Hypotonic stress induces E-cadherin expression in cultured human keratinocytes

Human epidermis marks the interface between internal and external environments with the major task being to maintain body hydration. Alternating exposure of skin to a dry or humid environment is likely to cause changes in the epidermal water gradient resulting in osmotic alterations of epidermal keratinocytes. The present in vitro approach studied the effect of hypotonicity on cell-cell contact. It was demonstrated that hypotonic stress applied to human epithelial cells (HaCaT, A-431) induced upregulation of E-cadherin at both, the protein and mRNA level. 5'-deletional mutants of the E-cadherin promoter identified an element ranging from -53 to +31 that conveyed strong transactivation under hypotonic stress. In order to define relevant upstream regulators members of the MAP kinase family, the epidermal growth factor receptor (EGFR) and protein kinase B/Akt (PKB/Akt) were investigated. Hypotonic conditions led to a fast activation of ERK1/2, SAPK/JNK, p38, EGFR and PKB/Akt with distinct activation patterns. Experiments using specific inhibitors showed that p38 contributes to the E-cadherin transactivation under hypotonic conditions. Further upstream, adhesion was found to be a prerequisite for E-cadherin transactivation in this model. In summary, the present study provides evidence that E-cadherin is an osmo-sensitive gene that responds to hypotonic stress. The function of this regulation may be found in morphological changes induced by cell swelling. It is likely that induction of E-cadherin contributes to the stabilization between adjacent cells in order to withstand the physical forces induced by hypotonicity.     

Remodeling of the adventitia during coronary arteriogenesis

We studied the role of the adventitia in adaptive arteriogenesis during the phase of active growth of coronary collateral vessels (CV) induced by chronic occlusion of the left circumflex coronary artery in canine hearts. We used electron microscopy and immunoconfocal (IF) labeling for bFGF, matrix metalloproteinase (MMP)-2, MMP-9, tissue-type plasminogen activator (tPA), its inhibitor (PAI-1), fibronectin (FN), and Ki-67. Proliferation of smooth muscle cells and adventitial fibroblasts was evident. Quantitative IF showed that adventitial MMP-2, MMP-9, and FN were 9.2-, 7.5-, and 8.6-fold, bFGF was 5.1-fold, and PAI-1 was 3.4-fold higher in CV than in normal vessels (NV). The number of fibroblasts was 5-fold elevated in CV, but the elastic fiber content was 25-fold greater in NV than in CV. Perivascular myocyte damage and induction of endothelial nitric oxide synthase in peri-CV capillaries indicate expansion of CV. It was concluded that adventitial activation is associated with the development of CV through cell proliferation, production of growth factors, and induction of extracellular proteolysis thereby contributing to remodeling during adaptive arteriogenesis.     

Epizootiologic and ecologic investigations of European brown hares (Lepus europaeus) in selected populations from Schleswig-Holstein, Germany

From 1997-99 European brown hare (Lepus europaeus) population densities were estimated by spotlight surveys within different areas in Schleswig-Holstein, Germany. These areas showed a wide variation in local hare population densities. In addition, red fox (Vulpes vulpes) densities were estimated in 1997 by surveys of fox dens and litters. Sera of 321 hares (shot between 1998-2000) from four study areas were examined for antibodies against European brown hare syndrome virus (EBHSV) by enzyme linked immunosorbent assay (ELISA), Yersinia spp. (n = 299) and Francisella tularensis (n = 299) by western blotting, Brucella spp. by Rose Bengal test, and Toxoplasma gondii by Sabin-Feldman test (n = 318). Tissue samples comprising lung, liver, spleen, kidney, heart, and adrenal glands were collected for histopathology. Liver (n = 201) and spleen (n = 201) samples were processed for the detection of T. gondii-antigen in tissue sections and 321 liver and spleen samples were investigated for EBHSV-antigen by ELISA. Furthermore, 116 hares were examined macro- and microscopically for lungworms. Significant negative correlations between hare and fox densities were found in spring and autumn 1997. Antibodies against EBHSV were detected in 92 of 321 (29%), against Yersinia spp. in 163 of 299 (55%), and against T. gondii in 147 of 318 (46%) hares. We evaluated the potential influence of origin and hunting season on exposure rates of hares using logistic regression analysis. A strong association between hare densities and exposure rates was observed for various agents. One hundred and eight of 201 (57%) hares were positive for T. gondii-antigen. All sera were negative for antibodies against Brucella spp. and F. tularensis and all lung samples were negative for lungworms. In conclusion, variation in red fox densities may have an impact on the hare populations examined and the infectious diseases we studied seem to play a subordinate role in the dynamics of European brown hare populations from Schleswig-Holstein.     

Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates.

Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Pacemaking by HCN channels requires interaction with phosphoinositides

Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels mediate the depolarizing cation current (termed I(h) or I(f)) that initiates spontaneous rhythmic activity in heart and brain. This function critically depends on the reliable opening of HCN channels in the subthreshold voltage-range. Here we show that activation of HCN channels at physiologically relevant voltages requires interaction with phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) acts as a ligand that allosterically opens HCN channels by shifting voltage-dependent channel activation approximately 20 mV toward depolarized potentials. Allosteric gating by PIP(2) occurs in all HCN subtypes and is independent of the action of cyclic nucleotides. In CNS neurons and cardiomyocytes, enzymatic degradation of phospholipids results in reduced channel activation and slowing of the spontaneous firing rate. These results demonstrate that gating by phospholipids is essential for the pacemaking activity of HCN channels in cardiac and neuronal rhythmogenesis.     

Changes in caridac output and hemolymph flow during hypoxic exposure in the gravid grass shrimp, <Emphasis Type="Italic">Palaemonetes pugio</Emphasis>

The cardiovascular response of decapod crustaceans to hypoxic exposure is well documented; however, information is limited concerning the influence of reproductive state on cardiovascular demands during hypoxic exposure. Given the additional metabolic demand of reproduction, we investigated the cardiovascular adjustments employed by gravid grass shrimp Palaemonetes pugio to maintain oxygen delivery during hypoxic stress. Cardiac output values were elevated in gravid compared to nongravid grass shrimp. Gravid grass shrimp were exposed to hypoxia and the stroke volume, heart rate, cardiac output and hemolymph flow were determined using video-microscopy and dimensional analysis. Oxygen consumption rates were determined using respirometry. There where no changes in the cardiac output values of gravid females until reaching 6.8 kPa O₂, with a significant redistribution of hemolymph flow at 13.7 kPa O₂. Flow was significantly decreased to the anterior lateral arteries that supply the ovaries and hepatopancreas, the anterior aorta and the posterior aorta. The redistribution of hemolymph flow away from these vessels results in an enhanced hemolymph flow to the sternal artery that supplies the ventral segmental system, the gills, the buccal apparatus and the ventral nerve cord. The data suggest that during hypoxic stress, gravid females place a priority on survival.