Neutral Serine Protease Research Articles

Aspartic protease supplementation enhancing the performance, carcass characteristics, nutrient digestibility and economic viability, without changing blood parameters and salivary cortisol of pigs

Aiming to study the performance, carcass characteristics, nutrient digestibility, blood parameters, salivary cortisol levels, and economic viability of pigs administered aspartic protease, a total of 135 pigs were housed in pens in a randomized block design, divided into five treatments with nine replications. The experimental diets were positive control (PC), basic diet with a 5.0% reduction in protein and amino acid requirements; negative control (NC) with a 7.5% reduction in protein and amino acid requirements; NC + 100 g/mT of aspartic protease (NC100); NC + 150 g/mT of aspartic protease (NC150); and NC + 200 g/mT of neutral serine protease (NC200). The inclusion of protease, independently of the source and amount, increased the average daily weight gain (P < 0.05) of animals compared with the control treatments (PC and NC), improved feed conversion (P < 0.05) in early stages, and improved diet digestibility (P < 0.05) compared with the PC. Treatment with NC150 and NC200 resulted in greater carcass weights (P < 0.05) than treatment with the PC. NC100 led to a greater carcass yield than PC (P < 0.05), and NC150 resulted in a greater loin eye area than PC (P < 0.05). No differences (P > 0.05) in the blood parameters or salivary cortisol levels were found. Regarding economic viability, proteases increased the profitability, with NC150 leading to the best results. Thus, the use of aspartic proteases is recommended to improve performance and further facilitate pork production.

Neutrophil proteinase 3 — An LDL- and HDL-proteolyzing enzyme with a potential to contribute to cholesterol accumulation in human atherosclerotic lesions

Neutrophil proteinase 3 (PR3) is a multifunctional neutral serine protease involved in the regulation of pro-inflammatory processes, but its potential causal roles in the lipid-driven responses in atherosclerosis have remained unexplored. This study aimed to investigate the presence of PR3 in human atherosclerotic lesions and the ability of this protease to modify the structure and functions of LDL and HDL particles in vitro. Coronary artery segments were collected from autopsied subjects and immunostained for PR3. Atherosclerotic lesions but not normal intima contained PR3. Incubation of LDL particles with the PR3 led to extensive degradation of their apoB-100 component and strongly increased their binding strength to isolated human aortic proteoglycans in vitro. Moreover, cultured human monocyte-derived macrophages avidly ingested the PR3-modified LDL particles and were converted into foam cells. Incubation of HDL particles with PR3 led to proteolysis of their major apolipoproteins (apoA-I, apoA-II, and apoE) and impaired their ability to promote cholesterol efflux from the macrophage foam cells. We conclude that PR3 is present in human atherosclerotic lesions and that this neutral serine protease has proatherogenic properties. Thus, by proteolytically modifying LDL and HDL particles, PR3 may promote cholesterol accumulation both extra- and intracellularly in atherosclerotic lesions, and so contribute to the lipid-driven component of atherogenesis.

Anticoagulant and antiplatelet activities of novel serine protease purified from seeds of Cucumis maderaspatensis

In this study, we report the biochemical characterization of a novel serine protease from seeds of Cucumis maderaspatensis, aimed with assessing the anticoagulant and antiplatelet activities. The purified serine protease was obtained by subjecting the seed extract to ammonium sulphate precipitation followed by anion exchange and gel filtration chromatography. Twenty seven-fold purification with the specific activity of 884.2 U/mg of protease activity was obtained. The characterization of the novel protease enzyme activity for optimum temperature, pH and effect of different protease inhibitors and metal ions were measured using caseinolytic assay and casein zymogram. The relative molecular mass of the novel neutral serine protease (CmSP) is ~ 32kDa. Its anticoagulant was determined by assessing the delay in plasma re-calcification time in both platelet-rich and platelet-poor plasma. The antiplatelet activity of serine protease was demonstrated by inhibition of agonists induced platelet aggregation; it was in the order of Epinephrine > Adenosine tri phosphate. Further studies would decipher the mechanism of action to understand its therapeutic potential as an antiplatelet and anticoagulant molecule.

