An efficient dehairing system supported by oxidative-enzymatic auxiliary towards sustainability.

A method of dehairing of goat skins using oxidative chemicals and protease enzymes has been attempted. The dehairing process is one of the important and essential steps in leather making, where hair is removed by lime and sodium sulphide in the conventional process. This conventional dehairing system generates a higher amount of pollution problem as compared to the other unit operations and unit processes. In this work, dehairing of the goat skins through oxidative agents namely magnesium peroxide and protease enzyme has been attempted. For this, protease has been produced from Bacillus sp. at the laboratory level and the activity was found. The dehairing of goat skins takes place for the duration of 14-16 h. The leather produced with the experimental sample showed comparable organoleptic and strength properties with the conventional sample. This method paved the way for the reduction of pollution loads especially BOD, COD, and TDS to the level of 59, 27, and 77%, respectively, in comparison with the control sample. The reaction kinetics for the formation of the ligand-macromolecular complex is found in the isothermal titration calorimetry (ITC) experiment and a mathematical model has been formulated. The dyed crust leather showed comparable colour properties. In addition to that, there is a reduction in processing time for leather making through skipping reliming and deliming processes which are said to be another advantage of this method. The physical strength properties of the experimental leather were also comparable with conventionally produced leather.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

BACKGROUND: Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS: Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION: Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

Recent Progress in the Molecular Imaging of Nonalcoholic Fatty Liver Disease.

Pathological fibrosis of the liver is a landmark feature in chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Diagnosis and assessment of progress or treatment efficacy today requires biopsy of the liver, which is a challenge in, e.g., longitudinal interventional studies. Molecular imaging techniques such as positron emission tomography (PET) have the potential to enable minimally invasive assessment of liver fibrosis. This review will summarize and discuss the current status of the development of innovative imaging markers for processes relevant for fibrogenesis in liver, e.g., certain immune cells, activated fibroblasts, and collagen depositions.

Stable and scalable SERS tags conjugated with neutravidin for the detection of fibroblast activation protein (FAP) in primary fibroblasts.

SERS tags are a class of nanoparticles with great potential in advanced imaging experiments. The preparation of SERS tags however is complex, as they suffer from the high variability of the SERS signals observed even at the slightest sign of aggregation. Here, we developed a method for the preparation of SERS tags based on the use of gold nanostars conjugated with neutravidin. The SERS tags here obtained are extremely stable in all biological buffers commonly employed and can be prepared at a relatively large scale in very mild conditions. The obtained SERS tags have been used to monitor the expression of fibroblast activation protein alpha (FAP) on the membrane of primary fibroblasts obtained from patients affected by Crohn's disease. The SERS tags allowed the unambiguous identification of FAP on the surface of cells thus suggesting the feasibility of semi-quantitative analysis of the target protein. Moreover, the use of the neutravidin-biotin system allows to apply the SERS tags for any other marker detection, for example, different cancer cell types, simply by changing the biotinylated antibody chosen in the analysis.

Gluten Degradation, Pharmacokinetics, Safety, and Tolerability of TAK-062, an Engineered Enzyme to Treat Celiac Disease.

BACKGROUND AND AIMS: Celiac disease (CeD) is an immune-mediated disorder triggered by the ingestion of gluten. Despite adhering to a gluten-free diet (the only management option available to patients with CeD), many patients continue to experience symptoms and intestinal injury. Degradation of immunogenic fractions of gluten peptides in the stomach has been proposed as an approach to reduce toxicity of ingested gluten; however, no enzymes evaluated to date have demonstrated sufficient gluten degradation in complex meals. TAK-062 is a novel, computationally designed endopeptidase under development for the treatment of patients with CeD. METHODS: Pharmacokinetics, safety, and tolerability of TAK-062 100-900 mg were evaluated in a phase I dose escalation study in healthy participants and patients with CeD. Gluten degradation by TAK-062 was evaluated under simulated gastric conditions in vitro and in healthy participants in the phase I study, with and without pretreatment with a proton pump inhibitor. Residual gluten (collected through gastric aspiration in the phase I study) was quantified using R5 and G12 monoclonal antibody enzyme-linked immunosorbent assays. RESULTS: In vitro, TAK-062 degraded more than 99% of gluten (3 g and 9 g) within 10 minutes. In the phase I study, administration of TAK-062 was well tolerated and resulted in a median gluten degradation ranging from 97% to more than 99% in complex meals containing 1-6 g gluten at 20-65 minutes postdose. CONCLUSIONS: TAK-062 is well tolerated and rapidly and effectively degrades large amounts of gluten, supporting the development of this novel enzyme as an oral therapeutic for patients with CeD. (ClinicalTrials.gov: NCT03701555, https://clinicaltrials.gov/ct2/show/NCT03701555.).

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria.

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( PhotobacteriumNlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG.IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.

Three Distinct Stroma Types in Human Pancreatic Cancer Identified by Image Analysis of Fibroblast Subpopulations and Collagen.

PURPOSE: Cancer-associated fibroblasts have emerged to be highly heterogenous and can play multifaceted roles in dictating pancreatic ductal adenocarcinoma (PDAC) progression, immunosuppression, and therapeutic response, highlighting the need for a deeper understanding of stromal heterogeneity between patients and even within a single tumor. We hypothesized that image analysis of fibroblast subpopulations and collagen in PDAC tissues might guide stroma-based patient stratification to predict clinical outcomes and tumor characteristics. EXPERIMENTAL DESIGN: A novel multiplex IHC-based image analysis system was established to digitally differentiate fibroblast subpopulations. Using whole-tissue slides from 215 treatment-naïve PDACs, we performed concurrent quantification of principal fibroblast subpopulations and collagen and defined three stroma types: collagen-rich stroma, fibroblast activation protein α (FAP)-dominant fibroblast-rich stroma, and α smooth muscle actin (ACTA2)-dominant fibroblast-rich stroma. These stroma types were assessed for the associations with cancer-specific survival by multivariable Cox regression analyses and with clinicopathologic factors, including CD8+ cell density. RESULTS: FAP-dominant fibroblasts and ACTA2-dominant fibroblasts represented the principal distinct fibroblast subpopulations in tumor stroma. Stroma types were associated with patient survival, SMAD4 status, and transcriptome signatures. Compared with FAP-dominant fibroblast-rich stroma, collagen-rich stroma correlated with prolonged survival [HR, 0.57; 95% confidence interval (CI), 0.33-0.99], while ACTA2-dominant fibroblast-rich stroma exhibited poorer prognosis (HR, 1.65; 95% CI, 1.06-2.58). FAP-dominant fibroblast-rich stroma was additionally characterized by restricted CD8+ cell infiltrates and intense neutrophil infiltration. CONCLUSIONS: This study identified three distinct stroma types differentially associated with survival, immunity, and molecular features, thereby underscoring the importance of stromal heterogeneity in subtyping pancreatic cancers and supporting the development of antistromal therapies.

Progress Curve Analysis of the one stage chromogenic assay for ecarin.

Snake venom prothrombin activators such as Ecarin are readily assayed by continuous spectrophotometric monitoring of p-nitroaniline production in a one step assay containing prothrombin and a p-nitroanilide peptide substrate for thrombin. The coupled reactions result in accelerating p-nitroaniline (pNA) production over the course of the assay giving non-linear progress curves, from which initial velocities are not readily obtained. Most studies therefore resort to approximate estimates of activity, based on the absorbance reached at an arbitrary time. A simple kinetic analysis of the coupled reactions shows that the early points of such curves should be fitted by second order polynomials, representing the accelerating reaction rate in µmol pNA/min/min. The first derivative of the polynomial then gives the increasing velocity of pNA production in µmol pNA/min over the time course of the assay. We demonstrate here that, with the substrate S2238, these rates can be converted to absolute thrombin concentrations using the Michaelis-Menten equation, substituted with values for kcat and Km. These thrombin concentrations increase linearly over the time course of the assay allowing the activity to be expressed in units, defined as µmol product/min, most commonly used to report enzyme activity.

Acoustic biosensors for ultrasound imaging of enzyme activity.

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors-molecules that 'light up' in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce nonlinear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.

Advances in understanding the potential therapeutic applications of gut microbiota and probiotic mediated therapies in celiac disease.

