appendages of vertebrates. Mature feathers consist of interconnected dead keratinocytes that are filled with heavily cross-linked proteins. Although the molecular architecture determines essential functions of feathers, only few feather proteins have been characterized with regard to their amino acid sequences and evolution. Here, we identify Epidermal Differentiation protein containing DPCC Motifs (EDDM) as a cysteine-rich protein that has co-evolved with other feather proteins. The EDDM gene is located within the avian epidermal differentiation complex (EDC), a cluster of genes that has originated and diversified in amniotes. EDDM shares the exon-intron organization with EDC genes of other amniotes, including humans, and a gene encoding an EDDM-like protein is present in crocodilians, suggesting that avian EDDM arose by sequence modification of an epidermal differentiation gene present in a common ancestor of archosaurs. The EDDM protein contains multiple sequence repeats and a higher number of cysteine residues than any other protein encoded in the EDC. Immunohistochemical analysis of chicken skin and skin appendages showed expression of EDDM in barb and barbules of feathers as well as in the subperiderm on embryonic scutate scales. These results suggest that the diversification and differential expression of EDDM, besides other EDC genes, was instrumental in facilitating the evolution of the most complex molecular architecture of feathers.

Oleic acid induces the novel apolipoprotein O and reduces mitochondrial membrane potential in chicken and human hepatoma cells.

Non-alcoholic fatty liver disease (NAFLD) is marked by hepatic fat accumulation and reflects a spectrum of chronic liver diseases associated with obesity, impaired insulin sensitivity and dyslipidemia. Apolipoprotein O (ApoO) is a new member of the plasma apolipoprotein family that may play a role in lipid metabolism and electron transport activity of the mitochondrium. However, its physiological functions have not been elucidated yet. Based on our previous data in a non-mammalian experimental system [1], we hypothesized that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to increased lipoprotein-production induced by, e.g., hormones and oxidative stress. To gain insight into a mammalian experimental system, we compared the effects of lipid loading on ApoO regulation in chicken hepatoma LMH cells with those in the human hepatoma cell line HepG2. Incubation of the cells with BSA-complexed oleic acid (OA-Alb) induced triglyceride accumulation, but did not affect cell viability. qPCR using specific primer pairs and Western blot analysis with in-house produced rabbit anti-ApoO antisera demonstrated significant increase in ApoO transcript and protein levels in both cell lines. ROS formation due to OA-Alb treatment was only slightly altered in LMH cells, indicating an intact antioxidant defense system of the cells. Oxidative stress applied by addition of H2O2 revealed induction of ApoO transcript and protein level in the same or even higher extent as monitored in the presence of OA-Alb. Upon treatment with estrogen for 24 h quantitative analysis of ApoO transcript and protein revealed increases of ApoO expression supporting the assumption that estrogen affects lipoprotein metabolism at various points. Furthermore, both cell lines showed a significant decrease of the mitochondrial membrane potential upon incubation with OA-Alb. Therefore, we assume that our findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of non-alcoholic fatty liver disease.

Estrogen-enhanced apical and basolateral secretion of apolipoprotein B-100 by polarized trophoblast-derived BeWo cells.

Cholesterol is an important nutrient for fetal development and transplacental transport occurs at all stages of human pregnancy. Furthermore, cholesterol is required for membrane building as well as steroid hormone synthesis. Therefore, all placental cell types require cholesterol for proper function. In human term placenta, the syncytiotrophoblast (STB) faces the maternal circulation. Uptake of maternal-derived cholesterol at the apical membrane of the STB is well understood, but the route by which cholesterol exits at the basal side for subsequent transfer across the fetal endothelial cells (FEC) or to other placental cell types remains not well characterized. Our aim was to provide evidence for basal secretion of apolipoprotein B-100 (apoB) containing lipoproteins. Furthermore, we investigated the placental localization of apolipoprotein receptors (LRP2, LDLR and LRP1) to identify cell targets of lipoprotein particles secreted in a polarized fashion by the STB. In trophoblast-derived BeWo cells grown on permeable filter supports, we demonstrate by immunoprecipitation apical as well as basolateral apoB secretion, which was significantly upregulated by estrogen-treatment for 24 or 48 h. Furthermore, we showed by immunofluorescence microscopy apoB and microsomal triglyceride transfer protein subunits localization in the STB and placental stromal cells in situ. All investigated receptors were detected by RT-qPCR and western blot in BeWo cells, but only expression of LRP2 was estrogen-inducible. In situ, the multi-ligand receptor LRP2 was expressed exclusively in the cytotrophoblast (CTB), the STB precursor cell type. LDLR and LRP1 localized to trophoblasts as well as stromal cells in situ. In summary, basal apoB secretion by BeWo cells supports the concept of basal lipoprotein particle secretion by placental STB. These lipoprotein particles may serve as cholesterol source for STB precursor cells, the CTBs, as well as all stromal cells of the chorionic villi including FECs, which were herein demonstrated to express apoB receptors, LRP2 and LDLR, respectively.

