Decrease In Protease Activity Research Articles

Purification and characterization of a serine protease secreted by Brevibacillus sp. KH3 for reducing waste activated sludge and biofilm formation

A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50°C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50°C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.

Tetrameric β를 이용한 고초균 포자에서의 미생물 표면 발현 모체 선별

Using tetrameric <TEX>${\beta}$</TEX>-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell <TEX>${\beta}$</TEX>-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. <TEX>${\beta}$</TEX>-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell <TEX>${\beta}$</TEX>-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell <TEX>${\beta}$</TEX>-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

Expression of Cystatin C in Human Esophageal Cancer

Cystatin C is a member of the cysteine protease inhibitors and its function is to decrease protease activity. A recent study showed that it was aberrantly expressed in many malignant tumors in association with tumor invasion and metastasis. We attempted to detect its expression in esophageal cancer tissues and adjacent reparative normal tissues. Samples of cancers and non-cancerous esophageal tissues were obtained as matched pairs from 30 surgery patients with esophageal cancer and paraffin embedded. The expression of cystatin C in tissues was investigated by immunohistochemistry. Fisher's exact test was used to analyze the relationship between esophageal cancer tissues and adjacent normal tissues. Furthermore, mRNA was extracted, and reverse transcriptase polymerase chain reaction was performed. The intensity of cystatin C immunostaining in tumor tissues was increased compared to that of adjacent normal tissues. mRNA expression of the cystatin C gene was greater in esophageal cancer than in normal tissues (P <0.05). Our results indicate that cystatin C may play an important role in the pathogenesis and metastasis of esophageal cancer.

Cerebral small-vessel disease protein HTRA1 controls the amount of TGF-β1 via cleavage of proTGF-β1

Cerebral small-vessel disease is a common disorder in elderly populations; however, its molecular basis is not well understood. We recently demonstrated that mutations in the high-temperature requirement A (HTRA) serine peptidase 1 (HTRA1) gene cause a hereditary cerebral small-vessel disease, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1 belongs to the HTRA protein family, whose members have dual activities as chaperones and serine proteases and also repress transforming growth factor-β (TGF-β) family signaling. We demonstrated that CARASIL-associated mutant HTRA1s decrease protease activity and fail to decrease TGF-β family signaling. However, the precise molecular mechanism for decreasing the signaling remains unknown. Here we show that increased expression of ED-A fibronectin is limited to cerebral small arteries and is not observed in coronary, renal arterial or aortic walls in patients with CARASIL. Using a cell-mixing assay, we found that HTRA1 decreases TGF-β1 signaling triggered by proTGF-β1 in the intracellular space. HTRA1 binds and cleaves the pro-domain of proTGF-β1 in the endoplasmic reticulum (ER), and cleaved proTGF-β1 is degraded by ER-associated degradation. Consequently, the amount of mature TGF-β1 is reduced. These results establish a novel mechanism for regulating the amount of TGF-β1, specifically, the intracellular cleavage of proTGF-β1 in the ER.

Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CARASIL): From Discovery to Gene Identification

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a single-gene disorder directly affecting the cerebral small blood vessels, that is caused by mutations in the HTRA1 gene encoding HtrA serine peptidase/protease 1 (HTRA1). CARASIL is the second known genetic form of ischemic, nonhypertensive, cerebral small-vessel disease with an identified gene, along with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The exact prevalence of CARASIL is currently unknown, and to date approximately 50 patients have been reported, most of them from Japan and two from China. Genetically, no founder haplotype has been identified, and thus the disease is expected to be found more widely. The main clinical manifestations of CARASIL are ischemic stroke or stepwise deterioration in brain functions, progressive dementia, premature baldness, and attacks of severe low back pain or spondylosis deformans/disk herniation. The most characteristic findings on brain magnetic resonance imaging are diffuse white matter changes and multiple lacunar infarctions in the basal ganglia and thalamus. Histopathologically, CARASIL is characterized by intense arteriosclerosis, mainly in the small penetrating arteries, without granular osmiophilic materials or amyloid deposition. CARASIL is a prototype single-gene disorder of cerebral small vessels secondary to and distinct from CADASIL. CARASIL-associated mutant HTRA1 exhibited decreased protease activity and failed to repress transforming growth factor-β family signaling, indicating that the increased signaling causes arteriopathy in CARASIL. Therefore, HTRA1 represents another new gene to be considered in future studies of cerebral small-vessel diseases, as well as alopecia and degenerative vertebral/disk diseases.