INTRODUCTION: Celiac Disease (CD) is an autoimmune enteropathy caused by exposure to gluten in genetically predisposed people. While gluten is the main driving force in CD, evidence has shown that microbiota might be involved in the pathogenesis, development, and clinical presentation of CD. Microbiota manipulation may modify its functional capacity and may be crucial for setting-up potential preventive or therapeutic application. Moreover, probiotics are an excellent source of endopeptidases for digesting gluten. AREAS COVERED: In this narrative review we illustrate all the recent scientific discoveries in this field including CD pathogenetic mechanism where gut microbiota might be involved and possible use of probiotics in CD prevention and treatment. EXPERT OPINION: In the future, probiotics could be used as an add-on medication for strengthening/facilitating the gluten-free diet (GFD) and improving symptoms; the prospect of using it for therapeutic purposes is to be sought in a more distant future.

Simple method for analyzing the purity of protease-containing samples by acid-treatment SDS-PAGE.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to analyze the purity of a protein. However, it is necessary to denature (via boiling) the samples before subjecting them to electrophoresis. In the case of protease-containing samples, autolysis of the protease can occur, affecting the accuracy of results. In this study, we investigated the methods for analyzing the purity of Dispase I, a thermolysin-like neutral protease. When we analyzed D protease, a neutral metalloprotease component of Dispase I and highly purified Dispase I using the conventional SDS-PAGE method, a large number of bands were detected in both cases. These bands (putative D protease fragments) were assumed to result from autolysis. To inactivate D protease (optimal pH 7-8), 0.05 M sulfuric acid was utilized (pH 0.7-2.5). Using a conventional sample preparation solution, acid-treated Dispase I samples (without boiling) were made, and SDS-PAGE (15% w/v gel) was carried out. Our findings show that autolysis was inhibited under strong acidic conditions, and protein denaturation was achieved by treatment with sulfuric acid and SDS without boiling. Using this modified SDS-PAGE method, the purities of Dispase I and the purified enzyme were determined to be approximately 80% and 98%, respectively. Furthermore, we demonstrated that this method can be applied for the analysis of other samples including non-acidic proteases (e.g., thermolysin, subtilisin, and trypsin) and protease-contaminated samples (a mixed solution of albumin and D protease).

Establishment of an HPLC-based method to identify key proteases of proteins in vitro.

Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.

Recent Advances in Activity-Based Protein Profiling of Proteases.

The activity of proteases is tightly regulated, and dysregulation is linked to a variety of human diseases. For this reason, ABPP is a well-suited method to study protease biology and the design of protease probes has pushed the boundaries of ABPP. The development of highly selective protease probes is still a challenging task. After an introduction, the first section of this chapter discusses several strategies to enable detection of a single active protease species. These range from the usage of non-natural amino acids, combination of probes with antibodies, and engineering of the target proteases. A next section describes the different types of detection tags that facilitate the read-out possibilities including various types of imaging methods and mass spectrometry-based target identification. The power of protease ABPP is illustrated by examples for a selected number of proteases. It is expected that some protease probes that have been evaluated in animal models of human disease will find translation into clinical application in the near future.

A Sensitive Assay for Proteases in Bioaerosol Samples: Characterization and Quantification of Airborne Proteases in Salmon Industry Work Environments.

Proteases are probably underestimated exposure agents in bioaerosols. Their roles as barrier disrupters in allergic sensitization and activators of innate inflammation call for more attention in exposure-response studies. The main objectives of this study was (i) to establish a suitable method for detection of small quantities of proteases in filtered air samples and (ii) to utilize the method to characterize exposure to proteases in a salmon industry work environment. Analysis of proteases in filtered air samples was based on zymography, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 0.1% gelatin as substrate added in the polyacrylamide gel. Gelatinase activity was evident as cleared (unstained) regions. The area of these regions was quantified using image analysis (UVP Vision Works®). Standard curves with known amounts of active porcine trypsin were added to each gel. Validation of 11 non-linear standard curves showed R2 (range) = 0.8989-0.9882, limit of detection = 0.056 nM, lower limit of quantification = 0.161 nM, and coefficients of variations (range) = 20-28%. Sampling of bioaerosols in salmon industry was performed using polytetrafluoretylene filters with an airflow of 3 l min-1. All samples contained visible bands close to the size of porcine trypsin (23.3 kDa). The bands did not disappear in the presence of EDTA but abolished by Pefabloc, demonstrating that the enzyme is a serine protease, most likely salmon trypsin. Airborne levels of active protease were below the statistical detection limit in the filleting department but quantifiable in extract samples from the slaughter department. Three filtered air samples from the slaughter department showed air concentrations of 6.2, 16.5, and 27.0 ng m-3 air. We conclude that zymography is a sensitive and reliable method for exposure assessment of active proteases in indoor environmental samples. We recommend this assay for use in occupational studies to characterize and quantify exposure to active proteases in bioaerosols.