Immunolocalization of a Histidine-Rich Epidermal Differentiation Protein in the Chicken Supports the Hypothesis of an Evolutionary Developmental Link between the Embryonic Subperiderm and Feather Barbs and Barbules.

The morphogenesis of feathers is a complex process that depends on a tight spatiotemporal regulation of gene expression and assembly of the protein components of mature feathers. Recent comparative genomics and gene transcription studies have indicated that genes within the epidermal differentiation complex (EDC) encode numerous structural proteins of cornifying skin cells in amniotes including birds. Here, we determined the localization of one of these proteins, termed EDMTFH (Epidermal Differentiation Protein starting with a MTF motif and rich in Histidine), which belongs to a group of EDC-encoded proteins rich in aromatic amino acid residues. We raised an antibody against an EDMTFH-specific epitope and performed immunohistochemical investigations by light microscopy and immunogold labeling by electron microscopy of chicken embryos at days 14-18 of development. EDMTFH was specifically present in the subperiderm, a transient layer of the embryonic epidermis, and in barbs and barbules of feathers. In the latter, it partially localized to bundles of so-called feather beta-keratins (corneous beta-proteins, CBPs). Cells of the embryonic periderm, the epidermis proper, and the feather sheath were immunonegative for EDMTFH. The results of this study indicate that EDMTFH may contribute to the unique mechanical properties of feathers and define EDMTFH as a common marker of the subperiderm and the feather barbules. This expression pattern of EDMTFH resembles that of epidermal differentiation cysteine-rich protein (EDCRP) and feather CBPs and is in accordance with the hypothesis that a major part of the cyclically regenerating feather follicle is topologically, developmentally and evolutionarily related to the embryonic subperiderm.

Hepatosteatosis and estrogen increase apolipoprotein O production in the chicken.

Apolipoprotein O (ApoO) is a recently discovered plasma apolipoprotein that may also play a role in the mitochondrial inner membrane. Possibly due to this complexity, its physiological functions have not been elucidated yet. To gain insight from a non-mammalian experimental system, we have investigated the regulation of ApoO levels in an alternative, well-suited model for studies on lipid metabolism, the chicken. qPCR using specific primer pairs and Western blot analysis with our rabbit anti-chicken ApoO antiserum demonstrated ApoO in the liver of chickens fed a control or a fat-enriched diet, as well as in 2 chicken hepatoma cell lines, LMH cells and the estrogen-responsive LMH-2A cells, under conditions of lipid loading by incubation with BSA-complexed oleic acid. Induced triglyceride accumulation in both the liver and the hepatic cells was associated with significantly increased levels of ApoO mRNA and protein. Furthermore, upon treatment for 24 h with estrogen of the estrogen receptor-expressing LMH-2A cells, quantitative analysis of ApoO transcripts and Western blotting revealed increases of ApoO expression. Finally, upon a single administration of estrogen to roosters that leads to hyperlipidemia, higher hepatic levels of both ApoO transcript and protein were observed within 24 h. Based on these data, we propose that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to increased lipoprotein-production induced by, e.g., hormones. The findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of non-alcoholic fatty liver disease.

Convergent evolution of cysteine-rich proteins in feathers and hair.

