Several diseases induce hypermetabolism, which is characterized by increases in resting energy expenditures (REE) and whole body protein loss. Exaggerated protein degradation is thought to be the driving force underlying this response. The effects of caspase and calpain inhibitors on REE in physiological and hypermetabolic conditions, however, are unknown. Thus, we studied whether MDL28170 (calpain inhibitor) or z-VAD-fmk (caspase inhibitor) affect REE under physiological conditions and during hypermetabolism post-burn. Rats were treated five times weekly and observed for 6 weeks. Treatment was started 2 h (early) or 48 h (late) after burn. In normal rats, MDL28170 transiently increased REE to 130 % of normal during week 2-4. z-VAD-fmk reduced REE by 20-25 % throughout the observation period. Within 14 days after burns, REE increased to 130+/-5 %. Whereas MDL28170/early treatment did not affect REE, MDL28170/late transiently increased REE to 180+/-10 % of normal by week 4 post-burn. In contrast, with z-VAD-fmk/early REE remained between 90-110 % of normal post-burn. z-VAD-fmk/late did not affect burn-induced increases in REE. These data suggest that caspase cascades contribute to the development of hypermetabolism and that burn-induced hypermetabolism can be pharmacologically modulated. Our data point towards caspase cascades as possible therapeutic targets to attenuate hypermetabolism after burns, and possibly in other catabolic disease processes.

• MeSH
• chloromethylketony aminokyselin farmakologie   terapeutické užití   MeSH
• dipeptidy farmakologie   terapeutické užití   MeSH
• energetický metabolismus účinky léků   MeSH
• inhibitory cysteinových proteinas farmakologie   terapeutické užití   MeSH
• inhibitory kaspas farmakologie   terapeutické užití   MeSH
• metabolické nemoci farmakoterapie   etiologie   MeSH
• pilotní projekty MeSH
• popálení komplikace   MeSH
• potkani Sprague-Dawley MeSH
• preklinické hodnocení léčiv MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.

• MeSH
• akrosin metabolismus   MeSH
• akrozom enzymologie   MeSH
• analýza rozptylu MeSH
• histologické techniky veterinární   MeSH
• imunoelektronová mikroskopie veterinární   MeSH
• inhibitory proteas farmakologie   MeSH
• motilita spermií účinky léků   fyziologie   MeSH
• neparametrická statistika MeSH
• proteasy farmakologie   MeSH
• rosanilinová barviva MeSH
• ryby fyziologie   MeSH
• sperma enzymologie   MeSH
• spermie účinky léků   enzymologie   fyziologie   MeSH
• tosylfenylalanylchlormethylketon farmakologie   MeSH
• tosyllysinchlormethylketon farmakologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the 'water retention site', suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.

• MeSH
• chloromethylketony aminokyselin chemická syntéza   farmakologie   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• endopeptidasy chemie   genetika   metabolismus   MeSH
• Escherichia coli chemie   enzymologie   genetika   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• membránové proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• molekulární modely * MeSH
• mutace MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• Providencia chemie   MeSH
• rekombinantní proteiny MeSH
• simulace molekulární dynamiky * MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Truncated tau protein at Asp(421) is associated with neurofibrillary pathology in Alzheimer disease (AD); however, little is known about its presence in the form of nonfibrillary aggregates. Here, we report immunohistochemical staining of the Tau-C3 antibody, which recognizes Asp(421)-truncated tau, in a group of AD cases with different extents of cognitive impairment. In the hippocampus, we found distinct nonfibrillary aggregates of Asp(421)-truncated tau. Unlike Asp(421)-composed neurofibrillary tangles, however, these nonfibrillary pathologies did not increase significantly with respect to the Braak staging and, therefore, make no significant contribution to cognitive impairment. On the other hand, despite in vitro evidence that caspase-3 cleaves monomeric tau at Asp(421), to date, this truncation has not been demonstrated to be executed by this protease in polymeric tau entities. We determined that Asp(421) truncation can be produced by caspase-3 in oligomeric and multimeric complexes of recombinant full-length tau in isolated native tau filaments in vitro and in situ in neurofibrillary tangles analyzed in fresh brain slices from AD cases. Our data suggest that generation of this pathologic Asp(421) truncation of tau in long-lasting fibrillary structures may produce further permanent toxicity for neurons in the brains of patients with AD.