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO(4))) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO(4)) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO(4)) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO(4)) residues in both CS and DS. IdoA-GalNAc(4,6-SO(4)) units that account for approximately 40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO(4)) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO(4)) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.

Effect of Iron Fortified Wheat Flour on the Biology and Physiology of Red Flour Beetle, Tribolium castaneum (Herbst)

Iron overload in the fortified flour can influence the life stages and physiology of the insects. The present study was carried out to evaluate the effect of commercially available premix iron fortified flour as well as effect of different concentrations of post-mix iron fortified flour (30–5 ppm) on biology of red flour beetle, Tribolium castaneum (Hebrst.). Larval and pupal duration, total developmental time, fecundity and larval weights in two consecutive generations of beetle were compared with control treatment. Amylase and protease activities of gut of the beetle were also measured in premix and postmix flours. Results showed that larval mortality increased in two sources of premix iron flour when compared with control. Larval weight was reduced in first generation only. The larval mortality was significantly higher in 30 ppm postmix iron fortified flour than in other postmix concentrations and control treatment. The larvae of T. castaneum fed on two sources of premix and in various concentrations of postmix iron fortified flour revealed an increase in amylases and decrease in protease activities.

Effect of fermentation ensilaging on recovery of oil from fresh water fish viscera

Fish viscera are an important source for biomolecules such as protein and lipids. Studies were carried out to assess fermentation ensilaging as a method for recovery of oil from fresh water fish viscera. The total lipid content in the viscera ranged from 19% to 21% and upto 85% of this could be recovered by fermentation. Fermentation using added lactic cultures ( Enterococcus faecium HAB01 and Pediococcus acidilactici K7) did not show any advantage over natural fermentation with respect to recovery of oil and no differences were observed in fatty acid composition of oil recovered by fermentation using different cultures. Activity of acidic, neutral and alkaline proteases decreased during fermentation. Eventhough degree of protein hydrolysis increased during fermentation with highest (62.3%) being in fermentation using Pediococcus acidilactici K7 no differences were found in oil recovery. With decrease in protease activity the rate of change in degree of hydrolysis also decreased.

SarZ Promotes the Expression of Virulence Factors and Represses Biofilm Formation by Modulating SarA and agr in Staphylococcus aureus

Staphylococcus aureus is a remarkably adaptable organism capable of multiple modes of growth in the human host, as a part of the normal flora, as a pathogen, or as a biofilm. Many of the regulatory pathways governing these modes of growth are centered on the activities of two regulatory molecules, the DNA binding protein SarA and the regulatory RNAIII effector molecule of the agr system. Here, we describe the modulation of these regulators and their downstream target genes by SarZ, a member of the SarA/MarR family of transcriptional regulators. Transcriptional and phenotypic analyses of a sarZ mutant demonstrated that the decreased transcription of mgrA and the agr RNAIII molecule was accompanied by increased transcription of spa (protein A) and downregulation of hla (alpha-hemolysin) and sspA (V8 protease) transcripts when compared to its isogenic parent. The decrease in protease activity was also associated with an increase in SarA expression. Consistent with an increase in SarA levels, the sarZ mutant displayed an enhanced ability to form biofilms. Together, our results indicate that SarZ may be an important regulator governing the dissemination phase of S. aureus infections, as it promotes toxin expression while repressing factors required for biofilm formation.

