The therapeutic potential of the insect metalloproteinase inhibitor against infections caused by Pseudomonas aeruginosa.

OBJECTIVES: The objective of this study was to investigate the therapeutic potential of the insect metalloproteinase inhibitor (IMPI) from Galleria mellonella, the only known specific inhibitor of M4 metalloproteinases. METHODS: The fusion protein IMPI-GST (glutathione-S-transferase) was produced by fermentation in Escherichia coli and was tested for its ability to inhibit the proteolytic activity of the M4 metalloproteinases thermolysin and Pseudomonas elastase (PE), the latter a key virulence factor of the wound-associated and antibiotic-resistant pathogen Pseudomonas aeruginosa. We also tested the ability of IMPI to inhibit the secretome (Sec) of a P. aeruginosa strain obtained from a wound. KEY FINDINGS: We found that IMPI-GST inhibited thermolysin and PE in vitro and increased the viability of human keratinocytes exposed to Sec by inhibiting detachment caused by changes in cytoskeletal morphology. IMPI-GST also improved the cell migration rate in an in vitro wound assay and reduced the severity of necrosis caused by Sec in an ex vivo porcine wound model. CONCLUSIONS: The inhibition of virulence factors is a novel therapeutic approach against antibiotic resistant bacteria. Our results indicate that IMPI is a promising drug candidate for the treatment of P. aeruginosa infections.

Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles.

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 ±â€¯6 years old), sized 20-210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 ±â€¯2.4% vs. 90.4 ±â€¯3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 ±â€¯3.4% vs. 89.7 ±â€¯4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 ±â€¯3.4%; Group 2: 91.8 ±â€¯6.0%; Group 3: 90.5 ±â€¯6.9%; Group 4: 87.3 ±â€¯2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 ±â€¯1.6%; Group 2: 4.4 ±â€¯1.6%; Group 3: 1.6 ±â€¯1.1%; Group 4: 2.4 ±â€¯1.5%), late apoptotic (Group 1: 2.7 ±â€¯2.4%; Group 2: 44.0 ±â€¯1.9%; Group 3: 3.1 ±â€¯1.1%; Group 4: 2.8 ±â€¯0.7% and necrotic (Group 1: 0.9% ±â€¯0.1%; Group 2: 2.9 ±â€¯0.8%; Group 3: 3.4 ±â€¯4.5%; Group 4: 1.1 ±â€¯0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex.

A Modified Protocol for the Isolation of Primary Human Hepatocytes with Improved Viability and Function from Normal and Diseased Human Liver.

Successful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver and the results of hepatocyte isolation from such tissue are inferior compared to normal tissue. Here we describe a modified method, combining the use of Liberase and N-acetylcysteine (NAC), for the isolation of primary human hepatocytes with high viability from normal and diseased liver.

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

Combinatorial enzymatic digestion with thermolysin and collagenase type I improved the isolation and culture effects of hair cell progenitors from rat cochleae.

The high incidence of hearing loss in human combined with the lack of hair cell regeneration in mammalian cochleae had got the attention to manipulate stem/progenitor cells to participate in hair cell regeneration for years. Cochlear progenitor cells are considered as the best candidate for hair cell regeneration. However, there is not any effective and feasible way to separate hair cell progenitors from rat cochleae, yet. In this study, we tried to isolate single epithelial cells from rat basilar membrane by combinatorial enzymatic digestion with thermolysin and collagenase type I. The results showed that the harvested single cells gave rise to otospheres with features of stem cells and could be induced to differentiate into hair cells. Significantly, more otospheres of epithelial origin were obtained by digesting with thermolysin and collagenase type I. The combinatorial enzymatic digestion would be a potential method for hair cell progenitor isolation and culture with broad applications.

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver.

BACKGROUND AIMS: Successful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues. METHODS: Hepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis. RESULTS: Baseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 10(4) to 28.3 × 10(4) cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group. CONCLUSIONS: NAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research.

Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors in the cochlea.

Auditory hair cells are surrounded on their basolateral aspects by supporting cells, and these two cell types together constitute the sensory epithelium of the organ of Corti, which is the hearing apparatus of the ear. We show here that Lgr5, a marker for adult stem cells, was expressed in a subset of supporting cells in the newborn and adult murine cochlea. Lgr5-expressing supporting cells, sorted by flow cytometry and cultured in a single-cell suspension, compared with unsorted cells, displayed an enhanced capacity for self-renewing neurosphere formation in response to Wnt and were converted to hair cells at a higher (>10-fold) rate. The greater differentiation of hair cells in the neurosphere assay showed that Lgr5-positive cells had the capacity to act as cochlear progenitor cells, and lineage tracing confirmed that Lgr5-expressing cells accounted for the cells that formed neurospheres and differentiated to hair cells. The responsiveness to Wnt of cells with a capacity for division and sensory cell formation suggests a potential route to new hair cell generation in the adult cochlea.

A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane.

Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C4 species with dimorphic chloroplasts in individual chlorenchyma cells. Fluorescent protein tagging and immunogold studies revealed that the localization patterns of Toc159 were distinctive from those of Toc34, suggesting different targeting pathways. Bioinformatics analyses indicated that the C-terminal tails (CTs) of Toc159 possess physicochemical and structural properties of chloroplast transit peptides (cTPs). These results were further confirmed by fluorescent protein tagging, which showed the targeting of CT fusion proteins to the chloroplast surface. The CT of Bs Toc159 in reverse orientation functioned as a cleavable cTP that guided the fluorescent protein to the stroma. Moreover, a Bs Toc34 mutant protein was retargeted to the chloroplast envelope using the CTs of Toc159 or reverse sequences of other cTPs, suggesting their conserved functions. Together, our data show that the C terminus and the central GTPase domain represent a novel dual domain-mediated sorting mechanism that might account for the partitioning of Toc159 between the cytosol and the chloroplast envelope for preprotein recognition.

