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K Orth

Publications and source records attributed to K Orth.

At least 37 records · Page 2Linked to original sources
PubMed1998

Photochemotherapy of experimental colonic tumours with intra-tumorally applied methylene blue.

INTRODUCTION: Phototoxicity of intra-tumoral injected methylene blue (MB+) was studied in 48 experimental colonic tumours in comparison with photosan-3, Zn-phthalocyanine and tetrasulphanated ClAl-phthalocyanine. METHODS: In mice. xenotransplanted subcutaneous tumours about 1 cm in diameter were treated photodynamically twice, with different sensitisers. The irradiation was performed at the sensitiser-specific wavelength, and a density of 100 mW/cm2 and a dose of 100 J/cm2. RESULTS: Light alone without sensitiser did not induce any effect in mice tumours. Surprisingly, Al-phthalocyanine could only be used for intratumoral injections because of toxic effects after intravenous applications in nude mice. Using MB+ (1%), 75% of the tumours were destroyed by a single photodynamic treatment (PDT). In addition, toxicity of MB+ was most intense when compared with Zn-phthalocyanine and photosan-3. However, after the second PDT, there was no statistically significant difference among these sensitisers. Dark toxicity of MB+ (1%) could be well demonstrated by sufficient sensitiser incorporation without irradiation, which led to a stationary tumour volume up to 3 weeks after injection. CONCLUSION: Intra-tumoral MB+ PDT is a potential treatment for inducing necrosis in vivo. With regard to tumour tissue, the selectivity of MB+ is high and depends on a precise local injection of the dye.

Animals↗
PubMed1998

Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade.

Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic up-regulation of cyclin A-associated cyclin-dependent kinase 2(cdk2) activity. In apoptotic cells, the C termini of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 are truncated by specific cleavage. The enzyme involved in this cleavage is CPP32 and/or a CPP32-like caspase. After cleavage, p21Cip1/Waf1 loses its nuclear localization sequence and exits the nucleus. Cleavage of p21Cip1/Waf1 and p27Kip1 results in a substantial reduction in their association with nuclear cyclin-cdk2 complexes, leading to a dramatic induction of cdk2 activity. Dominant-negative cdk2, as well as a mutant of p21Cip1/Waf1 resistant to caspase cleavage, partially suppress apoptosis. These data suggest that cdk2 activation, through caspase-mediated cleavage of cdk inhibitors, may be instrumental in the execution of apoptosis following caspase activation.

Apoptosis↗
PubMed1998

Proteolysis of poly(ADP-ribose) polymerase by caspase 3: kinetics of cleavage of mono(ADP-ribosyl)ated and DNA-bound substrates.

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which is responsible for synthesis of poly(ADP-ribose) in response to DNA damage caused by numerous agents and during DNA base excision repair. After DNA damage, the enzyme binds to nicks in DNA through its N-terminal zinc fingers and catalyzes the formation of poly(ADP-ribose) on various nuclear acceptors including itself. When DNA damage is extensive, cells induce their own demise by activating the proteases that induce apoptosis (caspases) which cleave PARP and other death substrates. Here we report the development of a new approach to investigate the sensitivity of mono(ADP-ribosyl)ated and DNA-bound PARP to cleavage during apoptosis. The development of a stoichiometric labeling procedure of the enzyme has allowed us to evaluate the catalytic properties of caspase 3 toward mono(ADP-ribosyl)ated PARP at various enzyme:substrate molar ratios. We show that low levels of automodification (< or = 3 U of ADP-ribose per chain) do not inhibit the proteolysis of the substrate. In addition, we demonstrate that binding of unmodified PARP to DNA influences the kinetics of its cleavage by caspase 3.

ADP Ribose Transferases↗
PubMed1997

Granule-mediated killing: pathways for granzyme B-initiated apoptosis.

We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.

Apoptosis↗
PubMed1997

Cleavage of focal adhesion kinase by caspases during apoptosis.

Apoptotic cells undergo characteristic morphological changes that include detachment of cell attachment from the substratum and loss of cell-cell interactions. Attachment of cells to the extracellular matrix and to other cells is mediated by integrins. The interactions of integrins with the extracellular matrix activates focal adhesion kinase (FAK) and suppresses apoptosis in diverse cell types. Members of the tumor necrosis family such as Fas and Apo-2L, also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induce apoptosis in both suspension and adherent cells through the activation of caspases. These caspases, when activated, cleave substrates that are important for the maintenance of nuclear and membrane integrity. In this study, we show that FAK is sequentially cleaved into two different fragments early in Apo-2L-induced apoptosis. We also demonstrate that FAK cleavage is mediated by caspases and that FAK shows unique sensitivity to different caspases. Our results suggest that disruption of FAK may contribute to the morphological changes observed in apoptotic suspension and adherent cells.

Apoptosis↗
PubMed1997

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Bik and Bak induce apoptosis downstream of CrmA but upstream of inhibitor of apoptosis.