Regulation of extracellular protease activity in soil in response to different sources and concentrations of nitrogen and carbon

A large proportion of the nitrogen (N) in soil is in the form of proteinaceous material. Its breakdown requires the activity of extracellular proteases and other decomposing enzymes. The goal of our study was to better understand how carbon (C) and N availability affect soil protease activity. Several aerobic incubations were carried out with ammonium (NH 4 +) and proteins as N sources and cellulose as the main C source. A strong increase in protease activity was observed when proteins were added, the increase depending on the amount of protein added and its solubility. Protease synthesis was clearly substrate induced, as NH 4 + had no effect. During this substrate induced phase, the addition of glucose but not NH 4 + resulted in protease repression, indicating that the level of protease synthesis was determined by the need for C rather than N. After 1 month of incubation, protease activity remained relatively constant over time and was closely related to microbial biomass N. Different concentrations of mineral N in soil solution had no direct effect on protease activity. However, during this stationary phase, protease activity could be repressed by glucose and NH 4 + in a treatment with low mineral N content while in treatments with a higher N availability no repression was observed. We hypothesize that the need for N determined protease activity in the treatment with limited N availability. The addition of NH 4 + allowed for reallocation of C and N away from protease synthesis, leading to the observed decrease in protease activity. The repression by glucose may be attributed to shifts in the pathway of microbial NH 4 + assimilation. The results emphasize the close links between the microbially mediated cycles of organic C and N.

DISCREPANCY BETWEEN PANCREATIC PROTEIN SECRETION AND CCK RELEASE IN RESPONSE TO BILE-PANCREATIC JUICE DIVERSION IN THE HYPERTROPHIED RAT PANCREAS INDUCED BY SYNTHETIC TRYPSIN INHIBITOR

Pancreatic exocrine secretion in conscious rats is mainly regulated by protease activities in the proximal intestine (luminal feedback regulation).1 Decrease in protease activities by ingestion of trypsin inhibitors (TI)s or diversion of bile-pancreatic juice (BPJ) stimulates pancreatic secretion2 and produces pancreatic hypertrophy via CCK release. Since CCK releasing factor, LCRF was purified from rat intestine,3 we determined whether the levels of LCRF, CCK, and protein secretion were synchronized to one another. Changes in pancreatic secretion, plasma CCK levels, and luminal LCRF contents produced by 2- and 4-h BPJ diversion, were determined in rats with 7-day TI treatment, and compared with controls. A diet containing 0.1% synthetic TI (ONO-3402) and a standard diet (CE-2) were fed in Wistar male rats for 7 days. After 7 day-treatment, the pancreatic wet weight was significantly higher in TI treated rats than the controls. For the experiments, on the third day of the feeding, rats were prepared with cannulas draining BPJ separately, and with a duodenal cannula and with an external jugular vein cannula. On the 7th day of the feeding, after 90 min of basal secretion with returning BPJ, BPJ were diverted. Changes in pancreatic secretions of fluid and protein were determined. After 2- and 4-h BPJ diversion, rats were sacrificed. Samples of 3-4 ml of blood were withdrawn through the venous cannula to measure plasma CCK concentrations. The proximal quarter of the small intestine was removed to measure LCRF content. The levels of LCRF and CCK were parallel (Fig. 1). Pancreatic protein secretion was higher in rats treated with TI than in controls throughout the experimental period. The plasma CCK and luminal LCRF content 4-h after BPJD was significantly lower in rats treated with TI than in controls. The hypertrophied pancreas may require a smaller dose of CCK to increase protein secretion.FIGURE 1: Changes in plasma CCK concentrations, LCRF content, and pancreatic protein secretion after 4-h BPJ diversion. *significantly different from the control values. TI; trypsin inhibitor. The numbers in the parentheses are the number of animals.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and β1 integrin

We studied the induction of protease activity by the laminin α1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin α1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and β1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing β1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and β1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and β1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and β1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.

