The PI3K regulatory subunits p55α and p50α regulate cell death in vivo.

The phosphatidylinositol 3-kinase (PI3K) regulatory subunits p55α and p50α are coordinately transcriptionally upregulated by signal transducer and activator of transcription 3 (Stat3) at the onset of mammary gland involution, a process that requires Stat3. Deletion of both p55α and p50α subunits in vivo abrogated mammary epithelial cell death during involution. This was associated also with reduced cytosolic levels and activity of the cysteine protease cathepsin L, which is implicated in lysosomal-mediated programmed cell death (LM-PCD) and is upregulated in involution. Furthermore, involution is delayed in cathepsin L-deficient mice suggesting that the p55α/p50α subunits mediate cell death in part by elevating the level of cathepsin L resulting in increased cytosolic activity. Surprisingly, we found that p55α/p50α localize to the nucleus where they bind to chromatin and regulate transcription of a subset of inflammatory/acute phase genes that are also Stat3 targets. Our findings reveal a novel role for these PI3K regulatory subunits as regulators of LM-PCD in vivo.

Cell type-dependent pathogenic functions of overexpressed human cathepsin B in murine breast cancer progression.

The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.

Stefin B deficiency reduces tumor growth via sensitization of tumor cells to oxidative stress in a breast cancer model.

Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (Stfb) in mammary cancer, Stfb null mice were crossed with transgenic polyoma virus middle T oncogene (PyMT) breast cancer mice. We show that ablation of Stfb resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of Stfb-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed no significant effects of ablation of Stfb expression on proliferation, angiogenesis, migration and spontaneous cell death as compared with control cells. However, upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking Stfb exhibited a significantly higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for Stfb in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.

STAT3 expression, activity and functional consequences of STAT3 inhibition in esophageal squamous cell carcinomas and Barrett's adenocarcinomas.

Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC, n=49) and Barrett's adenocarcinomas (BAC, n=61) revealed similar STAT3 expression in ESCCs and BACs (P=0.109), but preferentially activated P-STAT3 in ESCCs (P=0.013). In vitro, strong STAT3 activation was seen by epidermal growth factor (EGF) stimulation in OE21 (ESCC) cells, whereas OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 (P=0.0148) and OE33 (P=0.0243) cells. Importantly, STAT3 knockdown reduced cell migration of OE33 cells by 2.5-fold in two types of migration assays (P=0.073, P=0.015), but not in OE21 cells (P=0.1079, P=0.386). Investigation of transcriptome analysis of STAT3 knockdown revealed a reduced STAT3 level associated with significant downregulation of cell cycle genes in both OE21 (P<0.0001) and OE33 (P=0.01) cells. In contrast, genes promoting cell migration (CTHRC1) were markedly upregulated in OE21 cells, whereas a gene linked to tight-junction stabilization and restricted cell motility (SHROOM2) was downregulated in OE21 but upregulated in OE33 cells. This study shows frequent, but distinct, patterns of STAT3 expression and activation in ESCCs and BACs. STAT3 knockdown reduces cell proliferation in ESCC and BAC cells, inhibits migration of BAC cells and may support cell migration of ESCC cells. Thereby, novel STAT3-regulated genes involved in ESCC and BAC cell proliferation and cell migration were identified. Thus, STAT3 may be further exploited as a potential novel therapeutic target, however, by careful distinction between the two histotypes of esophageal cancers.

Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice.

Cysteine cathepsins are a family of proteases involved in intracellular protein turnover and extracellular matrix degradation. Cathepsin B (Ctsb) and cathepsin Z (Ctsz) promote tumorigenesis and Ctsb is a known modulator of tumor angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (laser-CNV). Ctsb(-/-), Ctsz(-/-), Ctsb/Ctsz double-knockout (Ctsb/z DKO), and wild type (WT) mice underwent argon laser treatment to induce choroidal neovascularization (CNV). The neovascularized area was quantified individually for each lesion at 14 days after laser coagulation. In vitro the effects of cathepsin inhibitors on angiogenesis were analysed by endothelial cell (EC) spheroid sprouting and EC invadosome assays. Retinas from cathepsin KO mice did not show gross morphological abnormalities. In the laser CNV model, however, Ctsb/z DKO mice displayed a significantly reduced neovascularized area compared to WT (0.027 mm(2) vs. 0.052 mm(2); p = 0.012), while single knockouts did not differ significantly from WT. In line, VEGF-induced EC spheroid sprouting and invadosome formation were not significantly altered by a specific cathepsin B inhibitor alone, but significantly suppressed when more than one cathepsin was inhibited. Our results demonstrate that laser-CNV formation is significantly reduced in Ctsb/z DKO mice. In line, EC sprouting and invadosome formation are blunted when more than one cathepsin is inhibited in vitro. These results reveal an angiomodulatory potential of cathepsins with partial functional redundancies between different cathepsin family members.