Recent studies have identified a number of cell death pathway components. In this study, we describe the role that two such components, Bik and Bak, play in initiating the apoptotic program. These Bcl-2 family members engage the death pathway downstream of the block imposed by the serpin CrmA, but upstream of the block initiated by cellular inhibitors of apoptosis, which are a family of molecules characterized by a conserved baculovirus inhibitor of apoptosis repeat motif. Distal death pathway components activated by Bik and Bak are similar to those activated by the CD-95 (Fas/Apo1) and tumor necrosis factor death receptors.

Apoptosis↗
PubMed1997

Target protease specificity of the viral serpin CrmA. Analysis of five caspases.

When ectopically expressed in animal cells, cytokine response modifier A (CrmA), a product of the cowpox virus, prevents programmed cell death initiated by a variety of stimuli. Since CrmA is a proteinase inhibitor, its target is probably a protease that promotes cell death. The identification of this target is crucial in delineating essential regulation points that modulate the apoptotic program. We have compared the kinetics of interaction of CrmA with five proteases that may play a role in apoptosis. Four of the proteases, all members of the caspase family, are inhibited with widely different rates and affinities ranging over 5 orders of magnitude. One is not inhibited at all under the experimental conditions. CrmA is quite selective in its ability to inhibit caspases, showing the highest affinity for interleukin-1beta-converting enzyme and the second highest for the caspase FLICE (Ki = 0.95 nM), identified as a component of the intracellular signaling complex recruited by ligation of the death receptor Fas. On the basis of comparative inhibitor kinetics, we propose that CrmA is unlikely to inhibit the caspases Yama, Mch2, or LAP3 in vivo but that its inhibition of FLICE is of a magnitude for this protease to be a key target of CrmA during Fas-mediated apoptosis. Therefore, our results support the hypothesis that FLICE catalyzes a crucial step in the promotion of cell death.

Amino Acid Sequence↗
PubMed1997

Thermo-controlled device for inducing deep coagulation in the liver with the Nd:YAG laser.

BACKGROUND AND OBJECTIVE: To increase the effectiveness of laser-induced interstitial thermotherapy (LITT), a new thermo-controlled application system for minimal invasive intervention was designed. Our system consists of a laser applicator of 2.5 mm in diameter, insertion equipment, and a Nd:YAG-laser source. STUDY DESIGN/MATERIALS AND METHODS: A cylindrical light emitting fiber (1-6 cm in length) was placed in the center of the applicator. The surrounding tissue was irradiated through a Duran window at the distal end of the applicator. The power of the laser source was controlled dynamically by thermosensors in a water-cooling system of the laser applicator. The temperature at the surface of the Duran window was kept constant at approximately 60 degrees C, without charring the surrounding tissue. RESULTS/CONCLUSION: We obtained homogeneous coagulation zones. In in vitro experiments with pig livers, we reached ellipsoid coagulation volumes of 3 and 5 cm in diameter within 10 minutes, corresponding to a volume of approximately 25 cm3.

Animals↗
PubMed1997

[Postoperative and post-traumatic acute cholecystitis. Value of ultrasound diagnosis].

An often unrecognized but potentially fatal complication, mostly seen in posttraumatic patients under intensive care, is reactive acute cholecystitis. On account of the high specificity of ultrasound diagnosis in the biliary system we decided to examine the ultrasound criteria for early detection of posttraumatic cholecystitis. Ultrasound of the abdomen was performed prospectively, seven times on different days, in each of 40 artificially respirated patients under intensive care conditions over a period of 12 months. The results show that artificial respiration, parenteral feeding and previous trauma can lead to tardive (28/40) wall-thickening or to a three-layered wall of the gallbladder (9/40). In 22.5% of patients (9/40) we found the sonographic signs of acute cholecystitis. In correlation with the clinical signs, cholecystectomy was indicated in only two patients. The preoperative ultrasonographic findings and clinical signs of 23 patients with the diagnosis of acute reactive cholecystitis were analysed retrospectively. We found good correlation between sonographic and clinical signs of acute cholecystitis in 21 of these 23 patients. Our study shows that the morphological correlate of a thickened three-layered gallbladder wall can occur in the context of systemic alterations, even if there is no underlying cholecystitis. The diagnosis of acute reactive cholecystitis and the indication for cholecystectomy should be based on the synopsis of pathologic and clinical findings.

Acute Disease↗
PubMed1997

[Dynamic interstitial laser application for therapy of secondary liver tumors. An animal experiment study].