Autoclaving kills soil microbes yet soil enzymes remain active

Biocidal treatment of soil is used to remove or inhibit soil microbial activity, and thus provides insight into the relationship between soil biology and soil processes. Chemical (soil pH, phosphodiesterase, protease) and biological (substrate induced respiration) characteristics of three contrasting soils from tropical savanna ecosystems in north Queensland, Australia were measured in field fresh samples and following autoclaving (121 °C/103 kPa for 30 min on two consecutive days). Autoclaving treatment killed the active soil microbial biomass and significantly decreased protease activity (∼90%) in all three soils. Phosphodiesterase activity in kaolinitic soils also significantly decreased by 78% and 92%. However, autoclave treatment of smectitic soil only decreased phosphodiesterase activity by 4% only. This study demonstrates phosphodiesterase can remain stable in extreme conditions. This might be a characteristic vital to the cycling of phosphorus in shrink–swell clays in Australian tropical savanna ecosystems.

Transcriptional and secretory responses of Entamoeba histolytica to mucins, epithelial cells and bacteria

Invasive intestinal amebiasis, caused by Entamoeba histolytica, is initiated with attachment of trophozoites to the colonic mucous layer, mucous disruption and/or depletion, and adherence to and cytolysis of host epithelial and inflammatory cells. A current working model of intestinal amebiasis suggests that the microenvironment of the host intestine, particularly intestinal mucins and the bacterial biofilm, may influence the behavior of pathogenic amebae. The invasive phenotype is dependent on expression of a number of virulence factors of which cysteine proteases provide the most convenient experimental probe because their activity is readily monitored. In the present study, we examined the interaction of E. histolytica with GalNAc, mucin, different epithelial cell lines and bacteria both by biochemical assays of protease release and transcriptional profiling using a previously validated genomic microarray. A significant down-regulation of released cysteine protease activity was observed when amebic trophozoites were grown with GalNAc, specific colonic cell lines and bacteria. Transcriptional profiling during GalNAc interaction revealed enhanced expression of the 170-kDa Gal/GalNAc lectin. Decreased protease activity during GalNAc interaction and enhanced expression of the Gal/GalNAc lectin gene are consistent with a program of commensal infection and mucus coat colonization mediated by the lectin. The down-regulation of cysteine protease activity following interaction with a colonic epithelial cell line parallels the presence of secretory mucin having a complex carbohydrate structure rich in Gal and GalNAc. In contrast, interaction of E. histolytica trophozoites with stomach porcine mucin enhanced cysteine protease (EhCP1 and EhCP2) secretion 3-fold. This suggests the specific composition of mucins may affect the Entamoeba phenotype. Transcriptional profiling revealed interaction of Entamoeba with intestinal bacteria induced protein kinase, ABC transporter, Rab family GTPase and hsp 90 gene expression. The enhanced expression of this gene cluster is consistent with enhanced phagocytosis of E. histolytica during interaction with bacteria.

The Effect of Epibrassinolide on Senescence in Wheat Leaves

ABSTRACTBrassinosteroids (BRs) are new groups of plant hormones with significant growth-promoting activity. Brassinolide (BL) is the most biologically active BR. BRs are considered as hormones, as they influence varied developmental processes like growth, germination of seeds, rhizogenesis, flowering and senescence. Senescence is regulated by phytohormones. This research shows that epibrassinolide (eBL) accelerates or delays senescence in wheat leaf segments. For this aim, epibrassinolide was applied exogenously into the excised wheat leaf segments. An increase in peroxidase activity and decrease in protease activity, chlorophyll content (at 10 and 0.1 μM. eBL) were detected. However, protein content decreased only at 10 μM. eBL. Finally, it was observed that eBR accelerated senescence specially at high concentration.