• MeSH
• Alzheimerova nemoc patologie   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• elektronová mikroskopie MeSH
• kaspasa 3 metabolismus   farmakologie   MeSH
• kyselina aspartová metabolismus   MeSH
• lidé MeSH
• molekulová hmotnost MeSH
• mozek metabolismus   patologie   ultrastruktura   MeSH
• neurofibrilární klubka metabolismus   patologie   ultrastruktura   MeSH
• neurofibrily metabolismus   patologie   ultrastruktura   MeSH
• neuroprotektivní látky farmakologie   MeSH
• oligopeptidy farmakologie   MeSH
• proteiny tau účinky léků   metabolismus   ultrastruktura   MeSH
• senioři nad 80 let MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05  µM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 µM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 µM Q-VD-OPh while 10 µM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.

• MeSH
• apoptóza účinky léků   MeSH
• buněčná adheze MeSH
• chinoliny farmakologie   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• kaspasy antagonisté a inhibitory   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Search for new substances with antiproliferative activity towards melanoma cells is important since malignant melanoma is notoriously resistant to conventional chemotherapy. Benzo[c]phenanthridine alkaloids (BAs) are natural products with significant anti-proliferative activities, therefore they are considered as agents promising for cancer therapy. OBJECTIVES: The effects of five BAs (sanguinarine, chelerythrine, chelidonine, sanguilutine, and chelilutine) on human malignant melanoma cell lines were compared. The study focused on BAs effects on DNA, anti-apoptotic and p53 protein levels; and the involvement of p53 in cellular responses to alkaloids treatment. METHODS: Melanoma cell lines, two wild types and two with dysfunctional p53 derived from one of them were used. The mechanism of anti-proliferative and pro-apoptotic effects and the effect on DNA was investigated using MTT assay, flow cytometry, Western blot analysis, fluorescence and electron microscopy. RESULTS: All tested alkaloids exhibit strong anti-proliferative activity. CHL, CHE and SA induced apoptosis, which was probably mediated by decreasing levels of anti-apoptotic proteins (Bcl-xL, Mcl-1, XIAP) and was accompanied by mitochondrial membrane potential decrease as well as caspase-3 and PARP cleavage. Although all alkaloids caused DNA damage, which was demonstrated by induction of H2AX phosphorylation, none of the tested alkaloids stabilised p53 and their toxicity in cells with non-functional p53 was comparable to wild type cells. CONCLUSION: Despite the profound similarity of BAs molecular structures, it is clear that the mechanism of cell death induction is different for each alkaloid. Our results indicate that BAs could be effective in malignant melanoma treatment, including tumours which have lost wild type p53.

• MeSH
• alkaloidy chemie   metabolismus   farmakologie   MeSH
• apoptóza MeSH
• benzofenantridiny farmakologie   MeSH
• biologické modely MeSH
• chemické modely MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• DNA metabolismus   MeSH
• geny p53 MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• melanom genetika   metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory kůže genetika   metabolismus   MeSH
• poškození DNA MeSH
• proliferace buněk MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti-leukemic therapy. We demonstrated previously that the co-treatment of HL-60 cells with Tumor necrosis factor-alpha (TNF-alpha) (1 ng/mL) and inhibitor of 5-lipoxygenase MK886 (5 microm) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen-activated protein kinases (MAPKs) (p38, c-Jun amino-terminal kinase [JNK], extracellular signal-regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro-apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF-alpha co-treatment. On the other hand, down-regulation of pro-survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF-alpha. Pan-caspase inhibitor ZVAD-fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL-60 cells.

• MeSH
• aktivace enzymů účinky léků   MeSH
• akutní promyelocytární leukemie farmakoterapie   metabolismus   patologie   MeSH
• apoptóza fyziologie   účinky léků   MeSH
• buněčná diferenciace účinky léků   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• HL-60 buňky MeSH
• indoly farmakologie   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• inhibitory lipoxygenas farmakologie   MeSH
• kaspasy antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém účinky léků   MeSH
• monocyty patologie   účinky léků   MeSH
• NF-kappa B antagonisté a inhibitory   MeSH
• TNF-alfa farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

A technique for coating surfaces with attached fibrin structures without the formation of fibrin gel in bulk solution was developed. It is based on the catalytic effect of the surface-bound thrombin on fibrinogen stabilized with inhibitor which inhibits thrombin in solution but not the thrombin on the surface. Such an inhibitor is antithrombin, the effect of which may be enhanced with heparin. Fibrinogen is first adsorbed on the substrate surface and then incubated with thrombin. The unbound thrombin is washed out and the surface is incubated with fibrinogen solution containing antithrombin III and heparin. A fibrin gel forms at the surface by the action of surface-bound thrombin on ambient fibrinogen solution; however, the gel formation in bulk solution catalyzed by thrombin partially released from the surface is suppressed. By utilizing antithrombin-independent inhibitors or repeating thrombin binding and incubation with fibrinogen solution, the amount of surface-attached fibrin gel can be controlled. The formation of immobilized fibrin networks was observed using surface plasmon resonance and turbidity measurements and morphology was observed by TEM, SEM, and AFM. Using this technique, a porous scaffold made of polylactide fibers was coated with fibrin without filling the space between fibers with a bulk fibrin gel. The technique makes it possible to coat the inner surface of porous scaffolds with surface-attached fibrin gel while preserving free volume for cell migration into the pores.

• MeSH
• biokompatibilní materiály chemie   MeSH
• chloromethylketony aminokyselin metabolismus   MeSH
• fibrin chemie   metabolismus   MeSH
• fibrinogen chemie   metabolismus   MeSH
• fibrinolytika metabolismus   MeSH
• financování organizované MeSH
• hirudiny metabolismus   MeSH
• inhibitory proteas metabolismus   MeSH
• lidé MeSH
• methylmetakryláty chemie   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• poréznost MeSH
• povrchové vlastnosti MeSH
• prasata MeSH
• testování materiálů MeSH
• thrombin antagonisté a inhibitory   metabolismus   MeSH
• tkáňové podpůrné struktury MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

We recently demonstrated that TLCK and TPCK could act as potent but nonspecific inhibitors of mature caspases [Frydrych and Mlejnek [2008] J Cell Biochem 103:1646-1656]. The question whether TLCK and TPCK inhibit simultaneously caspase activation and/or processing remained, however, open. In this article, we demonstrated that TPCK even enhanced caspase-3 and caspase-7 processing although it substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity in HL-60 cells exposed to various cell death inducing stimuli. Under the same conditions, TLCK had no effect or affected caspase-3 and caspase-7 processing marginally depending on cell treatment used. Importantly, TLCK substantially inhibited caspase-3 and caspase-7 enzymatic (DEVDase) activity irrespectively to the treatment used. Interestingly, treatment of cells with toxic concentrations of TPCK alone was accompanied by full caspase-3 and -7 processing even if it induced necrosis. In contrast, treatment of cells with concentrations of TLCK that caused necrosis was accompanied by only partial caspase-3 and caspase-7 processing. Our results clearly indicated that TPCK and TLCK did not inhibit caspase-3 and -7 enzymatic activity by prevention of their activation and/or processing.

• MeSH
• apoptóza MeSH
• HL-60 buňky MeSH
• inhibitory serinových proteinas farmakologie   MeSH
• kaspasa 3 antagonisté a inhibitory   metabolismus   MeSH
• kaspasa 7 antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• tosylfenylalanylchlormethylketon farmakologie   MeSH
• tosyllysinchlormethylketon farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.

• MeSH
• apoptóza účinky léků   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• financování organizované MeSH
• inhibitory enzymů farmakologie   MeSH
• kaspasa 3 antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• proliferace buněk účinky léků   MeSH
• spotřeba kyslíku účinky léků   MeSH
• U937 buňky enzymologie   patologie   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH