Sulpiride in the treatment of somatoform disorders: results of a European observational study to characterize the responder profile.

An open, observational study was conducted in five European countries to obtain information concerning the profile of patients responding to sulpiride. A total of 1,356 patients were evaluable for analysis. The majority of patients (81.1%) had at least three principal somatic complaints; asthenia being the most common, followed by dizziness and headache. Most patients (76.0%) were rated as moderately to extremely ill according to the Clinical Global Impression (CGI) severity score. All patients received oral sulpiride for 3-6 weeks (mean dose, 175 mg/day). Sulpiride demonstrated good efficacy as shown by a reduction in the incidence and severity of somatic complaints, and an improvement in CGI severity score and the Hopkins Symptom Checklist--58 items. Based on a CGI rating of very much or much improved, 58.2% of patients were rated as responders. Sulpiride was well tolerated. There were no serious adverse events and only 16 patients (1.2%) were withdrawn prematurely from the study due to adverse events. There were no differences between the countries regarding the patients' profile or their response to sulpiride. Thus, the prescription profile of sulpiride appears not to be culturally dependent.

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines.

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.

Plasmodium vivax and Plasmodium chabaudi: intraerythrocytic traffic of antigenically homologous proteins involves a brefeldin A-sensitive secretory pathway.

We have used a monoclonal antibody (mAb 7C5B71) raised against the erythrocytic stages of Plasmodium vivax to identify a 148-kDa P vivax protein antigen (Pv-148) which crossreacts with an antigenically homologous 190-kDa protein of P. chabaudi (Pc-190). During parasite intraerythrocytic development Pv-148 and Pc-190 are exported into the host cell cytosol and become located in the surface membrane of the infected erythrocyte. Immunofluorescence confocal microscopy and immunoelectron microscopy studies showed that both Pv-148 and Pc-190 are released from the parasite and exported to the host cell cytoplasm in association with tubovesicular membrane (TVM) structures. Fluorescent in vivo labelling of P. chabaudi with Bodipy-ceramide followed by immunofluorescence staining with the mAb supported the association of antigenically homologous Pc-190 with TVM structures. In the presence of brefeldin A (BFA), secretion of antigenically homologous Pc-190 into the host cell cytoplasm was inhibited and the antigen remained in the parasite cytoplasm. BFA also arrested the maturation of the parasite. Taken together these results suggest that Pv-148 and Pc-190 are related parasite proteins that are transported into the host cell through a BFA-sensitive secretory pathway.

UV-A-related alterations of young and adult lens water-insoluble alpha-crystallin, plasma membranous and cytoskeletal proteins.

The damaging effects of UV-A irradiation on lens water-insoluble alpha-crystallin, plasma membranous and cytoskeletal proteins derived from bovine lenses were studied. Young and adult bovine lenses were kept viable for 2 months in organ culture. After 24 h of incubation they were irradiated, and analyses of the proteins by one-dimensional and two-dimensional gel electrophoresis followed by Western blotting were carried out at several time intervals. RNA isolation, PCR and Northern blotting were also performed. We identified age-related changes in water-insoluble alpha-crystallin, the major membrane protein MP26 and the cytoskeletal proteins vimentin, phakinin and actin between control and UV-irradiated lenses. It appeared that adult lenses are more susceptible to UV light than young lenses, and protein modification occurred more frequently in adult lenses. UV-A irradiation affects not only the cytoskeletal structure, as deduced by the abnormal arrangement of actin in the fiber cells, but also leads to degradation of actin mRNA. Furthermore, analysis of the expression of hsp25 and hsp70 revealed some alteration in the protein pattern of adult lenses. We suggest that degradation of the cytoskeletal proteins following irradiation is due to, at least in part, the decreased protective ability of heat shock proteins upon aging.

Peptide secretion in the cutaneous glands of South American tree frog Phyllomedusa bicolor: an ultrastructural study.