Statin Treatment Is Not Associated With Consistent Alterations in Inflammatory Status of Carotid Atherosclerotic Plaques

Anti-inflammatory qualities are held partially responsible for the reduction of cardiovascular events after statin treatment. We examined the phenotype of carotid atherosclerotic plaques harvested during carotid endarterectomy in relation to the previous use of different statins prescribed in clinical practice. Three hundred and seventy-eight patients were included. Atherosclerotic plaques were harvested, immunohistochemically stained and semiquantitively examined for the presence of macrophages (CD68), smooth muscle cells, collagen and fat. Adjacent atherosclerotic plaques were used to study protease activity and interleukin levels. Patients' demographics were recorded and blood samples were stored. Serum cholesterol, low-density lipoprotein, apolipoprotein B, and C-reactive protein levels were lower in patients treated with statins compared with patients without statin treatment. Atheromatous plaques were less prevalent in patients receiving statins compared with patients without statin therapy (29% versus 42%, P=0.04). An increase of CD68 positive cells was observed in patients receiving statins compared with nonstatin treatment (P=0.05). This effect was specifically related to atorvastatin treatment. In patients treated with atorvastatin, the increased amount of CD68 positive cells were not associated with increased protease activity. In contrast, a dose-dependent decrease in protease activity was shown in the atorvastatin group. Interleukin 6 expression was lower in plaques obtained from patients treated with statins (P=0.04). Statin use may exert pleiotropic effects on plaque phenotype. However, not the presence of macrophages but activation with subsequent protease and cytokine release may be attenuated by statin use.

Effect of different metals on protease activity in sunflower cotyledons

Proteases are crucial for living cells and play a role in plant cell adaptation to environmental conditions. Oxidative stress produced oxidized proteins which are selectively degraded by proteases. To understand the role of proteolysis in response to metal stress, sunflower plants (a plant suitable for phytoremediation) were treated with 100 µM of CdCl 2 , CuCl 2 , AlCl 3 , CoCl 2 , PbCl 2 , CrCl 3 , NiCl 2 , HgCl 2 or ZnCl 2 . Changes in protease activity, gelatinase profile and protein oxidation were examined in sunflower cotyledons. Our results indicate that this tissue has mainly acid proteases belonging to different classes. Although all metals (except Zn) increased protein oxidation (62, 57, 112, 74, 74, 68, 64 and 40% for Pb, Al, Ni, Cd, Hg, Co, Cr and Cu over the control), they altered proteolysis in different ways. Pb, Al and Ni treatment decreased protease activity 22, 28 and 30% respect to control while Cd and Hg increased this activity in 23 and 27%. In Zn, Cu and Co treatments protease activity remained similar to control treatment. These results indicate that different proteases are involved in plant defence against metal toxicity. However, the identification of specific oxidized proteins involved in this process and the metal effect on class specific proteases should provide greater information.

Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation

The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.

Protein kinase C modulates the pulmonary inflammatory response in acute pancreatitis

The present study aims at evaluating the role of protein kinase C (PKC) in the development of acute lung injury, production of inflammatory mediators and expression of adhesion molecules on leukocytes after induction of acute pancreatitis (AP). AP was induced by the intraductal infusion of 5% sodium taurodeoxycholate in the rat. The animals had the PKC inhibitor polymyxin B administered intraperitoneally 30min prior to induction of AP. Levels of protein content, protease activity, cytokines and chemokines in bronchoalveolar lavage fluid (BALF) were assessed 1 and 6h after AP induction. Adhesion molecule expression on leukocytes were measured by flowcytometry. Pretreatment with polymyxin B prevented against acute pancreatitis-induced lung injury and the otherwise occurring increases in TNF-alpha, IL-1beta, MCP-1 and IL-10, as well as against the decreases in IL-2, IFNgamma and TIMP-1, decreased protease activity and down-regulation of CD31, CD54 and CD62L on recruited neutrophils and macrophages in BALF. The results indicate that the leukocyte response in acute pancreatitis vary depending on leukocyte subpopulation. It seems that activation of the PKC signalling pathway may play an important role in pancreatitis-associated lung injury.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets