The Actin-Binding Protein α-Adducin Modulates Desmosomal Turnover and Plasticity.

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules.

High metabolic activity of tissue-nonspecific alkaline phosphatase not only in young but also in adult bone as demonstrated using a new histochemical detection protocol.

Tissue-nonspecific alkaline phosphatase (TNAP) is playing a key role in bone calcification, as has been demonstrated in different mammalian species including human and rodents. However, to investigate age-related changes during life history, histochemical demonstration of TNAP is severely hampered, particularly in the elderly, by technical difficulties associated with sectioning calcified tissue. Sufficient fixation must precede decalcification since poorly fixed bone tissue is exposed to the deleterious effects of decalcification reagents. In order to find a method that would allow cryosectioning of bone without loss of TNAP activity, we assessed the efficacy of different fixation reagents regarding the effects on structural integrity and TNAP activity using liver and osseous tissue from younger and older horses. The results of this study reveal that glyoxal-based fixatives sufficiently preserved bone tissue for successful cryosectioning without compromising TNAP activity. The method described combines the demonstration of TNAP activity with optimal preservation of tissue morphology in osseous tissue of younger and even of older mammals. As a model species, we selected horse bones in light of potentially higher similarities to ageing history and lifelong locomotion in humans as compared to other, mostly smaller, experimental model species with a much shorter life span and artificial locomotive activity when kept in cages. This may serve as a basis for future studies addressing the impact of different life traits in iconic, domestic and companion animals, which are often patients in veterinary medicine, as well as for basic research on human physiology and pathologies of the musculoskeletal system.

Distinct presence of the tight junction protein claudin-3 in olfactory bulb and fila olfactoria of the mouse.

The tight junction protein claudin-3 is overexpressed in diverse epithelial tumours and is associated with increased survival, progression and motility of tumour cells. Claudin-3 expression profiles are being increasingly used for diagnostic and prognostic tumour classification. Claudin-3 has been identified as a receptor for Clostridium perfringens enterotoxin, which is under consideration for selective lysis of claudin-3-expressing tumours, particularly brain metastases, and other translational medicine uses. However, the localization of claudin-3 in the brain has not been completely elucidated. While claudin-3 in brain tissue adjacent to claudin-3-expressing metastases had been excluded and low or undetectable levels proposed in the CNS, under physiological conditions, in adult human, rat and mouse brains, claudin-3 was exclusively found in choroid plexus epithelium where it is considered an integral component of the blood-cerebrospinal-fluid barrier. We report here the pronounced presence of claudin-3 not only in the nasal region (as described for rat), but also in the mouse olfactory bulb and nerve using immunohistochemistry and Western blot. Claudin-3 was present in the fila olfactoria from the epithelium to the olfactory nerve and in the main and accessory olfactory bulb. We propose that the abundant presence of claudin-3 in the olfactory system, particularly in nerve fibres and the olfactory bulb cone, which we present here, may play a role at the interface of the central and peripheral nervous system, both as barrier and for axonal growth and communication. Thus, claudin-3 should be considered and further explored with regards to treatment approaches addressing the olfactory bulb and nasal region.

Novel polyvinyl alcohol (PVA) based cryoprotection method that facilitates cutting frozen sections of decalcified human trabecular bone.

Processing adult human trabecular bone to obtain tissue sections suitable for research or diagnostic purposes has always been challenging, particularly in the preparation of adult bone specimens for advanced immunohistochemistry applications. In contrast to the majority of soft tissues, decalcified bone samples perform poorly under standard paraffin embedding techniques and immunolabeling protocols fail frequently, due to the loss of protein antigenicity observed. We report on a new, PVA based infiltration method that avoids excessive heat exposure to tissue samples during embedding. The developed PVA based infiltration medium provides sufficient structural support to the heterogenic morphology and distinct architecture of subchondral trabecular bone and adjacent articular cartilage. Furthermore, the addition of bovine serum albumin (BSA) to this infiltration solution guaranteed safe attachment of cryosections to glass slides. The protocol allows the preparation of high quality sections of adult human trabecular bone tissues which can be used for both classical histochemical stains and for immunohistochemistry, since protein antigenicity is satisfactorily preserved.

Bile duct ligation in the rat causes upregulation of ZO-2 and decreased colocalization of claudins with ZO-1 and occludin.

