Phase transitions in plant cuticles

The effect of temperature on wet plant cuticles has been investigated with the following techniques: Calorimetry, densitometry, spin-label electron-spin-resonance-(ESR)-spectroscopy, photo bleaching, and light and electron microscopy. At low temperatures cuticles ofCitrus aurantium L. andHedera helix show, at 16.3°C, a sharp transition (T0.5°C) with a latent heat of 4.7±0.5 J g^-1-cuticle. Below transition: The main orientation of the polymer matrix is parallel to the normal of the cuticle and the main orientation of the layer with soluble lipids is perpendicular to the normal. The cuticle is in a rigid state. Above transition (between 16.3°C and 38°C): Only the orientation of the polymer matrix has changed (tilted in parts). There exist several very sharp (T0.1°C) transitions (38°C, 41°C, 45°C, 49°C, ...) with a latent heat in the order of 0.4 J g^-1-cuticle. Above 38°C: The lamella of the soluble lipids is in a fluid state. Above 45°C there is a change in the molecular orientation of the soluble lipids as well as in the polymer matrix. The soluble lipids are mainly oriented parallel to the normal. The dry cuticles show no phase transition between 0°C and 200°C. At room temperature a dry/wet transition can be observed.Abbreviations ESR-spectroscopy electron-spin-resonance-spectroscopy     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

Iron-mediated oxidative stress plays an essential role in ferritin-induced cell death

Previously, we have demonstrated an apoptosis-inducing activity of an acidic, H-chain-rich isoferritin secreted from primary rat hepatocytes in vitro. Because this proapoptotic property may be responsible for the growth-inhibitory and immunosuppressive effects described for certain ferritin species, we aimed to address the mechanism by which ferritin can trigger cell death. Suggesting a pivotal role for iron, iron chelation by desferrioxamine significantly abrogates ferritin-mediated apoptosis and necrosis in primary rat hepatocytes and substantially lowers the extent of protein modification by 4-hydroxynonenal (HNE)—a major lipid peroxidation (LPO) product. Furthermore, supplementing the cultures with the radical-scavenging compound trolox also provided significant protection from ferritin-mediated apoptosis. Moreover, a significant increase in micronucleated cells upon exposure to ferritin indicates that ferritin also introduces damage to DNA. Based on these observations we therefore propose that endocytosis of extracellular ferritin increases the level of free ferrous iron in the lysosomal compartment, promoting Fenton chemistry-based oxidative stress involving LPO and increased lysosomal membrane permeability. Subsequently, the release of reactive lysosomal content leads to cellular damage, in particular modification of protein and DNA induced by HNE and other reactive aldehydic LPO products. Together, these effects will trigger apoptosis and necrosis based on the upregulation of p53, increased mitochondrial membrane permeability, and proapoptotic Fas signaling as described recently. In conclusion, based on their iron-storing ability, secreted acidic isoferritins may act as soluble mediators of oxidative stress under certain physiological and pathophysiological conditions.     

Role of epidermis-type lipoxygenases for skin barrier function and adipocyte differentiation

12R-lipoxygenase (12R-LOX) and epidermis-type LOX-3 (eLOX-3) are novel members of the multigene family of mammalian LOX. A considerable gap exists between the identification of these enzymes and their biologic function. Here, we present evidence that 12R-LOX and eLOX-3, acting in sequence, and eLOX-3 in combination with another, not yet identified LOX are critically involved in terminal differentiation of keratinocytes and adipocytes, respectively. Mutational inactivation of 12R-LOX and/or eLOX-3 has been found to be associated with development of an inherited ichthyosiform skin disorder in humans and genetic ablation of 12R-LOX causes a severe impairment of the epidermal lipid barrier in mice leading to post-natal death of the animals. In preadipocytes, a LOX-dependent PPARgamma activating ligand is released into the cell supernatant early upon induction of differentiation and available evidence indicates that this ligand is an eLOX-3-derived product. In accordance with this data is the observation that forced expression of eLOX-3 enhances adipocyte differentiation.     

Long-term evaluation of a selective retrograde coronary venous perfusion model in pigs (Sus scrofa domestica)

The lack of suitable target vessels remains a challenge for aortocoronary bypass grafting in end-stage coronary heart disease. This study aimed to investigate the arterialization of cardiac veins as an alternative myocardial revascularization strategy in an experimental long-term model in pigs. Selective retrograde perfusion of a coronary vein (aorta to coronary vein bypass, retrobypass) before ligation of the ramus interventricularis paraconalis (equivalent to the left anterior descending artery in humans) was performed in 20 German Landrace pigs (Sus scrofa domestica). Retroperfusion of the left anterior descending vein was performed in 10 pigs (RP+) but not in the other 10 (RP-), and the vena cordis magna was ligated (L+) in 5 pigs in each of these groups but left open (L-) in the remaining animals. Hemodynamic performance (for example, cardiac output) was significantly better in the group that underwent selective retroperfusion with proximal ligation of vena cordis magna (RP+L+; 4.1 L/min) compared with the other groups (RP+L-, 2.5 L/min; RP-L+, 2.2 L/min; RP-L-, 1.9 L/min). Long-term survival was significantly better in RP+L+ pigs (112±16 d) than in all other groups. Histologic follow-up studies showed significantly less necrosis in the RP+L+ group compared with all other groups. Venous retroperfusion is an effective technique to achieve long-term survival after acute occlusion of the left anterior descending artery in a pig model. In this model, proximal ligation of vena cordis magna is essential.     

Effect of Oxidative Stress on the Junctional Proteins of Cultured Cerebral Endothelial Cells

Summary 1. There is increasing evidence that the cerebral endothelium and the blood–brain barrier (BBB) plays an important role in the oxidative stress-induced brain damage. The aim of the present study was to investigate the role of interendothelial junctional proteins in the BBB permeability increase induced by oxidative stress.2. For the experiments, we have used cultured cerebral endothelial cells exposed to hypoxia/reoxygenation or treated with the redox cycling quinone 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of glucose. The expression of junctional proteins and activation of mitogen activated protein kinases (MAPK) was followed by Western-blotting, the interaction of junctional proteins was investigated using coimmunoprecipitation.3. Oxidative stress induces a downregulation of the tight junction protein occludin expression which is more pronounced in the absence of glucose. Furthermore, oxidative stress leads to disruption of the cadherin--catenin complex and an activation of extracellular signal-regulated kinase (ERK1/2), which is more intense in the absence of glucose.4. We have shown that one of the causes of the BBB breakdown is probably the structural alteration of the junctional complex caused by oxidative stress, a process in which ERK1/2 may play an important role.This revised article was published online in May 2005 with a February 2005 cover date.     

Krankheitsspezifische Hautmodelle

A variety of skin equivalent systems have been developed recently mainly for burn therapy but also for studies of the cell and molecular biology of dermatologic and immunologic disorders and for cosmetic and pharmaceutical research. Since European regulation forbids the use of animals to prove product safety in cosmetic products, several commercially available three-dimensional skin models were developed by the cosmetic and chemical industry and validated according to OECD and ECVAM regulations. Three-dimensional skin models consist of two compartments: one serves as a dermal equivalent, usually consisting of fibroblasts in type I collagen, onto which a terminally differentiating epidermis is placed. Up-to-date models are missing that mimic monogenic skin disorders or signs of disease in the skin caused by a systemic autoimmune disorder. We recently developed a three-dimensional skin model for congenital ichthyosis as an example for a keratinization disorder. The system is being validated and will be fundamental for studies of disturbed epidermal differentiation and pharmaceutical intervention.     

Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion

Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of nonerythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homozygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prominent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis.