Role of illness representations and coping in patients with atopic dermatitis: a cross-sectional study.

BACKGROUND: Atopic dermatitis (AD) is a skin disease accompanied by psychological burden. It has been shown for other chronic diseases that illness representations and coping strategies are associated with disease-related burden and other outcome variables like time until patients return to work or health care use. OBJECTIVE: The goal of this cross-sectional study was to investigate whether illness representations and coping strategies are correlated with the severity of AD and self-rated physical impairment of the patients. METHODS: A total of 109 AD patients were examined at the beginning of their stay at a rehabilitation centre. They filled in validated questionnaires to measure illness perceptions (IPQ), coping strategies (EBS) and self-rated physical well-being (FEW). In addition, the severity of AD (SCORAD) was determined by a doctor. RESULTS: Linear regression analysis revealed that a considerable amount of the variance in self-rated physical well-being (51%) could be predicted by particular illness perceptions and coping. Subsequent multiple mediation analyses indicated that certain coping strategies (active problem solving and depressive reactions) mediated the effect of illness representations on self-rated physical well-being. In contrast, only 7.4% of the SCORAD could be predicted by the IPQ scale illness identity. CONCLUSION: This study showed that illness representations and coping are highly associated with self-rated physical impairment in AD patients. Therefore, this patient group might profit from cognitive behavioural interventions designed to alter patients' illness perceptions. The hypothesis that a modification in illness perceptions leads to a faster recovery and a more rapid return to work should be tested in future randomized controlled trials.

Quantification of ZP1, ZP2 and ZP3 mRNA of marmoset monkey (Callithrix jacchus) oocytes from periantral and antral follicles.

In mammals, the oocyte and preimplantation embryo are protected by the zona pellucida (ZP) consisting mainly of ZP glycoproteins, which are responsible for sperm binding, induction of the acrosome reaction and zona pellucida hardening to prevent polyspermia. The ZP proteins become increasingly important as possible predictors for in vitro cultured oocytes competence. As little is known about the stage-dependent expression of ZP1, ZP2 and ZP3 in marmoset monkey (Callithrix jacchus) oocytes, mRNA expression was investigated with real-time RT-PCR. Total-RNA was isolated from three different classes of marmoset oocytes; Class 1 oocytes from periantral follicles (<600 µm, n = 10), Class 2 oocytes from small antral follicles (600-1000 µm, n = 10) and Class 3 oocytes from large antral follicles (>1000 µm, n = 9). Compared with Class 1 oocytes mRNA expression of ZP1, ZP2 and ZP3 in Class 2 oocytes was significantly decreased. In Class 3 oocytes, the transcription of ZP1, ZP2 and ZP3 genes showed also a significant decrease compared with Class 1 oocytes. In this study a differently regulated expression of the ZP genes during late folliculogenesis with an obvious downregulation of ZP1, ZP2 and ZP3 could be demonstrated for the first time in the marmoset monkey.

[International Classification of Function, Disability and Health (ICF). Basis for cutaneous rehabilitation management]. / Internationale Klassifikation der Funktionsfähigkeit, Behinderung und Gesundheit (ICF). Grundlage des Handelns und Denkens in der dermatologischen Rehabilitation.

Functioning and health are central issues in medical rehabilitation. With the International Classification of Functioning, Disability and Health (ICF), the World Health Organization implemented in 2001 a system for the description and classification of functioning that is accepted worldwide. Applying the bio-psycho-social perspective, this model based on interdependencies, comprises the components Body Function, Body Structure and Activity and Participation as well as contextual influences of health: the Environmental and Personal factors. The ICF is of prime importance in clinical practice, teaching, and research. It provides a universal language to be used in health care permitting the characterization of the specific functional problems of individuals. The rehabilitation of skin diseases takes advantage on the ICF in rehabilitation management. In this paper we describe the philosophy of the ICF and its implementation in rehabilitation of patients with chronic skin problems on the basis of a case study.

VDAC2 (porin-2) expression pattern and localization in the bovine testis.

In this study, sequencing of voltage-dependent anion channel 2 (VDAC2, porin-2) cDNA from bovine testis is reported. High identity to the murine, rabbit, and human subtypes at both the nucleotide and amino acid levels is demonstrated. mRNA analysis revealed expression of VDAC2 in bovine testis, whereas high levels of VDAC2 proteins were found in late spermatocytes, spermatids, and spermatozoa. In contrast, VDAC1 (porin-1) is exclusively localized in Sertoli cells. The possible role of testicular VDAC2 in providing energy metabolites and in germ cell apoptosis is discussed.

Tissue distribution of beta 1- and beta 2-subunits of regulatory guanine nucleotide-binding proteins.

The beta-subunit of G-proteins occurs in two forms (beta 1 and beta 2), which differ in their primary structure as derived from cDNA clones and in their mobilities on SDS gels (36 and 35 kDa, respectively). To assess the tissue distribution of the two forms of beta-subunits, we synthesized peptides corresponding to defined regions of beta 1- and beta 2-subunits and injected them into rabbits; the antisera obtained reacted either with both beta-subunits or specifically with the beta 1- or the beta 2-subunit. They were used to identify the two beta-subunits in membranes prepared from various rat tissues and from human placenta. The concentration of total beta-subunits was high in rat brain and lung, human placenta, rat kidney, liver and spleen; it was much lower in rat erythrocytes, cardiac and skeletal muscle. In all tissues studied, both beta 1- and beta 2-subunits were detectable. In most tested tissues, the two forms were about equally distributed, whereas in the placenta, the beta 2-subunit was found to occur in approx. 2-fold excess over the beta 1-subunit. Our results demonstrate that both beta-subunits are widely distributed. In the majority of tissues, levels of beta 2-subunits are very similar to those of beta 1-subunits. Thus, the abundance of beta 2-subunits as compared to that of the beta 1-subunit is considerably higher than was previously estimated by measuring the respective mRNA levels.

A synthetic decapeptide from a conserved ZP3 protein domain induces the G protein-regulated acrosome reaction in bovine spermatozoa.

In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm-oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm-egg interaction in bovine and other species.

Stimulatory effect of insulin and insulin-like growth factor I on Gi proteins and angiotensin-II-induced phosphoinositide breakdown in cultured bovine adrenal cells.

Recent data have shown that pretreatment of bovine adrenal fasciculata cells with insulin-like growth factor I (IGF-I) or insulin enhances the steroidogenic response to angiotensin II (A-II). In the present work we have studied the effects of both peptides on the first steps of the mechanism of action of A-II and on the amounts of pertussis toxin (PT)-sensitive guanine nucleotide binding proteins (Gi proteins). Both peptides increased A-II-induced phosphoinositide breakdown without modification of either A-II-induced Ca2+ uptake or the A-II-potentiating effect on ACTH-induced cAMP production. The effects of IGF-I at a nanomolar concentration were higher than those induced by insulin at a micromolar concentration, which in turn was higher than those induced by a nanomolar concentration of this peptide. Treatment of cells with pertussis toxin (0.5 microgram/ml) for 24 h reduced by 25% of the A-II-induced phosphoinositide breakdown in control cells and 32% and 28% in cells pretreated with insulin at nanomolar and micromolar concentrations, respectively, but had no significant effect in cells pretreated with IGF-I. No effect of pertussis toxin was observed on A-II-induced Ca2+ uptake or on its potentiating action on ACTH-induced cAMP production. Moreover, both IGF-I and insulin enhanced the amounts of Gi protein(s) evaluated by pertussis toxin ADP-ribosylation or immunoblotting. Again, the effects of insulin at nanomolar concentrations were lower than those induced by the same concentrations of IGF-I or insulin at micromolar concentrations. These results suggest that, in bovine adrenal fasciculata cells, A-II receptors are coupled to the phosphoinositide pathway through pertussis toxin sensitive and insensitive Gp protein(s). Moreover, the findings also indicate that the enhanced A-II responsiveness of IGF-I or insulin treated cells is in part mediated through an increase in the amount of G protein(s).

Differential sensitivity to guanine nucleotides of basal and insulin-stimulated glucose transporter activity reconstituted from adipocyte membrane fractions.

The effects of GTP gamma S on glucose transport activity reconstituted from adipocyte membrane fractions were studied in order to test the hypothesis that intrinsic activity changes of the insulin-sensitive glucose transporter may be mediated by guanine nucleotide-dependent mechanisms. GTP gamma S and GTP inhibited reconstituted glucose transport activity by 50% in membrane fractions from insulin-treated cells in a concentration-dependent manner; no inhibitory effect was observed in membrane fractions obtained from basal cells. GDP, GMP and guanosine were less effective than GTP, whereas the adenine nucleotides ATP gamma S and AMP failed to reduce the reconstituted transport activity. The data indicate that guanine nucleotides may modulate the activity of the adipocyte glucose transporter. Since the effect is dependent on treatment of cells with insulin, the hormone appears to induce a specific functional alteration of the glucose transporter.

ADP-ribosylation of Rho proteins inhibits sperm motility.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells.

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, Gi2, requires Mg2+ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go.

Antisera generated against synthetic peptides were used to identify G-protein alpha-subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs alpha-subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi alpha-subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2 alpha-subunit. A serum specific for the Go alpha-subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go.

The primary structure of the 70 kDa subunit of bovine soluble guanylate cyclase.

The primary structure of the 70 kDa subunit of soluble bovine guanylate cyclase, which catalyzes the formation of cyclic GMP from GTP, has been determined. The alignment of six different clones out of two bovine libraries yielded a total of 3.1 kb with a coding region of 1857 bases. The open reading frame encodes a protein of 619 amino acids and a molecular mass of 70.5 kDa. Antibodies raised against a synthetic peptide, which corresponded to the C-terminus of the deduced sequence precipitated guanylate cyclase activity from guanylate cyclase-enriched preparations.

G-protein alpha-subunits in cytosolic and membranous fractions of human neutrophils.

In plasma membranes of human neutrophils, we identified two major pertussis toxin substrates of 40 kDa Mr with pI values of 5.30 and 5.37. Only the acidic of the two substrates was also present in neutrophil cytosol. Two-dimensional tryptic peptide maps revealed a high degree of homology of cytosolic and particulate substrates. Purified G-protein beta gamma-complex stimulated pertussis toxin-catalyzed [32P]ADP-ribosylation of membranous and cytosolic substrates of neutrophils less than 2-fold and 6-fold, respectively. Hydrodynamic properties of the cytosolic substrate strongly suggested that it exists as a monomer. Purified G-protein beta gamma-complex increased the s20,w value of the cytosolic substrate from 3.3 S to 4.0 S. The GTP analogue, guanosine 5'-O-(3-thiotriphosphate), promoted the release of pertussis toxin substrates from plasma membranes. An antiserum raised against a sequence specific for the Gi2 alpha-subunit reacted with 39-40 kDa proteins in plasma membranes and with an apparently single 40 kDa protein in cytosol. We conclude that neutrophil cytosol contains monomeric Gi2 alpha-subunits which--by interacting with hydrophobic beta gamma-complexes--may reversibly bind to the plasma membrane.

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats.

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.

Phosphorylation of G-protein alpha-subunits in intact adipose cells: evidence against a mediating role in insulin-dependent metabolic effects.

The phosphorylation of G-protein alpha-subunits was studied in plasma membranes prepared from isolated, intact adipocytes equilibrated with [32P]phosphate and subsequently incubated in the presence or absence of insulin. In iodinated or unlabeled plasma membranes, antiserum generated against a peptide corresponding to a region common to G-protein alpha-subunits immunoprecipitated two major proteins of 45 and 40 kDa, which were identified as Gs and Gi alpha-subunit, respectively, by comparison with [32P]ADP-ribosylated G-proteins. In membranes prepared from cells equilibrated with [32P]phosphate, the antiserum precipitated a 45 kDa phosphoprotein. Pre-immune serum failed to immunoprecipitate the phosphoprotein. Insulin stimulated [32P]phosphate incorporation into the 45 kDa protein approximately 2-fold. Control experiments suggested that the 45 kDa phosphoprotein was not identical with G alpha s, since (1) the peptide used to raise the antiserum failed to inhibit significantly immunoprecipitation of the 45 kDa phosphoprotein with the antiserum, (2) in contrast to the Gs alpha-subunit, the phosphoprotein was readily removed from the immunocomplex by washing with sodium dodecyl sulfate (SDS), and (3) the subcellular localization of the phosphoprotein differed considerably from that of the Gs alpha-subunit. No phosphate was detected in immunoprecipitates from either basal or insulin-treated cells after the 45 kDa phosphoprotein had been removed. These data argue against a mediating role of phosphorylated G-protein alpha-subunits in the action of insulin.

Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function.

OBJECTIVE: To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. DESIGN: Prospectively designed, blinded, internally controlled study. SETTING: Tertiary care academic center. PATIENTS: Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. INTERVENTIONS: Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. RESULTS: A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6-treated hemizonae than patients with sperm factor. CONCLUSIONS: These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

The zona pellucida "receptors".

Binding of mammalian sperm to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. In the mouse model, the zona pellucida consists of three sulfated glycoproteins, ZP1, ZP2, and ZP3. Zona pellucida proteins are secreted to form a filamentous zona matrix in which ZP2 and ZP3 complex into co-polymers cross-linked by ZP1. ZP3 is the ligand for primary sperm binding and important for the induction of the acrosome reaction. The zona pellucida glycoprotein ZP2 is also crucially involved in the process of fertilization. Previous reports suggest that ZP2 mediates secondary binding of spermatozoa and that cleavage of ZP2 by proteases released through cortical granule reaction causes zona "hardening" and thus prevents polyspermy. Human and mouse ZP2 proteins differ in the primary structure as derived from cDNA clones. We designed an immunological approach to search for ZP2 domains with functional relevance. Antisera were generated against synthetic peptides derived (a) from ZP2 amino acid sequences that are homologous in human and mouse ZP2 amino acid sequences (AS ZP2-20) or (b) from human ZP2 amino acid sequences that differ from the mouse ZP2 sequence (AS ZP2-26). Immunochemical studies with microbisected bovine zonae pellucidae demonstrated that both antisera, AS ZP2-20 and AS ZP2-26, specifically detected ZP2 protein. Using the competition-hemizona-assay, sperm binding to antibody treated bovine hemizonae pellucidae were compared with control hemizonae (given as hemizona index). Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae (p < 0.0001), whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-egg interaction. Our results show that antibodies against ZP2 peptides react with bovine zonae pellucidae and can be used as markers for ZP2. Furthermore, AS ZP2-20 identifies a ZP2 epitope that is possibly of functional relevance for sperm-egg interaction.

Holographic interferometry of surface deformations of transparent fluids.

Holographic interferometry is employed to study deformations of a water surface by various disturbances. By using nondiffuse illumination and observing fringe patterns in the holographically reconstructed real image, high resolution even in the presence of large gradients in the surface deformation is achieved. Mathematical procedures for evaluating the fringe patterns are outlined. Several interesting applications (determination of surface tension, surface deformation by floating bodies) demonstrate the accuracy and versatility of the method.

Generation of antisera against mouse and human synthetic ZP3 peptides.

The ZP3 protein is a zona pellucida glycoprotein which plays a major role in sperm binding and induction of the acrosome reaction. ZP3 proteins occur in various mammalian zonae pellucidae; their primary structures are highly conserved as revealed by cDNA cloning. We generated antisera against synthetic peptides that are specific either for mouse ZP3 (AS ZP3-9) or for human ZP3 (AS ZP3-14). The antisera specifically recognized their respective peptide employed as immunogen and did not cross-react with control peptides. AS ZP3-9 detected ZP3 protein in isolated mouse zona pellucida and oocytes, whereas AS ZP3-14 did not react with proteins in murine egg cells or zona pellucida. Our results indicate that antisera against synthetic ZP3 peptides can be used as specific markers for the identification of individual ZP3 proteins in mouse and human oocytes. The antisera might be useful tools for the evaluation of ZP3 location and function.