Increasing Neutrophil Elastase and Decreasing Its Inhibitor, Alpha 1-Antitrypsin, in Patients with Non-Alcoholic Fatty Liver Disease

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in modern human life. Serum protease and protease inhibitor concentrations can be potentially considered the important risk factors for NAFLD, together with the main risk factors of this disorder, such as diabetes, obesity, and dyslipidemia. Objectives: The present study aimed to investigate the plasma level of a neutral serine protease family called neutrophil elastase (NE), as well as its inhibitor, alpha1-antitrypsin (A1AT), in NAFLD patients compared to normal subjects. Additionally, the most common deficient variants of A1AT (S and Z) were determined in both patient and healthy groups. Methods: The study included 54 consecutive unrelated NAFLD patients and 120 matched healthy subjects as controls over two years. Plasma concentrations of A1AT and neutrophil elastase activity were determined for all patients and controls using the enzyme immunoassay and alpha1-proteinase inhibitor-immunoglobulin A complex, respectively. The A1AT variants of PiZ (rs28929474) and PiS (rs17580) were analyzed by the polymerase chain reaction. Results: The NAFLD patient group had more plasma elastase activity than the healthy control group (1.7 (0.5) vs. 1.3 (0.5) (U/mL), P < 0.01). Furthermore, the plasma A1AT level was significantly lower in NAFLD patients than in controls (216.1 (171.3) vs. 244.6 (152.9) (mg/dL), P = 0.01). The heterozygous carriages of PiS and PiZ variants were not statistically different between NAFLD patients and controls. Conclusions: Higher elastase activity and lower A1AT concentration in plasma can be considered the potential prognostic and diagnostic biomarkers in patients with NAFLD.

Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Abstract Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage.

Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate.

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40°C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

Neutral serine proteases of neutrophils.

Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications.

Vacuolar proteases from Candida glabrata: Acid aspartic protease PrA, neutral serine protease PrB and serine carboxypeptidase CpY. The nitrogen source influences their level of expression

BackgroundThe Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. AimsThe present paper is the first report on proteolytic activity in the C. glabrata vacuole. MethodsBiochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. ResultsOur results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. ConclusionsThe proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen.

Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. pl antarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physicochemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metaldependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen). These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

Neutral serine protease from Penicillium italicum. Purification, biochemical characterization, and use for antioxidative peptide preparation from Scorpaena notata muscle.

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0-9.0. The protease was activated by divalent cations such as Ca(2+) and Mg(2+). Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.

PURIFICATION AND CHARACTERIZATION OF THE NEWLY THERMOSTABLE PROTEASE PRODUCED BY Brevibacillus thermoruber LII ISOLATED FROM PADANG CERMIN HOTSPRING, INDONESIA

Thermo stability is among of the vital enzyme characteristics for industrial application. Brevibacillus thermoruber LII was obtained as a potential isolate from the previous researchwhich screened the potential thermostable protease producing bacteria from Indonesian hotspring.The newly thermostable protease produced by thermophilic Brevibacillus thermoruber LII hadbeen purified and characterized. It was predicted that the pure enzyme obtained from Brevibacillusthermoruber LII was homo hexameric, having molecular weight of 36 kDa unit protein and itsnative was 215 kDa. In addition, it was also a neutral metalo serine protease according tobiochemical tests that it was totaly inhibited by PMSF (Phenylmethanesulfonyl fluoride) and EDTA(Ethylenediaminetetraacetic acid). It showed optimum activity at pH of 8 and active in acidic buffer(up to pH of 4). All of metal ion in the form of chloride salt (2.5 mM) which were tested on theenzyme enhanced the enzyme activity but Li2+. Ca2+ion increased the activity and the stability ofenzyme against thermal. The enzyme also showed the stability against solvent. The protease LIIhad optimum temperature at 60oC without CaCl 2and 80 – 85oC with addition of 2.5 mM CaCl 2. TheK Mand V maxvalues for the purified protease LII were 27.2 mg/ml or 0.362 – 0.272 M for substrateHammersteinCasein (MM 75–100 kDa) and 261.1 µg/minute/ml, respectively.

Chryseobacterium sp. JK1이 분비하는 세포외 단백질분해효소 특성

이전의 연구에서 토양으로부터 많은 양의 세포외 단백질분해 효소를 생산하는 신종 중온세균 Chryseobacterium sp. JK1를 분리하였다. 이 균주가 생산하는 단백질 분해효소의 특성조사 결과 최적반응온도와 pH는 각각 <TEX>$40^{\circ}C$</TEX>와 7.0이였으며, 좁은 최적온도 구간과 비교적 넓은 pH 구간인 pH 6.0-9.0에서 높은 활성을 보여주었다. 그리고 단백질 분해효소는 EDTA 또는 EGTA, PMSF와 금속이온 <TEX>$Ag^+$</TEX> 또는 <TEX>$Cu^{2+}$</TEX>의 첨가에 의해 강하게 저해 되었으며, <TEX>$Al^{3+}$</TEX>의 첨가에 의해 약하게 저해되었다. Pepstatin과 금 속이온 <TEX>$K^+$</TEX>, <TEX>$Ca^{2+}$</TEX>, <TEX>$Na^+$</TEX>, <TEX>$Fe^{2+}$</TEX> 또는 <TEX>$Mg^{2+}$</TEX>의 첨가는 저해에 큰 영향을 주지 않았다. 이와 반대로 단백질분해효소는 이가 금속이온인 <TEX>$Mn^{2+}$</TEX> (5 mM)의 첨가에 의해 효소활성이 향상되었다. 농축된 배양 상등액의 활성염색 분석으로 67과 145 kDa 크기의 주요 밴드 두 개가 관찰되었다. 이러한 결과들로 Chryseobacterium sp. JK1 균주가 식품산업에 응용 가능한 세포외 중성의 serine 단백질 분해효소를 생산한다는 것을 알 수 있었다. A novel Chryseobacterium sp. JK1 strain isolated from soil had been reported that this isolate produced large amount of extracellular protease at mesophilic temperature in previous study. The optimal temperature and pH of extracellular protease were <TEX>$40^{\circ}C$</TEX> and 7.0, respectively, showing narrow range of optimal temperature and relatively broad activity from pH 6.0 to 9.0. In addition, the protease showed greatest activity against skim milk and lowest against bovine serum albumin (BSA). The protease strongly inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) or phenylmethylsulfonyl fluoride (PMSF), and addition of cation <TEX>$Ag^+$</TEX> or <TEX>$Cu^{2+}$</TEX>, and slightly inhibited by <TEX>$Al^{3+}$</TEX>. No significant inhibition was found with pepstatin, and addition of cation, <TEX>$K^+$</TEX>, <TEX>$Ca^{2+}$</TEX>, <TEX>$Na^+$</TEX>, <TEX>$Fe^{2+}$</TEX> or <TEX>$Mg^{2+}$</TEX>. On the contrary, protease was enhanced by addition of divalent cation <TEX>$Mn^{2+}$</TEX> (5 mM). Zymography analysis of concentrated culture supernatant revealed two major bands at 67 and 145 kDa. These results suggest that Chryseobacterium sp. JK1 strain produced extracellular neutral serine proteases which could apply in food industry.

Cutaneous vasculitis: Report of 117 cases

Proteinase-3 mRNA expressed by glomerular epithelial cells correlates with crescent formation in Wegener's granulomatosis.Wegener's granulomatosis (WG) is characterized by systemic vasculitis with crescentic glomerulonephritis (CGN) and circulating autoantibodies directed against neutrophil cytoplasmic antigens (ANCA). Proteinase 3 (PR-3), a neutral serine proteinase in neutrophils implicated in the growth control of myeloid cells, has been identified as the target antigen for ANCA in WG. Since the kidneys are frequently involved in WG, we studied the in situ expression of PR-3 by renal parenchymal cells.We assessed the expression of PR-3 in kidney biopsies of 15 patients with WG by immunohistochemistry (IHC) and in situ hybridization (ISH). Normal kidney tissue served as the control.We detected PR-3 mRNA and PR-3 protein in distal tubular epithelial cells (TECs) and glomerular epithelial cells (GECs) in normal kidney tissue and in CGN. Furthermore, a strong glomerular PR-3mRNA expression restricted to the site of cellular crescents was detected in patients with WG. The analysis of 144 glomeruli with cellular or sclerotic crescents revealed a positive correlation of glomerular PR-3mRNA expression with the percentage of cellular crescents per glomerulus. The capability of human TECs and GECs to synthesize PR-3 was confirmed by Northern blot and ISH on cultured cells.These data provide evidence that nonhematopoetic renal parenchymal cells express PR-3 and that glomerular expression of PR-3 is associated with crescent formation in WG. Our findings suggest that renal parenchymal cells may directly be involved in the pathogenesis of CGN in WG.

Cathepsin G and neutrophil elastase play critical and nonredundant roles in lung-protective immunity against Streptococcus pneumoniae in mice.

Neutrophil serine proteases cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3) have recently been shown to contribute to killing of Streptococcus pneumoniae in vitro. However, their relevance in lung-protective immunity against different serotypes of S. pneumoniae in vivo has not been determined so far. Here, we examined the effect of CG and CG/NE deficiency on the lung host defense against S. pneumoniae in mice. Despite similar neutrophil recruitment, both CG knockout (KO) mice and CG/NE double-KO mice infected with focal pneumonia-inducing serotype 19 S. pneumoniae demonstrated a severely impaired bacterial clearance, which was accompanied by lack of CG and NE but not PR3 proteolytic activity in recruited neutrophils, as determined using fluorescence resonance energy transfer (FRET) substrates. Moreover, both CG and CG/NE KO mice but not wild-type mice responded with increased lung permeability to infection with S. pneumoniae, resulting in severe respiratory distress and progressive mortality. Both neutrophil depletion and ablation of hematopoietic CG/NE in bone marrow chimeras abolished intra-alveolar CG and NE immunoreactivity and led to bacterial outgrowth in the lungs of mice, thereby identifying recruited neutrophils as the primary cellular source of intra-alveolar CG and NE. This is the first study showing a contribution of neutrophil-derived neutral serine proteases CG and NE to lung-protective immunity against focal pneumonia-inducing serotype 19 S. pneumoniae in mice. These data may be important for the development of novel intervention strategies to improve lung-protective immune mechanisms in critically ill patients suffering from severe pneumococcal pneumonia.

