Biomedical subjects

Thomas Ott

Publications and source records attributed to Thomas Ott.

28 records · Page 2Linked to original sources

PubMed2003

Altered connexin expression and wound healing in the epidermis of connexin-deficient mice.

To analyze the effect of connexin loss on the repair of wounded tail skin, we have studied the following transgenic mouse mutants: connexin30-/-, connexin31-/- and connexin43Cre-ER(T)/fl (for inducible deletion of the connexin43 coding region). Connexin43 and connexin31 are expressed in the basal and spinous layers of wild-type epidermis, whereas connexin31 and small amounts of connexin30, as well as connexin26 proteins, were found in the granulous layer. Connexin43 was downregulated in connexin31-deficient mice, whereas mice with reduced connexin43 exhibited an upregulation of connexin30. During wound healing, connexin30 and connexin26 proteins were upregulated in all epidermal layers, whereas connexin43 and connexin31 protein expression were downregulated. In connexin31-/- mice, reduced levels of connexin30 protein were observed on days 1 and 2 after wounding. The closure of epidermal wounds in mice with decreased amounts of connexin43 protein occurred one day earlier. Under these conditions the expression profiles of connexin30 and connexin31 were also temporarily shifted by one day. Furthermore, dye transfer between keratinocytes in skin sections from connexin43-deficient mice was decreased by 40%. These results suggest that downregulation of connexin43 appears to be a prerequisite for the coordinated proliferation and mobilization of keratinocytes during wound healing.

Animals↗

PubMed2003

Severe acute pancreatitis and reduced acinar cell apoptosis in the exocrine pancreas of mice deficient for the Cx32 gene.

BACKGROUND & AIMS: The early events leading to acinar cell injury during acute pancreatitis are poorly characterized. Signaling through gap junction channels contributes to the homeostasis of the exocrine pancreas by coordinating acinar cell activity within an acinus. To explore the role of gap junctional communication in acinar cell response to injury, we analyzed the course of acute pancreatitis induced by injection of cerulein in mice deficient for Cx32, the major gap junction protein expressed in the exocrine pancreas. METHODS: The severity of pancreatitis was evidenced by measuring serum amylase activity, pancreatic edema, acinar cell necrosis, pancreatic tumor necrosis factor alpha concentration, and myeloperoxidase activity. Acinar cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), caspase-3 activity, and Bax/Bcl-2 expression. Expression and function of connexin were evaluated by immunofluorescence and dye coupling. RESULTS: Cx32-deficient mice exhibited a deleterious course of acute pancreatitis with increased necrosis, edema, and inflammation of the exocrine pancreas. In addition, the exocrine pancreas of Cx32-deficient mice showed a decreased number of TUNEL-positive acinar cells and decreased caspase-3 activity but no change in Bax or Bcl-2 pancreatic expression. Interestingly, chemicals known to induce apoptosis in vivo had no effect on Cx32-deficient pancreatic acinar cells. CONCLUSIONS: Deficiency of a pancreatic connexin converts a mild reversible form of acute pancreatitis into a severe disease and decreases the sensitivity of acinar cells to apoptotic stimuli. The results show that acinar cell-to-cell communication plays a key role in the modulation of severity of acute pancreatitis.

Acute Disease↗

PubMed2002

Essential myosin light chain as a target for caspase-3 in failing myocardium.

Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE(135)G motif. We demonstrated that vMLC1 cleavage in failing myocardium in vivo is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35 in vivo prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death.

Animals↗

PubMed2002

Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death.

Mutations in the gene encoding the gap junction protein connexin26 (Cx26) are responsible for the autosomal recessive isolated deafness, DFNB1, which accounts for half of the cases of prelingual profound hereditary deafness in Caucasian populations. To date, in vivo approaches to decipher the role of Cx26 in the inner ear have been hampered by the embryonic lethality of the Cx26 knockout mice. To overcome this difficulty, we performed targeted ablation of Cx26 specifically in one of the two cellular networks that it underlies in the inner ear, namely, the epithelial network. We show that homozygous mutant mice, Cx26(OtogCre), have hearing impairment, but no vestibular dysfunction. The inner ear developed normally. However, on postnatal day 14 (P14), i.e., soon after the onset of hearing, cell death appeared and eventually extended to the cochlear epithelial network and sensory hair cells. Cell death initially affected only the supporting cells of the genuine sensory cell (inner hair cell, IHC), thus suggesting that it could be triggered by the IHC response to sound stimulation. Altogether, our results demonstrate that the Cx26-containing epithelial gap junction network is essential for cochlear function and cell survival. We conclude that prevention of cell death in the sensory epithelium is essential for any attempt to restore the auditory function in DFNB1 patients.

Animals↗

PubMed2002

Human connexin26 (GJB2) deafness mutations affect the function of gap junction channels at different levels of protein expression.