As the only barrier between blood and bile compartments hepatocellular tight junctions play a crucial role in cholestasis-induced increase of biliary permeability. The molecular basis of this reversible defect is not known. We, therefore, examined expression, phosphorylation, distribution and colocalization of the junctional proteins occludin, claudin-1-3, ZO-1 and ZO-2 in rats after bile duct ligation and release of ligation. In control rats, claudin-1 and ZO-2 displayed a lobular gradient with highest expression levels in periportal cells, whereas claudin-2 showed a reciprocal distribution. Other proteins were evenly expressed in the liver lobule. Ligation resulted in upregulation of ZO-2 (2.7-fold), ZO-1 (1.4-fold) and occludin (1.2-fold) but not of claudins. Only ZO-2 showed increased phosphorylation. Distribution patterns were unchanged except for a strong accumulation of ZO-2 in perivenous hepatocytes. Colocalization analysis demonstrated that perivenous ZO-2 was the only protein examined revealing strongly increased overlap with occludin and ZO-1, whereas claudins and other proteins displayed a decrease. All changes were partially reversed by release of ligation. We conclude that differential expression of claudin-1-2 and ZO-2 has functional implications for bile formation. The moderately increased ZO-1 and occludin levels account for the known elongation of tight junction strands. The highly increased expression and changed distribution of ZO-2 suggests that ZO-1 is partly substituted by ZO-2, an alteration possibly causing impaired barrier function.

SDS disc electrophoresis of proteins in homogeneous, low-concentrated polyacrylamide gels.

In the attempt to separate in a single gel run low- and high-molecular-weight proteins, we present here a multiphasic buffer system designed for this purpose. It avoids the continuous stacking of SDS as it occurs in the 'classical' SDS-PAGE. The system allows complete stacking and destacking of proteins in the 3.5-250 kDa range at acrylamide concentrations as low as 4.5% T (total acrylamide concentration in %) and 2.6% C (degree of cross-linking in %). Taurine is used as the trailing ion in the cathode buffer and in the resolving zone of the gel, and two different counterions (Tris and imidazole) in the stacking zone. The gel system is easy to prepare and, due to the very low acrylamide concentrations, it is ideal for analytical as well as for preparative tasks.

Ultrathin-layer sodium dodecyl sulfate disc electrophoresis of proteins in the range from 10 to 220 kDa in homogeneous, low-concentrated polyacrylamide gels.

This study describes an ultrathin-layer sodium dodecyl sulfate (SDS) disc electrophoresis in polyacrylamide gels of a thickness of only 150 microm. By use of 2-amino-2-methyl-1,3-propanediol/glycine instead of traditional Tris/HCl buffer in the resolving phase of the gel, proteins with a wide range of molecular sizes (10 kDa to over 220 kDa) are separated in unusually low-concentrated gels (4%T, 3.3%C). 2-Amino-2-methyl-1,3-propanediol in the resolving part of the gel contributes to stabilization of the pH value at 8.8, while glycine improves destacking as well as separation of small proteins from the bulk of stacked SDS. This method combines both the advantages of conventional slab-gel electrophoresis and capillary gel electrophoresis. It is easy to apply and well suited for all further miniaturization attempts.

The so-called "testis-specific aldehyde dehydrogenase" corresponds to type 2 retinaldehyde dehydrogenase in the mouse.

The distribution pattern of "testis-specific aldehyde dehydrogenase" in mouse tissues was investigated. Because of the broad substrate specificity and the high degree of sequence identity of the large aldehyde dehydrogenase family a specific detection of single isoforms is not possible by histochemical means. Therefore, the technique of native isoelectric focusing was used. Thus, the expression of four to five banded "testis-specific aldehyde dehydrogenase" in the mouse testis was confirmed. However, the activity of this enzyme with the same pattern of multiplicity was found not only in the testis but also in the uterus and in embryonic tissues. At 9.5 and 10.5 days of embryonic development the enzyme activity was restricted to tissues of the embryonic trunk and absent in extracts from cranial tissues. The tissue distribution as well as substrate specificity and isoelectric points indicate that the "testis-specific aldehyde dehydrogenase" corresponds to mouse type 2 retinaldehyde dehydrogenase.