Laser-induced interstitial thermotherapy (LITT) is a promising method of treating different types of tumors. To increase the effectiveness of LITT, a new thermo-controlled application system for minimal invasive intervention was designed. This laser system consists of a laser applicator 3 mm in diameter, insertion equipment and an Nd:YAG laser source. The power of the laser source is controlled dynamically by thermosensors in a water-cooling system of the laser applicator. In in vivo experiments in five pigs, within 10 min we found homogeneous coagulation regions in the liver that were of 3 cm and 5 cm in diameter. Via ultrasound we observed in real time the increasing coagulation zone that appeared as a hyperechogenic halo. However, due to perifocal edema, the real coagulation area was smaller than detected by ultrasound. Macroscopically and microscopically, the coagulation zones showed well-demarcated borders of the coagulation lesions, and the surrounding tissue appeared vital. The reparative reaction after irradiation was early fibrosis. In the course of 4 weeks, the surrounding scar capsule, containing fibrocytes, biliary ductules and collagen fibers enlarged. As a result, the coagulation necrosis became more and more fragmented between collagen fibers and was largely resorbed. The absence of complications in our animal experiments suggest that the technique of thermo-controlled laser light application is safe and useful in therapy for metastases that are not resectable.

Animals↗
PubMed1996

New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis.

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

Apoptosis↗
PubMed1996

Molecular ordering of apoptotic mammalian CED-3/ICE-like proteases.

Apoptosis is executed by cysteine proteases belonging to the CED-3/ICE family, which, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Proteases belonging to this family exist in the cytosol as zymogens that require accurate processing at internal aspartate residues to generate the two-chain active enzymes. As such, CED-3/ICE family members are capable of activating each other in a manner analogous to the protease zymogens of the coagulation or complement cascades. At present, it is unknown whether such mutual processing exists in vivo, and if so whether it is sequential, implying an order to the death pathway. Using a cell-free apoptosis system, recombinant ICE proteases and both biochemical and morphological criteria, we demonstrate an ordering of the mammalian ICEs that are most related to the Caenorhabditis elegans death protease CED-3.

Animals↗
PubMed1996

ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B.

Members of the ICE/Ced-3 gene family are likely effector components of the cell death machinery. Here, we characterize a novel member of this family designated ICE-LAP6. By phylogenetic analysis, ICE-LAP6 is classified into the Ced-3 subfamily which includes Ced-3, Yama/CPP32/apopain, Mch2, and ICE-LAP3/Mch3/CMH-1. Interestingly, ICE-LAP6 contains an active site QACGG pentapeptide, rather than the QACRG pentapeptide shared by other family members. Overexpression of ICE-LAP6 induces apoptosis in MCF7 breast carcinoma cells. More importantly, ICE-LAP6 is proteolytically processed into an active cysteine protease by granzyme B, an important component of cytotoxic T cell-mediated apoptosis. Once activated, ICE-LAP6 is able to cleave the death substrate poly(ADP-ribose) polymerase into signature apoptotic fragments.

Amino Acid Sequence↗
PubMed1996

The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A.

Phylogenetic analysis of the CED-3/ICE family of cysteine proteases suggests the existence of a subfamily most related to the Caenorhabditis elegans death gene ced-3 and includes Yama (CPP32, apopain), LAP3 (Mch3, CMH1), and Mch2. Here, we show that Mch2 is processed from its zymogen form to a proteolytically active dimeric species during execution of the apoptotic program and by the cytotoxic T cell death protease granzyme B. Additionally, like Yama and LAP3, Mch2 functions downstream of the death inhibitors Bcl-2, Bcl-xL, and CrmA. Importantly, Mch2, but not Yama or LAP3, is capable of cleaving lamin A to its signature apoptotic fragment, indicating that Mch2 is an apoptotic laminase.

Alkaloids↗
PubMed1996

Cytotoxic T-cell-derived granzyme B activates the apoptotic protease ICE-LAP3.

Cytotoxic T lymphocytes (CTLs) and natural killers (NK) cells provide immune surveillance against viruses and neoplasms, and play a central role in the pathogenesis of autoimmune disease, AIDS and graft rejection. Thus, it is important to understand the precise molecular mechanism(s) whereby cytotoxic lymphocytes destroy susceptible target cells. Granule-mediated cytotoxicity requires a combination of both perforin and granzyme B. Perforin polymerizes to form transmembrane channels and presumably allows granzyme B access to target cell substrates, which until recently, were unknown. One clue to the identity of the physiological substrate(s) activated by granzyme B comes from its unusual specificity for cleaving synthetic substrates after aspartate residues. Members of the ICE/CED-3 family of cysteine proteases are prime candidates as they are important apoptotic effectors and are expressed as zymogens, which can be processed to form active heterodimeric enzymes after cleavage at specific aspartate residues. Previous studies have shown that granzyme B proteolytically activates the cell death effector Yama/CPP32/apopain (referred to here as Yama). Here we report that granzyme B also activates ICE-LAP3/Mch3/CMH-1 (referred to here as ICE-LAP3), which, along with Yama and Mch2, forms a subset of the ICE/CED-3 family of cysteine proteases most closely related to the Caenorhabditis elegans cell death gene, CED-3. Importantly, Jurkat T cells incubated with granzyme B and a sublytic concentration of perforin undergo apoptosis, which is preceded by the activation of endogenous ICE-LAP3. Thus, we propose that granzyme B mediates apoptosis by directly engaging the target cell's death effector machinery, which is probably composed of an arsenal of intracellular, CED-3-like cysteine proteases.

Apoptosis↗