Peter Kreutzmann

LEKTI, A NOVEL 15-DOMAIN TYPE OF HUMAN SERINE PROTEINASE INHIBITOR

Proteinase inhibitors are important negative regulators of proteinase action in vivo. We have succeeded in isolating two previously unknown polypeptides (HF6478 and HF7665) from human blood filtrate that are parts of a larger precursor protein containing two typical Kazal-type serine proteinase inhibitor motifs. The entire precursor protein, as deduced from the nucleotide sequence of the cloned cDNA, exhibits 15 potential inhibitory domains, including the Kazal-type domains, HF6478, HF7665, and 11 additional similar domains. An inhibitory effect of HF7665 on trypsin activity is demonstrated. Because all of the 13 HF6478- and HF7665-related domains share partial homology to the typical Kazal-type domain but lack one of the three conserved disulfide bonds, they may represent a novel type of serine proteinase inhibitor. The gene encoding the multidomain proteinase inhibitor, which we have termed LEKTI, was localized on human chromosome 5q31-32. As shown by reverse transcriptase-polymerase chain reaction and Northern blot analysis, it is expressed in the thymus, vaginal epithelium, Bartholin’s glands, oral mucosa, tonsils, and the parathyroid glands. From these results, we assume that LEKTI may play a role in anti-inflammatory and/or antimicrobial protection of mucous epithelia.

Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2

The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6-8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity.

Toxoplasma gondii Actively Inhibits Neuronal Function in Chronically Infected Mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii–infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca2+) imaging studies revealed that tachyzoites actively manipulated Ca2+ signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca2+ uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca2+ stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host.

LEKTI: a multidomain serine proteinase inhibitor with pathophysiological relevance

Proteinase inhibitors are important negative regulators of proteinase action in vivo and are thus involved in several pathophysiological processes. Starting with the isolation of two new peptides from human blood filtrate, we succeeded in cloning a cDNA encoding the precursor protein for a novel 15-domain Kazal-type-related serine proteinase inhibitor. Two of the 15 domains almost exactly match the Kazal-type pattern, whereas the other 13 domains exhibit only four instead of six cysteine residues. Since the corresponding gene is expressed in several lympho-epithelial tissues, we termed this inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI). For three of the 15 LEKTI domains, we demonstrated a significant trypsin-inhibiting activity. Recent results of another group show a relation between mutations within the LEKTI gene and the severe congenital disorder Netherton syndrome. In this review article, we give an overview of the already known data on the structure, processing, gene expression, and pathophysiological role of LEKTI.

Inhibitory modulation of the mitochondrial permeability transition by minocycline.

The semi-synthetic tetracycline derivative minocycline exerts neuroprotective properties in various animal models of neurodegenerative disorders. Although anti-inflammatory and anti-apoptotic effects are reported to contribute to the neuroprotective action, the exact molecular mechanisms underlying the beneficial properties of minocycline remain to be clarified. We analyzed the effects of minocycline in a cell culture model of neuronal damage and in single-channel measurements on isolated mitoplasts. Treatment of neuron-enriched cortical cultures with rotenone, a high affinity inhibitor of the mitochondrial complex I, resulted in a deregulation of the intracellular Ca2+-dynamics, as recorded by live cell imaging. Minocycline (100 microM) and cyclosporin A (2 microM), a known inhibitor of the mitochondrial permeability transition pore, decreased the rotenone-induced Ca2+-deregulation by 60.9% and 37.6%, respectively. Investigations of the mitochondrial permeability transition pore by patch-clamp techniques revealed for the first time a dose-dependent reduction of the open probability by minocycline (IC(50)=190 nM). Additionally, we provide evidence for the high antioxidant potential of MC in our model. In conclusion, the present data substantiate the beneficial properties of minocycline as promising neuroprotectant by its inhibitory activity on the mitochondrial permeability transition pore.

Impairment of mitochondrial function by minocycline

There is an ongoing debate on the presence of beneficial effects of minocycline (MC), a tetracycline‐like antibiotic, on the preservation of mitochondrial functions under conditions promoting mitochondria‐mediated apoptosis. Here, we present a multiparameter study on the effects of MC on isolated rat liver mitochondria (RLM) suspended either in a KCl‐based or in a sucrose‐based medium. We found that the incubation medium used strongly affects the response of RLM to MC. In KCl‐based medium, but not in sucrose‐based medium, MC triggered mitochondrial swelling and cytochrome c release. MC‐dependent swelling was associated with mitochondrial depolarization and a decrease in state 3 as well as uncoupled respiration. Swelling of RLM in KCl‐based medium indicates that MC permeabilizes the inner mitochondrial membrane (IMM) to K+ and Cl−. This view is supported by our findings that MC‐induced swelling in the KCl‐based medium was partly suppressed by N,N′‐dicyclohexylcarbodiimide (an inhibitor of IMM‐linked K+‐transport) and tributyltin (an inhibitor of the inner membrane anion channel) and that swelling was less pronounced when RLM were suspended in choline chloride‐based medium. In addition, we observed a rapid MC‐induced depletion of endogenous Mg2+ from RLM, an event that is known to activate ion‐conducting pathways within the IMM. Moreover, MC abolished the Ca2+ retention capacity of RLM irrespective of the incubation medium used, most likely by triggering permeability transition. In summary, we found that MC at low micromolar concentrations impairs several energy‐dependent functions of mitochondria in vitro.