BACKGROUND: Feathers and hair consist of cornified epidermal keratinocytes in which proteins are crosslinked via disulfide bonds between cysteine residues of structural proteins to establish mechanical resilience. Cysteine-rich keratin-associated proteins (KRTAPs) are important components of hair whereas the molecular components of feathers have remained incompletely known. Recently, we have identified a chicken gene, named epidermal differentiation cysteine-rich protein (EDCRP), that encodes a protein with a cysteine content of 36%. Here we have investigated the putative role of EDCRP in the molecular architecture and evolution of feathers. RESULTS: Comparative genomics showed that the presence of an EDCRP gene and the high cysteine content of the encoded proteins are conserved among birds. Avian EDCRPs contain a species-specific number of sequence repeats with the consensus sequence CCDPCQ(K/Q)(S/P)V, thus resembling mammalian cysteine-rich KRTAPs which also contain sequence repeats of similar sequence. However, differences in gene loci and exon-intron structures suggest that EDCRP and KRTAPs have not evolved from a common gene ancestor but represent the products of convergent sequence evolution. mRNA in situ hybridization demonstrated that chicken EDCRP is expressed in the subperiderm layer of the embryonic epidermis and in the barbule cells of growing feathers. This expression pattern supports the hypothesis that feathers are evolutionarily derived from the subperiderm. CONCLUSIONS: The results of this study suggest that convergent sequence evolution of avian EDCRP and mammalian KRTAPs has contributed to independent evolution of feathers and hair, respectively.

Estrogen enhances secretion of apolipoprotein B-100 containing lipoproteins by BeWo cells.

Although the early human embryo is capable of covering its cholesterol demand by endogenous synthesis, during later stages of development the fetus may become dependent on transplacental cholesterol transport. On one hand, this conclusion is based on the severe developmental abnormalities of embryos with mutations in the gene specifying the enzyme catalyzing the last step of cholesterol synthesis, 7-dehydrocholesterol reductase, causing Smith-Lemli-Opitz Syndrome. On the other hand, increased total maternal plasma cholesterol levels may reflect the requirement by the growing fetus and/or the placenta for cholesterol. Various molecules and complexes must cross the placental barrier consisting of trophoblasts and fetal endothelial cells to reach the fetal circulation. The de novo synthesis of apolipoprotein B (apoB)-containing lipoproteins coupled to secretion from trophoblasts towards the fetal side is one efficient pathway for cholesterol supply. ApoB and the microsomal triglyceride transfer protein (MTP) are essential components for the assembly of apoB-containing lipoproteins. The aim of this study was to evaluate functional properties of the human placental cell line BeWo as an in vitro model for placental synthesis of apoB-containing lipoproteins by focusing on components required for lipoprotein assembly and secretion. We demonstrate mRNA and protein production of apoB-100, MTP, and protein disulfide isomerase (PDI) in BeWo cells. In addition, metabolic radiolabeling and apoB-immunoprecipitation of cell extracts and media revealed that synthesis and secretion of apoB-containing lipoproteins are enhanced by estrogen. The expression of apoB-100, MTP, and PDI, and the estrogen-stimulated lipoprotein secretion by BeWo cells suggest that these cells are a useful system to study aspects of lipoprotein metabolism at the placental barrier.

Trichohyalin-like proteins have evolutionarily conserved roles in the morphogenesis of skin appendages.

S100 fused-type proteins (SFTPs) such as filaggrin, trichohyalin, and cornulin are differentially expressed in cornifying keratinocytes of the epidermis and various skin appendages. To determine evolutionarily conserved, and thus presumably important, features of SFTPs, we characterized nonmammalian SFTPs and compared their amino acid sequences and expression patterns with those of mammalian SFTPs. We identified an ortholog of cornulin and a previously unknown SFTP, termed scaffoldin, in reptiles and birds, whereas filaggrin was confined to mammals. In contrast to mammalian SFTPs, both cornulin and scaffoldin of the chicken are expressed in the embryonic periderm. However, scaffoldin resembles mammalian trichohyalin with regard to its expression in the filiform papillae of the tongue and in the epithelium underneath the forming tips of the claws. Furthermore, scaffoldin is expressed in the epithelial sheath around growing feathers, reminiscent of trichohyalin expression in the inner root sheath of hair. The results of this study show that SFTP-positive epithelia function as scaffolds for the growth of diverse skin appendages such as claws, nails, hair, and feathers, indicating a common evolutionary origin.

Carbamoylation abrogates the antioxidant potential of hydrogen sulfide.

Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein.

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.