Speckle-type POZ protein suppresses hepatocellular carcinoma cell migration and invasion via ubiquitin-dependent proteolysis of SUMO1/sentrin specific peptidase 7.

Hepatocellular carcinoma (HCC) is associated with high metastatic potential and high mortality. Accumulating evidence has demonstrated that speckle-type POZ protein (SPOP) is a key adaptor molecule of ubiquitination. However, the molecular mechanism of SPOP-mediated ubiquitination in HCC metastasis remains obscure. In the present study, our results indicated that SPOP expression was significantly downregulated in HCC and was associated with tumor size, differentiation and metastasis. Cox regression model showed that low SPOP expression was a risk factor related to the prognosis of HCC patients. Loss- and gain-of-function assays demonstrated that SPOP inhibited HCC cell migration and invasion in vitro. Mechanisitically, co-immunoprecipitation and ubiquitination assays revealed that SPOP recognized and bound SENP7 and promoted its degradation via ubiquitin-dependent proteolysis. Analysis of immunohistochemistry showed that vimentin expression was correlated negatively with SPOP and positively with SENP7. These results implied that increased degradation of SENP7 by overexpression of SPOP decreased vimentin levels, which in turn attenuated HCC cell metastasis. Moreover, in vivo assays showed that SPOP overexpression also significantly suppressed liver and lung metastases. In summary, SPOP inhibits HCC cell metastasis via ubiquitin-dependent SENP7 proteolysis and may thus serve as a new opinion for the prevention of HCC metastasis.

EndoProteoFASP as a Tool to Unveil the Peptidome-Protease Profile: Application to Salivary Diagnostics.

In the quest to fully comprehend the proteolytic events leading to the generation of the salivary peptidome, we have developed a method for the sequential elution of salivary peptides throughout progressive endogenous proteolysis. By screening the time-dependent changes in the salivary peptidome we can predict the activity pattern of salivary proteases responsible for such peptide fingerprint and identify susceptible protein targets. Herein, we describe a step-by-step tutorial based on a filter-aided sample preparation (FASP) method, taking advantage of the endogenous salivary proteases armamentarium (endoProteoFASP), to produce new peptides from the salivary proteins, adding to those present in the sample at the time of collection. In this protocol, the different sets of peptides retrieved after sample elution are identified following a liquid chromatography-tandem mass spectrometry approach. The likelihood of a large set of endogenous proteases (collected from several public sources) to be responsible for the generation of such peptides can be predicted by the analysis of the cleavage site specificity by Proteasix ( http://proteasix.cs.man.ac.uk /) algorithm. The attained peptidome-protease profile can be useful to elucidate the peptidome dynamics and the proteolytic events underpinning pathophysiological phenomena taking place locally within the oral cavity. This may help clinicians to diagnose oral pathologies and develop preventive therapeutic plans.

[Fibrinogen/fibrin-specific enzymes from copperhead (Agkistrodon halys halys) and cobra (Naja oxiana eichwald) snake venoms]. / Fibrinogen/fibrin-spetsifichnye fermenty iadov kobry (Naja oxiana eichwald) i shchitomordnika (Agkistrodon halys halys).

Ability of fractions of cobra's (Naja oxiana Eichwald) and copperhead snake's (Agkistrodon halys halys) venoms to hydrolyze fibrinogen/fibrin was studied. In cobra's snake a component with molecular mass of nearly 60 kDa was found to hydrolyze a-chain of fibrinogen but failed to hydrolyze casein/azocasein and fibrin. A fibrinogen-specific metalloproteinase, the enzyme was inhibited by EDTA. Cobra's venom reduced the mass of donor's fresh blood clots. The copperhead snake's venom and the fractions obtained by gel-filtration (HW-50) and ion exchange chromatography (DEAE-650) were found to hydrolyze casein/azocasein, a- and b-chains of fibrinogen/fibrin and donor's blood clots. The results from the study of the venom and proteolytically active fractions are the evidence for a thrombolytic potential in a copperhead snake's venom.