Minocycline, a possible neuroprotective agent in Leber’s hereditary optic neuropathy (LHON): Studies of cybrid cells bearing 11778 mutation

Lebers hereditary optic neuropathy (LHON) is a retinal neurodegenerative disorder caused by mitochondrial DNA point mutations. Complex I of the respiratory chain affected by the mutation results in a decrease in ATP and an increase of reactive oxygen species production. Evaluating the efficacy of minocycline in LHON, the drug increased the survival of cybrid cells in contrast to the parental cells after thapsigargin-induced calcium overload. Similar protection was observed by treatment with cyclosporine A, a blocker of the mitochondrial permeability transition pore (mPTP). Ratiometric Ca(2+) imaging reveals that acetylcholine/thapsigargin triggered elevation of the cytosolic calcium concentration is alleviated by minocycline and cyclosporine A. The mitochondrial membrane potential of LHON cybrids was significantly conserved and the active-caspase-3/procaspase-3 ratio was decreased in both treatments. Our observations show that minocycline inhibits permeability transition induced by thapsigargin in addition to its antioxidant effects. In relation with its high safety profile, these results would suggest minocycline as a promising neuroprotective agent in LHON.

Biochemical Features, Molecular Biology and Clinical Relevance of the Human 15-Domain Serine Proteinase Inhibitor LEKTI

Abstract Based on the isolation of a 55 amino acid peptide from human hemofiltrate, we cloned the cDNA for a novel human 15-domain serine proteinase inhibitor termed LEKTI. A trypsininhibiting activity was demonstrated for three different domains. High levels of expression of the corresponding gene were detected in oral mucosa, followed by the tonsils, parathyroid glands, thymus, and trachea. Hovnanian and coworkers recently found that certain mutations within the LEKTI gene are linked to the severe congenital disease Netherton syndrome and atopic manifestations (including asthma). Thus, a future therapeutic use of LEKTI is conceivable.

Interaction of the antibiotic minocycline with liver mitochondria – role of membrane permeabilization in the impairment of respiration

Several studies have proposed that the antibiotic minocycline (MC) has cytoprotective activities. Nevertheless, when cells have been exposed to MC at micromolar concentrations, detrimental effects have been also observed. Despite the known inhibitory activity of MC on ATP synthesis and the Ca2+ retention capacity of isolated rat liver and brain mitochondria, the underlying mechanism is still debated. Here, we present further arguments supporting our concept that MC acting on rat liver mitochondria suspended in KCl medium permeabilizes the inner membrane. Supplementation of the medium with cytochrome c and NAD+ strongly enhanced the respiration of MC‐treated mitochondria, thus partly preventing or reversing the inhibitory effect of MC on state 3 or uncoupled respiration. These results indicate that MC produced depletion of mitochondrial cytochrome c and NAD+, thus impairing mitochondrial respiration. In addition, NADH oxidation by alamethicin‐permeabilized mitochondria supplemented with cytochrome c was insensitive to 200 μm MC, arguing against direct impairment of respiratory chain complexes by MC. Finally, a surprising feature of MC was its accumulation or binding by intact rat liver mitochondria, but not by mitochondria permeabilized with alamethicin or disrupted by freezing and thawing.

Hydroxy-1-aryl-isochromans: protective compounds against lipid peroxidation and cellular nitrosative stress.

Abstract Hydroxy-1-aryl-isochromans are a novel class of polyphenolic heterocyclic compounds with potent antioxidative potential. Here we investigated the protective effects of hydroxy-1-aryl-isochroman derivatives against lipid peroxidation and nitrosative stress as a function of their degree of hydroxylation. Measurements of thiobarbituric acid reactive substances (TBARS) in rat brain mitochondria, nitric oxide analysis in microglia cultures, and cell viability analysis were performed. We found that hydroxyl-1-aryl-isochroman derivatives with two, three, and four hydroxyl groups (ISO-2, ISO-3, and ISO-4, respectively) display a higher activity against mitochondrial lipid peroxidation than the corresponding derivative without hydroxyl groups (ISO-0) as well as the reference antioxidant Trolox. However, ISO-0 strongly reduced microglial nitric oxide production, whereas ISO-2 and ISO-4 had a less pronounced effect and ISO-3 was even ineffective. ISO-0 and ISO-3 increased the cell viability of activated microglia, whereas a minor effect of ISO-2 and no significant increase after treatment with ISO-4 could be observed. In conclusion, hydroxy-1-aryl-isochromans exhibit cytoprotective properties, besides their action against reactive oxygen/nitrogen species.