The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of its endodermal epithelial cells.

During chicken yolk sac (YS) growth, mesodermal cells in the area vasculosa follow the migrating endodermal epithelial cell (EEC) layer in the area vitellina. Ultimately, these cells form the vascularized YS that functions in nutrient transfer to the embryo. How and when EECs, with their apical aspect directly contacting the oocytic yolk, acquire the ability to take up yolk macromolecules during the vitellina-to-vasculosa transition has not been investigated. In addressing these questions, we found that with progressive vascularization, the expression level in EECs of the nutrient receptor triad, LRP2-cubilin-amnionless, changes significantly. The receptor complex, competent for uptake of yolk proteins, is produced by EECs in the area vasculosa but not in the area vitellina. Yolk components endocytosed by LRP2-cubilin-amnionless, preformed and newly formed lipid droplets, and yolk-derived very low density lipoprotein, shown to be efficiently endocytosed and lysosomally processed by EECs, probably provide substrates for resynthesis and secretion of nutrients, such as lipoproteins. In fact, as directly demonstrated by pulse-chase experiments, EECs in the vascularized, but not in the avascular, region efficiently produce and secrete lipoproteins containing apolipoprotein A-I (apoA-I), apoB, and/or apoA-V. In contrast, perilipin 2, a lipid droplet-stabilizing protein, is produced exclusively by the EECs of the area vitellina. These data suggest a differentiation process that orchestrates the vascularization of the developing YS with the induction of yolk uptake and lipoprotein secretion by EECs to ensure embryo nutrition.

Renal LRP2 expression in man and chicken is estrogen-responsive.

In mammals, low-density lipoprotein receptor-related protein-2 (LRP2) is an endocytic receptor that binds multiple ligands and is essential for a wide range of physiological processes. To gain new insights into the biology of this complex protein, we have initiated the molecular characterization of the LRP2 homolog from an oviparous species, the chicken (Gallus gallus). The galline LRP2 cDNA encodes a membrane protein of 4658 residues. Overall, the galline and human proteins are 73% identical, indicating that the avian gene has been well conserved over 300 million years. Unexpectedly, LRP2 transcript and protein levels in the kidney of females and estrogen-treated roosters were significantly higher than those in untreated males. The estrogen-responsiveness of avian LRP2 may be related to the dramatic differences in lipoprotein metabolism between mature roosters and laying hens. Newly identified potential estrogen-responsive elements (ERE) in the human and galline LRP2 gene, and additional Sp1 sites present in the promoter of the chicken gene, are compatible with both direct estrogen induction via the classical ligand-induced ERE pathway and the indirect transcription factor crosstalk pathway engaging the Sp1 sites. In agreement with this assumption, estrogen induction of LRP2 was observed not only in primary cultured chicken kidney cells, but also human kidney cell lines. These findings point to novel regulatory features of the LRP2 gene resulting in sex-specific receptor expression.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia.

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.

Scavenger receptor, Class B, Type I provides an alternative means for beta-VLDL uptake independent of the LDL receptor in tissue culture.

Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate beta-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from beta-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of beta-VLDL was approximately 3 times higher after SR-BI over-expression, which was competed by beta-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated beta-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled beta-VLDL. beta-VLDL was degraded at a rate of 6 ng beta-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, beta-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in beta-VLDL uptake. beta-VLDL degradation was similar to ldlA7-SRBI cells. When beta-VLDL uptake was followed using fluorescence microscopy, beta-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.

Hydrogen sulfide scavenges the cytotoxic lipid oxidation product 4-HNE.

Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.

Identification of reptilian genes encoding hair keratin-like proteins suggests a new scenario for the evolutionary origin of hair.

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 alpha-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 alpha-keratins are not restricted to mammals and suggest that the evolution of mammalian hair involved the co-option of pre-existing structural proteins.

Deregulation of the activin/follistatin system in hepatocarcinogenesis.

BACKGROUND/AIMS: Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to investigate, whether activins and follistatins may play a role in hepatocarcinogenesis. METHODS: Expression levels of follistatin, follistatin-like 3, and activin subunits beta(A) as well as beta(E) were investigated in chemically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed. RESULTS: Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in contrast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic hepatocytes stimulated DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it. CONCLUSIONS: The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for therapeutic intervention.