Cathepsin G and neutrophil elastase play critical and non-redundant roles in lung protective immunity against S. pneumoniae in mice

Neutrophil serine proteases cathepsin G (CtsG), neutrophil elastase (NE) and proteinase-3 (PR3) have recently been shown to contribute to killing of Streptococcus pneumoniae in vitro. However, their relevance in lung protective immunity against different serotypes of S. pneumoniae in vivo has not been determined so far. We here infected CtsG-deficient mice and CtsG/NE deficient mice with two serotypes of S. pneumoniae exhibiting different virulence profiles. Despite similar neutrophil recruitment, both CtsG KO mice and, even more accentuated, CtsG/NE KO mice infected with serotype 19 S. pneumoniae demonstrated a severely impaired bacterial clearance, which was accompanied by lack of CtsG and NE but not PR-3 proteolytic activity in recruited neutrophils, as determined by FRET analysis. Moreover, both CtsG and CtsG/NE KO mice but not wild-type mice responded with increased lung permeability to infection with S. pneumoniae, resulting in severe respiratory distress and progressive mortality. On the other hand, CtsG/NE deficient mice demonstrated a similar mortality after infection with highly invasive serotype 2 S. pneumoniae as compared to wild-type mice. This is the first study to show a differenzial contribution of neutral serine proteases CtsG and NE to regulate lung protective immunity against focal pneumonia-inducing serotype 19 but not sepsis-inducing serotype 2 S. pneumoniae in mice. These data may be important for the development of novel intervention strategies to improve lung protective immune mechanisms in critically ill patients suffering from severe pneumococcal pneumonia.

Neutrophil Elastase Inhibition: A New Cancer Therapy

Cancer cells enter the circulation and attach to endothelial cells to pass through them and migrate over a distance to enter the tissue of the metastatic organ to proliferate there. In the same way, neutrophils drift in blood and adhere loosely to adhesive molecules on the endothelial cells in an inflamed area. They roll along the endothelial cells and then adhere closely to the endothelial cells to penetrate vessel wall. Neutrophils can destroy the basement membrane and migrate over a distance to fight against foreign bodies. Thus, the process that both of them follow is quite the same. Neutrophil elastase (NE) is a neutral serine protease which has broad substrate specificity under the physiological conditions, and its excessive production results in digestion of not only elastin, but also other extracellular matrix proteins. This minireview summarizes our recent experimental and clinical studies on NE/NE inhibition and cancer/cancer treatment based on our original view point. Keywords: Neutrophil Elastase, Cancer Therapy, endothelial cells, metastatic organ, neutral serine protease, plasminogen activator (PA)

Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45-50 degrees C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65 degrees C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.

Enamel maturation.

Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.

Azurocidin is a novel antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in systemic vasculitis.

We isolated a 27-kD protein using cation exchange chromatography from an acid extract of neutrophil granules. N-terminal amino acid sequence analysis of the first 10 residues showed that this protein is azurocidin, a member of the family of neutral serine proteinase found in the neutrophil, which shares amino acid sequence homology with the three other neutral serine proteinases, elastase, proteinase 3 (PR3) and cathepsin G, but unlike them is without proteolytic activity. To test whether, in addition to these proteases, azurocidin might be a target for the humoral autoimmune responses associated with human vasculitis, 185 indirect immunofluorescence (IIF)-positive ANCA sera, made up of four groups of sera with specificities for PR3 (n=37), myeloperoxidase (MPO; n=50), bactericidal/permeability-increasing protein (BPI; n=41) and sera that recognized none of them (triple negative, n=57), and 46 normal sera were screened for IgG anti-azurocidin antibodies using an ELISA incorporating purified azurocidin. Twenty of the 185 IIF-positive sera and 2/46 normal sera displayed reactivity with azurocidin. Positive sera could blot the 27-kD band by Western blot analysis. Further study of the 20 positive sera revealed that: (i) 10 also had autoreactivity for MPO, of which six additionally recognized lactoferrin; (ii) two had reactivity with BPI; (iii) the remaining eight sera were positive only for azurocidin. All 20 sera were from patients with systemic vasculitis, and four of the six sera with triple reactivity (for azurocidin, MPO and lactoferrin) were from patients with hydralazine-induced vasculitis. We concluded that: (i) azurocidin is a novel ANCA antigen; (ii) anti-azurocidin antibodies from a subgroup of patients might represent the consequence of a drug-induced multi-clone activation.