Transgenic expression of human cathepsin B promotes progression and metastasis of polyoma-middle-T-induced breast cancer in mice.

Elevated expression of the cysteine protease cathepsin B (CTSB) has been correlated with a poor prognosis for cancer patients. In order to model high CTSB expression in mammary cancer, transgenic mice expressing human CTSB were crossed with transgenic polyoma virus middle T oncogene breast cancer mice (mouse mammary tumor virus-PymT), resulting in a 20-fold increase in cathepsin B activity in the tumors of double-transgenic animals. CTSB expression did not affect tumor onset, but CTSB transgenic mice showed accelerated tumor growth with significant increase in weight for end-stage tumors, as well as an overall worsening in their histopathological grades. Notably, the lung metastases in the CTSB transgenic animals were found to be both significantly larger and to occur at a significantly higher frequency. Ex vivo analysis of primary PymT tumor cells revealed no significant effects from elevated CTSB levels on tumor cell characteristics, that is, the formation of tumor cell colonies and the sprouting of invasive strands from PymT cell spheroids. However, tumors from CTSB-overexpressing mice showed increased numbers of tumor-associated B cells and mast cells. In addition, more CD31+ endothelial cells were detected in these tumors, correlating with higher levels of vascular endothelial growth factor (VEGF) being present in the tumor and serum. We conclude that elevated proteolytic CTSB activity facilitates progression and metastasis of PymT-induced mammary carcinomas, and is associated with increased immune cell infiltration, enhanced VEGF levels and the promotion of tumor angiogenesis.

Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes.

Apoptotic stimuli have been shown to trigger lysosomal membrane permeability (LMP), leading to the release of cathepsins, which activate death signaling pathways in the cytosol. However, it is unknown whether this process is an initiating or amplifying event in apoptosis. In this study, we used fibroblasts and monocytes exposed to etoposide, ultraviolet light, FasL or deprived of interleukin-3 (IL-3) to show that LMP and the cytosolic release of cathepsins B, L and D consistently depends on Bax/Bak and components of the apoptosome. Neither Bax nor Bak resided on the lysosomes, indicating that lysosomes were not directly perforated by Bax/Bak but by effectors downstream of the apoptosome. Detailed kinetic analysis of cells lacking cathepsin B or L or treated with the cysteine protease inhibitor, E64d, revealed a delay in these cells in etoposide- and IL-3 deprivation-induced caspase-3 activation and apoptosis induction but not clonogenic survival, indicating that cathepsins amplify rather than initiate apoptosis.

Deficiency for the cysteine protease cathepsin L promotes tumor progression in mouse epidermis.

To define a functional role for the endosomal/lysosomal cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Ctsl deficiency in addition to epithelial expression of the human papillomavirus type 16 oncogenes (human cytokeratin 14 (K14)-HPV16). We found enhanced tumor progression and metastasis in the absence of Ctsl. As tumor progression in K14-HPV16 mice is dependent on inflammation and angiogenesis, we examined immune cell infiltration and vascularization without finding any effect of the Ctsl genotype. In contrast, keratinocyte-specific transgenic expression of cathepsin V, the human orthologue of mouse Ctsl, in otherwise Ctsl-deficient K14-HPV16 mice restored the phenotype observed in the control HPV16 skin. To better understand this phenotype at the molecular level, we measured several oncogenic signal transduction pathways in primary keratinocytes on stimulation with keratinocyte-conditioned cell culture medium. We found increased activation of protein kinase B/Akt and mitogen-activated protein kinase pathways in protease-deficient cells, especially if treated with media conditioned by Ctsl-deficient keratinocytes. Similarly, the level of active GTP-Ras was increased in Ctsl-deficient epidermis. We conclude that Ctsl is critical for the termination of growth factor signaling in the endosomal/lysosomal compartment of keratinocytes and, therefore, functions as an anti-tumor protease.

Reduced tumour cell proliferation and delayed development of high-grade mammary carcinomas in cathepsin B-deficient mice.

Expression levels of the papain-like cysteine protease cathepsin B (Ctsb) have been positively correlated with mammary tumour progression and metastasis; however, its roles in the hallmark processes of malignant growth remain poorly defined. Using Ctsb-deficient mice we investigated tumour cell differentiation, proliferation and apoptosis in the Tg(MMTV-PyMT) mouse mammary cancer model. Absence of Ctsb significantly impaired development of high-grade invasive ductal carcinomas and reduced the metastatic burden in the lungs. Mice lacking Ctsb exhibited reduced cell proliferation in mammary carcinomas and their lung metastases. Notably, intravenous injection of primarily isolated, Ctsb-expressing tumour cells into congenic Ctsb-deficient mice revealed impaired cell proliferation in the resulting experimental lung metastases, providing evidence for the involvement of Ctsb in paracrine regulation of cancer cell proliferation. No Ctsb genotype-dependent difference in tumour cell death was observed in vivo or by treatment of isolated PyMT cancer cells with tumour necrosis factor-alpha. However, cancer cells lacking Ctsb exhibited significantly higher resistance to apoptosis induction by the lysosomotropic agent Leu-Leu-OMe. Thus, our results indicate an in vivo role for Ctsb in promoting cellular anaplasia in mammary cancers and proliferation in lung metastases.

Cathepsin protease activity modulates amyloid load in extracerebral amyloidosis.

In cerebral amyloidoses, such as Alzheimer's disease, proteolytic processing of the precursor protein is a fundamental mechanism of the disease, since it generates the amyloid protein. However, the putative significance of proteases in extracerebral amyloidoses is less well defined. In this study, we investigated the biological significance of cathepsin (Cath) B, CathK, and CathL in the pathology and pathogenesis of extracerebral amyloidoses by using the murine model of reactive or secondary AA amyloidosis with three different cathepsin-deficient mouse strains. Extracerebral AA amyloid was induced by injecting amyloid-enhancing factor and silver nitrate into CathB(-/-), CathK(-/-), and CathL(-/-) mice. Wild-type mice served as a control. CathK(-/-) mice deposited over 90% more amyloid and CathL(-/-) mice 60% less amyloid than the control (p < 0.0001). The amyloid load in CathB(-/-) mice did not differ from that in wild-type mice. In vitro degradation experiments with recombinant human and murine serum amyloid A (SAA) 1.1 and CathK and CathL showed that CathL generates a large number of differently sized SAA cleavage products. One of these fragments spans the heparin/heparan sulphate binding site and the neutral cholesterol ester hydrolase activating region of SAA. CathK showed only endoproteolytic activity and did not generate any AA amyloid-like peptides. This study provides unequivocal evidence that proteases modulate amyloid load in extracerebral amyloidosis. CathL was identified as an amyloid-promoting and CathK as an amyloid-retarding cysteine protease. CathB may only modulate the primary structure of the amyloid peptide without affecting amyloid load.

Calvarial osteoclasts express a higher level of tartrate-resistant acid phosphatase than long bone osteoclasts and activation does not depend on cathepsin K or L activity.

Bone resorption by osteoclasts depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Next to these enzymes, tartrate-resistant acid phosphatase (TRAP) is considered to participate in this process. TRAP is synthesized as an inactive proenzyme, and in vitro studies have shown its activation by cysteine proteinases. In the present study, the possible involvement of the latter enzyme class in the in vivo modulation of TRAP was investigated using mice deficient for cathepsin K and/or L and in bones that express a high (long bone) or low (calvaria) level of cysteine proteinase activity. The results demonstrated, in mice lacking cathepsin K but not in those deficient for cathepsin L, significantly higher levels of TRAP activity in long bone. This higher activity was due to a higher number of osteoclasts. Next, we found considerable differences in TRAP activity between calvarial and long bones. Calvarial bones contained a 25-fold higher level of activity than long bones. This difference was seen in all mice, irrespective of genotype. Osteoclasts isolated from the two types of bone revealed that calvarial osteoclasts expressed higher enzyme activity as well as a higher level of mRNA for the enzyme. Analysis of TRAP-deficient mice revealed higher levels of nondigested bone matrix components in and around calvarial osteoclasts than in long bone osteoclasts. Finally, inhibition of cysteine proteinase activity by specific inhibitors resulted in increased TRAP activity. Our data suggest that neither cathepsin K nor L is essential in activating TRAP. The findings also point to functional differences between osteoclasts from different bone sites in terms of participation of TRAP in degradation of bone matrix. We propose that the higher level of TRAP activity in calvarial osteoclasts compared to that in long bone cells may partially compensate for the lower cysteine proteinase activity found in calvarial osteoclasts and TRAP may contribute to the degradation of noncollagenous proteins during the digestion of this type of bone.

Towards specific functions of lysosomal cysteine peptidases: phenotypes of mice deficient for cathepsin B or cathepsin L.

The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of 'knock out' mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class II-mediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNF-alpha induced hepatocyte apoptosis.

Differential oxidative injury in extrapancreatic tissues during experimental pancreatitis: modification of lung proteins by 4-hydroxynonenal.

Oxidative stress is considered to be a pathogenic factor for multisystem organ failure during acute pancreatitis. Infusion of 3% and 5% sodium taurocholate into the pancreatic duct of rats resulted in a 24-hr lethality of 8% and 82%, respectively. Kidney tissue showed a long-lasting significant elevation of malondialdehyde (lipid peroxidation). Only small amounts of this aldehyde were formed in the liver. In the lung malondialdehyde was increased during the first 6 hr after pancreatitis induction. Malondialdehyde levels were not different for pancreatitis initiated by 3% or 5% taurocholate. Protein-bound carbonyls (protein oxidation) in the tissues were not significantly changed at any time point. However, after infusion of 5% taurocholate, lung proteins were oxidatively modified by the product of lipid peroxidation, 4-hydroxynonenal. Another parameter characteristic for pancreatitis with high lethality was the high number of neutrophils in the lungs. We conclude that oxidative stress is important for the injury of extrapancreatic tissues during pancreatitis, but survival is determined by the degree of systemic inflammation.

Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis.

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb(-/-) animals was more than 80% lower than in ctsb(+/+) animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb(-/-) animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress.

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H(2)O(2) treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H(2)O(2) but not after 2 mM H(2)O(2). In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered.

Adaptation of protein carbonyl detection to the requirements of proteome analysis demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria.

The key technique in proteome analysis is high-resolution two-dimensional (2D) electrophoretic separation of proteins from biological samples. This method combines isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Derivatization of protein carbonyls with 2, 4-dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) immunoblotting is widely used for the detection of oxidatively modified proteins. In previous studies on adapting this method to 2D electrophoresis the derivatization step was carried out before and after the 2D procedure, resulting in an altered spot pattern and high background staining, respectively. The aim of the present experiments was to develop a method for protein derivatization with DNPH between the IEF and the SDS-PAGE steps. Mitochondria were exposed to 10 min hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and neutralized, and SDS-PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots were scanned and matched to a master gel, and the relative carbonyl content of each spot was calculated and compared for five experiments. Importantly, the spot patterns in DNPH-treated and untreated gels were not different. Protein carbonyls could be detected in 59 of the 125 matched spots. Although there was no significant increase in the total protein carbonyl content after hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl content. However, most protein spots did not show a change or even a decline (4 spots) in protein carbonyls.

Endoscopic treatment of clinically symptomatic leaks of thoracic esophageal anastomoses.

BACKGROUND: The mortality of thoracic anastomotic leakage following esophageal reconstruction has been reported to be as high as 60%. Early septic fulminant suture line leaks require rethoracotomy. In addition, however, clinically symptomatic leaks may also occur 2 to 7 days after resection of the esophagus. METHODS: Among 80 esophageal reconstructions performed between January 1994 and July 1998, a total of 7 (8.75%) clinically apparent leaks of thoracic anastomoses were observed. The standard treatment consisted of endoscopic lavage, drainage and subsequent closure of the defect by repeated intraluminal and submucosal applications of fibrin glue. In 2 patients a novel approach permitting rapid closure by plugging the fistula with a Vicryl-cylinder was tried. In 4 patients the effect of endoscopic treatment on the HLA-DR expression on monocytes was investigated and compared to 6 patients with intact anastomoses. RESULTS: All 7 patients were successfully treated via endoscopy. The cylinder plug achieved immediate closure of the leak. The measured change in HLA-DR expression reflected the improvement in the inflammatory response and thus documented the success of endoscopic treatment. CONCLUSIONS: Endoscopic management of thoracic leakages represents a safe and relatively noninvasive therapeutic option.

Oxidative stress in lung tissue induced by CO(2) pneumoperitoneum in the rat.

BACKGROUND: Clinical trials have found that the pneumoperitoneum has potentially hazardous side effects. The biochemical basis of organ injury induced by pneumoperitoneum is, however, not well defined. Since oxidative stress is believed to play an important role in many pathological conditions, we set out to examine oxidative stress markers in the lung, liver, kidney, and pancreas by using a rat model of laparoscopy with CO(2) pneumoperitoneum and comparing it to a group with gasless laparoscopy. METHODS: Malondialdehyde (for lipid peroxidation), protein-bound carbonyls (for protein oxidation), reduced and oxidized glutathione, and the neutrophil marker myeloperoxidase were evaluated in tissue homogenates at 2 h, 6 h, and 18 h after laparoscopy. Immunoblotting was used to analyze the modification of lung proteins by 4-hydroxynonenal at 6 h. RESULTS: Significant lipid peroxidation was found selectively in lungs at 2 h and 6 h after CO(2) pneumoperitoneum. This was accompanied by a loss of glutathione but only minor protein oxidation. Further, lung proteins were clearly modified by the aldehydic product of lipid peroxidation 4-hydroxynonenal. Myeloperoxidase in lungs increased continuously up to 18 h in both experimental groups, but there were higher levels in the group with pneumoperitoneum. CONCLUSION: Oxidative stress is likely to contribute to the impairment of pulmonary function after laparoscopic operations using a CO(2) pneumoperitoneum.

Degradation of hypochlorite-damaged glucose-6-phosphate dehydrogenase by the 20S proteasome.

Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.