Mutations in the connexin26 (GJB2) gene account for about half of inherited non-syndromic deafness cases in Western countries. The connexin26 protein is a subunit of gap junctions that form a network of intercellular communication among supporting cells and fibrocytes in the mammalian inner ear. Here we describe functional implications of mutations in the coding region of connexin26 genes (M1V, M34T, L90P, R127H, F161S, P173R, and R184P), identified in patients and stably transfected in human HeLa cells. While all mutated connexin26 cDNAs were transcribed, only M34T, L90P, R127H, F161S, and R184P were translated in HeLa cells. Analysis of indirect immunofluorescence showed membranous localization, strong for M34T, L90P, R127H, and very weak for F161S, but no signal corresponding to M1V, P173R and R184P. Tracer coupling experiments revealed diffusion of microinjected neurobiotin into neighbouring cells in the case of M34T and R127H, whereas M1V, L90P, F161S, P173R and R184P mutants did not show intercellular coupling. The results of oligomerisation studies suggested a partly disturbed assembly of hemichannels in M34T and L90P mutants but complete absence of hemichannel formation in the R184P mutant. The R127H mutation did not affect channel formation and is likely to represent a polymorphism. Our results show that mutations in the connexin26 gene can affect gap junctional intercellular communication at the level of protein translation, trafficking or assembly of hemichannels.

Biotin↗

PubMed2002

Macrophage-related demyelination in peripheral nerves of mice deficient in the gap junction protein connexin 32.

Mice deficient in the gap junction protein connexin 32 (Cx32) develop a slowly progressing demyelinating neuropathy, with enlarged periaxonal collars, abnormal non-compacted myelin domains and axonal sprouts. These mice serve as a model for the X-linked form of inherited demyelinating neuropathies in humans. Based on our previous findings that macrophages are involved in demyelination in other myelin mutants (i.e. mice heterozygously deficient in P0), we considered the possibility that macrophages might be also mediators of demyelination in Cx32-deficient mice. Indeed, we detected an age-related increase in the number of macrophages in demyelinating nerves of Cx32-deficient mice. In addition, immunoelectron microscopy revealed macrophages in an apposition to degenerating myelin reminiscent of a macrophage-mediated demyelinating neuropathy. We conclude that involvement of macrophages might be a widespread phenomenon in genetically-determined demyelination.

Animals↗

PubMed2002

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices.

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.

Animals↗

PubMed2002

Morphology and morphometric investigation of hepatocellular preneoplastic lesions and neoplasms in connexin32-deficient mice.

Gap junctions are composed of protein subunits, called connexins, and provide a pathway for the exchange of ions and small molecules between contacting cells. This transfer of molecules is thought to be an important pathway for direct cell communication, and is involved in tissue homeostasis, growth control and embryonic development. Impairment of gap junctional intercellular communication (GJIC) via different mechanisms may therefore contribute to dysregulated cellular proliferation and subsequent tumor development. We investigated the effect of Connexin32-deficiency on liver histology and the formation of preneoplastic foci and hepatocellular neoplasms in transgenic knockout mice, as Connexin32 (Cx32) is the major gap junction protein in the liver. Loss of Cx32 does not alter the morphology of extrafocal liver tissue. However, after administration of a single dose of diethylnitrosamine (DEN), given 2 weeks after birth, the number and volume fraction of preneoplastic foci showed a 3.3-fold to 12.8-fold increase in the Cx32-deficient mice as compared with the corresponding wildtype groups, regardless of sex and age of the animals. Number and volume fraction of hepatocellular adenomas and carcinomas also increased significantly in these animals. The experimental groups did not differ in the morphology of the different types of preneoplastic foci and neoplasms. On the other hand, Cx32-deficiency without DEN treatment did not lead to an increase in the spontaneous development of any type of preneoplastic hepatic foci or hepatocellular neoplasms in up to 18-month-old Cx32-deficient mice as compared with wildtype controls. In conclusion, our results indicate that impairment of GJIC in mouse liver due to deletion of the Cx32 coding DNA clearly promotes the carcinogenic effect of DEN administration and results in a higher susceptibility to hepatocellular neoplasms, but does not appear to initiate hepatic tumor development.

Animals↗

PubMed2002

Role of gap junctions in fluid secretion of lacrimal glands.

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.

Analysis of Variance↗

PubMedDate not supplied

Sequential superparamagnetic clustering for unbiased classification of high-dimensional chemical data.

For the clustering of chemical structures that are described by the Similog, ISIS count, and ISIS binary fingerprints, we propose a sequential superparamagnetic clustering approach. To appropriately handle nonbinary feature keys, we introduce an extension of the binary Tanimoto similarity measure. In our applications, data sets composed of structures from seven chemically distinct compound classes are evaluated and correctly clustered. The comparison, with results from leading methods, indicates the superiority of our sequential superparamagnetic clustering approach.

Journal Article↗