Identification of Group B Streptococci Using 16S rRNA, cfb, scpB, and atr Genes in Pregnant Women by PCR.

Streptococcus agalactiae is acommensalorganism, but it may cause infection in susceptible hosts. The aim of this study was to evaluate PCR assay compared with conventional culture method for direct detection of Streptococcus agalactiae. Total of 203 paired low vaginal swabs were collected from women at 35-37 weeks of pregnancy from June 2013 through February 2014 for detection of Streptococcus agalactiae using PCR assay targeting 16S rRNA, cfb, scpB, and atr genes and culture method following broth enrichment. The results were recorded and evaluated for determining of sensitivity, specificity, positive and negative predictive values of PCR assaycompared with culture method. Prevalence of Streptococcus agalactiae was determined as 7.39% (n=15) using culture method; 19.70% (n=40) by PCR targeting 16S rRNA gene; 18.23% (n=37) by targeting atr gene; 17.24% (n=35) by cfb gene; and 8.87% (n=18) by scpB gene. Generally, a total of 49 specimens were considered true positive (27 samples by PCR assay using the four genes in sum, 4 samples only by atr gene PCR, 3 samples only by cfb gene PCR, 2 samples only by culture method, and 13 samples by PCR assay and culture method in common) and prevalence of Streptococcus agalactiae determined 24.14% in Hamadan. The current data demonstrated that performing only culture method for detecting GBS from pregnant women leads to missed false negative carrier individuals. Thus, it is recommended that both the PCR assay and conventional culture method to be performed in order to detect Streptococcus agalactiae.

Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer.

Ras and a-factor-converting enzyme 1 (Rce1) have been reported to play a key role in the proteolysis processing of Ras proteins. The present study investigated the prognostic significance of Rce1 in patients with prostate cancer (PCa). The expressions of the mRNA and protein of Rce1 were analyzed in 12 pairs of PCa and benign prostatic hyperplasia (BPH) by quantitative real-time polymerase chain reaction and Western blotting, respectively. Immunohistochemistry was used to examine expression of Rce1 protein in 74 PCa tissues and 30 BPH tissues. The association between Rce1 expression and the specific clinicopathologic features was evaluated by χ(2) tests. Kaplan-Meier and Cox proportional hazards regression models were used to analyze the data. We found that expression of Rce1 mRNA and protein was markedly higher in PCa tissues than in paired BPH tissues. Expression of Rce1 in PCa was strongly associated with clinicopathologic features. It was detected in 69 (93.24%) of 74 PCa tissues by immunohistochemistry, and it was found to be associated with Gleason score (P = .013), T class (P = .015), and distant metastasis (P = .044). Patients with PCa having higher Rce1 expression had substantially shorter survival times than patients with lower Rce1 expression. Univariate and multivariate analysis revealed that Rce1 was an independent prognostic factor. In conclusion, our study suggests that expression of Rce1 can serve as an independent biomarker for the prognosis of PCa patients.

Biogenesis of Lysobacter sp. XL1 vesicles.

The Gram-negative bacterium Lysobacter sp. XL1 forms vesicles and, using them, secretes an extracellular protein, bacteriolytic endopeptidase L5. Fractionation of a Lysobacter sp. XL1 vesicle preparation in a sucrose density gradient yielded four vesicle fractions of 30%, 35%, 40% and 45% sucrose. The size of most vesicles concentrated in 30% and 35% sucrose fractions were 40-65 and 65-100 nm, respectively. Electrophoresis and immunoblotting showed vesicles of the 30% fraction differed from those in the other fractions not only in density but also in protein content. Protein L5 was found to be secreted into the extracellular medium only by means of vesicles of the 30% sucrose fraction. Electron microscopic immunocytochemistry of Lysobacter sp. XL1 cells showed protein L5 to be distributed unevenly along the periplasmic space and to be concentrated in certain periplasmic loci adjacent to the outer membrane. It was in those loci where vesiculation occurred. A model of the formation of Lysobacter sp. XL1 vesicles is proposed based on the data obtained.