I. Piotr Maly

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.

DISTRIBUTION OF ALCOHOL DEHYDROGENASE ISOENZYMES IN THE HUMAN LIVER ACINUS

Abstract By the use of a newly developed technique of ultrathin-layer electrophoresis, class I and class II alcohol dehydrogenase activity could be demonstrated in microdissected samples of the periportal, intermediate, and perivenous zones of the liver acinus in men and women. It could be demonstrated that both classes exhibit low activity in the periportal zone. From there, a rising gradient in the direction of the perivenous end was apparent. This increase, however, was found to be significant only in women. The analysis of class I alcohol dehydrogenase isoenzymes showed that the expression of α-, β-, and γ-containing isoforms did not differ in relation to the intraacinar position. The constant proportions of the isoenzymes to the maxima and minima of the total alcohol dehydrogenase activity support the view that the adult liver-specific isoenzyme pattern is determined during postnatal development.

Evidence for the Colocalization of Parvalbumin and Glutamate, but Not GABA, in the Perforant Path of the Gerbil Hippocampal Formation: A Combined Immunocytochemical and Microquantitative Analysis

Abstract: Gerbils (Meriones unguiculatus) are known for their seizure sensitivity, which is dependent on an intact perforant path from the entorhinal cortex to the hippocampus. In contrast with other species, the perforant path in gerbils contains parvalbumin, a cytosolic high‐affinity calcium‐binding protein. Parvalbumin is known to be present in a subpopulation of GABA‐containing neurons and is thought to be responsible for their physiological characteristics of fast spiking activity and lack of spike adaptation. Therefore, the question arose of whether this projection in gerbils is GABAergic or glutamatergic as in other species. In a first approach to this question, the effect of lesioning the origin of the perforant path, the entorhinal cortex, on levels of GABA and glutamate was determined by enzymatic‐luminometric assay in single layers of the dentate gyrus of lyophilized brain sections. Parallel sections were cryofixed using an acidified acetone‐formaldehyde mixture at ‐20°C for 48 h, and subsequently stained for parvalbumin immunocytochemistry. Seven days after ablation of the entorhinal cortex, parvalbumin staining was undetectable in the termination zone of the perforant path, the outer two‐thirds of the stratum moleculare. In parallel, glutamate content was reduced to 80% of controls (and of the unoperated contralateral side) but unchanged in the inner third of the stratum moleculare and in stratum granulare. GABA content was not significantly altered by the lesion. From these results, we conclude that in the gerbil as in other species, the perforant path contains glutamate. The association of glutamate with parvalbumin suggests, however, that in the gerbil the calcium‐dependent release of the transmitter is carefully controlled in the entorhinal perforant path.

SPATIAL DISTRIBUTION OF HUMAN LIVER ALDEHYDE DEHYDROGENASE ISOENZYMES

Abstract To elucidate the pattern of lesions in the liver parenchyma after ethanol ingestion, the quantitative distribution profiles of both the cytosolic and the mitochondrial aldehyde dehydrogenase isoenzyme activities were determined by the use of ultrathin-layer electrophoresis. It was found that in human liver parenchyma, both isoforms of aldehyde dehydrogenase are almost homogeneously represented in the liver acinus. These quantitative data are supported by the results of an improved histochemical technique. Moreover, sex differences were not detected either in activity or in the distribution pattern. Consequently, it can be assumed that it is not the activity of total aldehyde dehydrogenase or its isoforms which is responsible for the higher susceptibility of the perivenous zone to alcohol-dependent damage.

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.

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.

Changes in the activity of alcohol dehydrogenase isoenzymes during the estrus cycle in the vagina of the rat.

In rodents, the vaginal epithelium undergoes cyclical changes with an alternating pattern of keratinization and mucification. It has been known for decades that vitamin A and its active form retinoic acid are responsible for normal epithelial homeostasis. However, it has not so far been certain which enzymes catalyze the first and rate-limiting step in retinoic acid synthesis. By means of microdissection and ultrathin-layer gel electrophoresis, alcohol dehydrogenase isoenzyme activity was determined quantitatively in the various layers of the vaginal mucous membrane. It was found that, in the rat, only alcohol dehydrogenase 3 and 4 are expressed. Marked cyclical changes of alcohol dehydrogenase 4 activity in the stratum germinativum of the vaginal epithelium strongly support the assumption that this isoenzyme is responsible for retinoic acid synthesis, and that it is essential for the changes accompanying keratinization and mucification.

Microquantitative determination of lactate dehydrogenase isoenzymes in the myocardium and the conducting system

SummaryA newly developed technique was used for the electrophoretic separation of lactate dehydrogenase (LDH) isoenzymes from lyophilized tissue samples in the nanogram range. In this study portions of 10–200 ng from the myocardium and the conducting system of cattle, sheep, pig and man were microdissected and analysed.In the heart tissues of cattle, sheep and pig, the isoforms LDH1, LDH2 and LDH3 were detected in species-specific varying amounts. In all these animals, the conducting system is marked by high LDH1 activity, which is present at a ratio of about 2:1 compared with the myocardium. The values in man, however, differ from these values, but this might be due to post-mortem changes. The findings are discussed with respect to possible aerobic-anaerobic functions.