The development of the dermal glands of the arboreal frog Phyllomedusa bicolor was investigated by immunocytochemistry and electron microscopy. The 3 types of glands (mucous, lipid and serous) differed in size and secretory activity. The mucous and serous glands were apparent in the tadpole skin, whereas the lipid glands developed later in ontogenesis. The peptide antibiotics dermaseptins and the D-amino acid-containing peptide opioids dermorphins and deltorphins are abundant in the skin secretions of P. bicolor. Although these peptides differ in their structure and activity they are derived from precursors that have very similar preproregions. We used an antibody to the common preproregion of preprodermaseptins and preprodeltorphins and immunofluorescence analysis to show that only the serous glands are specifically involved in the biosynthesis and secretion of dermaseptins and deltorphins. Scanning and transmission electron microscopy revealed that the serous glands of P bicolor have morphological features, especially the secretory granules, which differ from those of the glands in Xenopus laevis skin.

Assembly of connexins and MP26 in lens fiber plasma membranes studied by SDS-fracture immunolabeling.

The SDS-fracture immunolabeling technique, unlike conventional freeze-fracture, provides direct evidence for the biochemical nature of membrane constituents. SDS-fracture immunolabeling shows that during differentiation of lens fiber cells the onset of junctional assembly is characterized by the presence of small clusters and linear arrays comprising connexins alpha3 and alpha8. At this initial stage MP26, a major fiber membrane constituent, appears to be colocalized with these two connexins. The application of double-immunogold labeling reveals that when large junctional plaques are assembled MP26 becomes mainly associated with the periphery of the junctional domains. This type of distribution suggests that MP26 may play a role in the clustering and gathering of connexons. In aged nuclear fiber membranes connexins, MP26 and their proteolytic derivatives form an orthogonal lattice of repeating subunits.

Agonist-induced redistribution of bradykinin B2 receptor in caveolae.

Redistribution of receptors within the plasma membrane as well as between the plasma membrane and various cell compartments presents an important way of regulating the cellular responsiveness to their cognate agonists. We have applied immunocytochemical methods to localize the bradykinin B2 receptor and to examine its agonist induced redistribution in A431 cells. In situ labeling with antibodies to ectodomain-2 of the receptor which do not interfere with bradykinin binding of the receptor showed a random distribution of the B2 receptor on the plasma membrane. Stimulation of cells with 20 nM bradykinin markedly reduced the accessibility of the antibody to its corresponding epitope in non-permeabilized cells. Immuno-electron microscopy revealed the presence of receptors in membrane-near vesicles that are surrounded by an electron-transparent halo. Fluorescence microscopic double labeling co-localized the B2 receptor protein with caveolin-1 by a convergent pattern of punctate staining. At the ultrastructural level the B2 receptor protein was found in vesicles that bear the immunolabel of caveolin-1 and display the morphological characteristics of caveolae. We conclude that stimulation of B2 receptors results in their redistribution and sequestration in caveolae, an event that is likely to be implicated in receptor signaling and/or desensitization. The localization of B2 receptors in endosome-like structures after prolonged exposure to bradykinin might indicate that the internalization through caveolae may communicate with other endocytotic pathways of A431 cells.

Transgenic mice carrying chimeric or mutated type III intermediate filament (IF) genes.

Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.

Towards a human crystallin map. Two-dimensional gel electrophoresis and computer analysis of water-soluble crystallins from normal and cataractous human lenses.

This paper describes a first approach to establish a master data base of human lens crystallins obtained by computer analysis of standardized two-dimensional lenticular protein patterns. To facilitate the eventual identification of the spots, the major crystallins have been separated into alpha-, beta H-, beta L- and gamma-crystallin fractions by gel filtration. The authors encourage colleague investigators to collaborate in a common effort in order to arrive eventually at a two-dimensional gel data base of all lenticular proteins.

Muscle-specific expression of a dominant negative desmin mutant in transgenic mice.

To extend our knowledge of the functions of desmin and vimentin intermediate filaments in the developing organism, a construct encoding a truncated desmin subunit driven by the desmin promoter (pDDV), was introduced into the murine germ line. The resulting mutant desmin subunit was assembly-incompetent and capable of disrupting both preexisting desmin and vimentin filaments in a dominant negative fashion in transfected C2C12 muscle cells and in transgenic mouse muscle tissue. Expression of the pDDV was tissue-specific in transgenic mice. High level expression of pDDV occurred in a small percentage of desmin-containing muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse desmin pattern instead of the dots and clumps into which mutant desmin typically accumulates in undifferentiated C2C12 muscle cells in tissue culture. Disruption of the endogenous desmin filaments in Sartorius muscle results in ultrastructural abnormalities.

Human multidrug resistance 3-P-glycoprotein expression in transgenic mice induces lens membrane alterations leading to cataract.

We have generated mice transgenic for a human multidrug resistance (MDR)3 mini-gene driven by a hamster vimentin promoter. The MDR3 gene encodes a P-Glycoprotein that resembles the mouse multidrug resistance 2 P-Glycoprotein shown to be involved in the translocation of the phospholipid phosphatidylcholine through the hepatocyte canalicular membrane (Smit et al., 1993. Cell. 75:451-462). The vimentin promoter drives expression of the MDR3 transgene in mesenchymal tissues and in the eye lens. We show here that the presence of human multidrug resistance 3 P-Glycoprotein in the lens results in a severe lenticular pathology. Lens structural abnormalities initiate at a late embryonic stage and increase during postnatal lens development. Differentiation of the primary fibers is affected, and the terminal differentiation of the lens epithelium into secondary fibers is also perturbed. The ultrastructural alterations, particularly of the lens plasma membranes, resemble those identified in congenital mouse osmotic cataract.

Transgenic mice: models for the study of cataractogenesis. A minireview.

Genetic manipulation followed by (over)expression of the transgene product in lens tissue results in: (I) inhibition of denucleation of lens fibers; (II) interference with lens cell differentiation; (III) cataract formation. The events reviewed are preceded by membrane damage, perturbed assembly of the IF cytoskeleton and distortion of the cytoskeleton-membrane complex.

Disruption of vimentin intermediate filaments in transgenic mice by expression of a dominant negative mutant desmin subunit.

To investigate putative functions of vimentin intermediate filaments in the context of intact tissues and the developing organism, a construct (pVDV), driven by the vimentin promoter and encoding a truncated desmin subunit, was introduced into the murine germ line. The mutant desmin was assembly-incompetent and capable of disrupting preexisting vimentin filaments in a dominant negative fashion, both in transgenic mouse tissues and in fibroblast cultures derived from these mice. Mutant desmin expression strongly enhanced vimentin turnover. In tissues of some transgenic mouse lines, high level expression of pVDV occurred in 10 to 40% of vimentin-containing cells and, surprisingly, in 1 to 10% of the skeletal and tongue muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse staining pattern instead of the punctated aggregates into which mutant desmin typically accumulates in other cell types. The overexpression of pVDV and the concomitant disruption of the endogenous vimentin filament network and enhanced vimentin turnover in a significant percentage of cells did not cause detectable developmental abnormalities.

Mice lacking vimentin develop and reproduce without an obvious phenotype.

To address the biological role of vimentin in the context of the living organism, we have introduced a null mutation of the vimentin gene into the germ line of mice. Surprisingly, animals homozygous for this mutation developed and reproduced without an obvious phenotype. Immunoblotting, immunofluorescence, and immunogold labeling analysis confirmed the absence of vimentin and of the corresponding filament network. Furthermore, no compensatory expression of another intermediate filament could be demonstrated. While these results leave open the question of the possible role of vimentin in unusual situations or pathological conditions, they show that a conspicuous developmental and cell-specific structure that is an integral part of the cytoskeleton can be eliminated without apparent effect on mouse reproduction and development.

Distribution of bradykinin B2 receptors in target cells of kinin action. Visualization of the receptor protein in A431 cells, neutrophils and kidney sections.

Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.

Distribution of bradykinin B2 receptors in target cells of kinin action: visualization of the receptor protein in A431 cells, neutrophils and kidney sections

Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Apllication of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue

Abnormal incisor-tooth differentiation in transgenic mice expressing the muscle-specific desmin gene.

Immunocytochemistry and electron microscopic observations on the incisor-tooth organ of transgenic mice expressing the muscle-specific desmin gene under the direction of the vimentin promoter, reveal that the expression of the hybrid transgene occurs both in mesenchymal cells and differentiating odontoblasts. The muscle-specific desmin, as estimated by fluorescence intensity, is more expressed in immature mesenchymal cells than in postmitotic differentiated odontoblasts. The expression of the transgene generates alteration of the odontoblast-intermediate filament network and interferes with the secretory activity of both odontoblasts and ameloblasts. Our results are consistent with the hypothesis that odontoblasts have inductive properties on the differentiation of ameloblasts and that intermediate filaments among other factors play the role of cell and tissue organizer.