Propeptide processing and proteolytic activity of proenzymes of the staphylococcal and enterococcal GluV8-family protease.

Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser_1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn.1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family proenzymes with shortened propeptides intrinsically possess proteolytic activity and are involved in the propeptide shortening that facilitates the final hetero-catalytic maturation.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3.

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60 percent of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation.

For more than a decade Liberase HI was commonly used as the standard enzyme blend for clinical human islet isolation until enforced replacement by collagenase NB1 (NB1). This change resulted initially in a reduction in islet isolation outcome and transplant activities worldwide. This retrospective study was initiated to compare the efficiency of NB1 premium grade with Liberase in 197 human islet isolations. All pancreata were processed between January 2006 and June 2008 utilizing the same procedures for isolation and quality assessment except the administration of preselected lots of either Liberase (n = 101) or NB1 (n = 96). Utilizing Liberase, significantly more digested tissue and purified islet yield was produced compared to NB1. In contrast, the use of NB1 was associated with significantly higher purity and glucose stimulation index during dynamic perifusion. The expression of proinflammatory markers was almost identical except tissue factor expression, which was higher after utilization of Liberase. No difference was found in the percentage of pancreata fulfilling the criteria for clinical islet transplantation. The results suggest that Liberase is more efficient for pancreas dissociation than collagenase NB1 but seems to be more harmful to exocrine cells and islet tissue.

A 1-megadalton translocation complex containing Tic20 and Tic21 mediates chloroplast protein import at the inner envelope membrane.

Chloroplast protein import is mediated by two hetero-oligomeric protein complexes, the Tic and Toc translocons, which are located in the inner and outer envelope membranes. At the inner membrane, many Tic components have been identified and characterized, but it remains unclear how these Tic proteins are organized to form a protein-conducting channel or whether a stable Tic core complex that binds translocating preproteins exists. Here, we report the identification of a 1-megadalton (MD) translocation complex as an intermediate during protein translocation across the inner membrane in Arabidopsis thaliana and pea (Pisum sativum). This complex can be detected by blue native PAGE using the mild detergent digitonin without any chemical cross-linkers. The preprotein arrested in the 1-MD complex can be chased into its fully translocated form after a subsequent incubation. While Tic20 and Tic21 appear to be involved in the 1-MD complex, Tic110, a well-characterized Tic component, exists as a distinct entity from the complex. Several lines of evidence suggest that the 1-MD complex functions in between the Toc and Tic110-containing complexes, most likely as a protein-conducting channel at the inner envelope.

The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation.

BACKGROUND: The isolation of islets from the human pancreas critically depends on an efficient enzyme blend. Previous studies have solely focused on the presence of collagenase and neutral protease/thermolysin. Despite improved characterization of these components, the lot-related variability in efficacy still persists suggesting that additional so far disregarded enzymes are required for efficient islet cleavage. METHODS: Varying activities of a tryptic-like enzyme were identified within collagenase NB1 lots, which were selected according to a matched ratio between tryptic-like and collagenase activity (TLA-ratio). Rat and human pancreata were processed with current standard procedures. RESULTS: Increasing the TLA-ratio from 1.3% to 10% reduced pancreas dissociation time in rats by 50% without affecting islet yield, viability, or posttransplant function in diabetic nude mice. Enhancing the TLA-ratio from 1.3% to 12.6% for human pancreas processing resulted in a significant reduction of recirculation time and increased incrementally human islet yield without affecting purity, in vitro function or recovery after culture. Optimized pancreas digestion correlated with a higher percentage of islet preparations fulfilling quality criteria for clinical transplantation. CONCLUSIONS: We conclude that TLA is an effective component that should be included in moderate amounts in enzyme blends for human islet isolation to optimize the efficiency and minimize the lot-related variability.

Parameters for successful pig islet isolation as determined using 68 specific-pathogen-free miniature pigs.

BACKGROUND: Islet cell transplantation is a novel therapeutic modality for the cure of diabetes. Pig islet cells are an attractive substitute for human islet cells; however, they are known to be particularly difficult to isolate because of a weak islet capsule and a tendency to be fragmented during enzymatic digestion. Therefore, parameters favoring successful pig islet isolation were investigated using specific-pathogen-free (SPF) miniature pigs. METHODS: Sixty-eight SPF miniature pigs were used for islet isolation. Birth weight, body weight, age, sex, pregnancy history, and the fasting blood glucose levels of each pig were determined. Each pig's general condition was assessed with regard to feeding status and physical activity. Pancreas procurement was performed by one surgical team. Anesthesia duration, operation duration, procedure quality, and perfusate type were recorded. After pancreatectomy, a biopsy was performed for islet density analysis. Decapsulation, cannulation duration, degree of distension, and cold ischemic time were assessed. During islet isolation, pancreas weight, digestion time, and digested tissue proportion were recorded. Isolation results were evaluated by total islet equivalents (IEQ), islet equivalents per gram of pancreas (IEQ/g), isolation index, islet recovery rate, purity, and visual grade. To identify the predictors of higher islet isolation yield, we performed binary logistic regression analysis with significant (P < 0.05) variables from the univariate analysis. RESULTS: The pigs were categorized into high (n = 34) and low yield (n = 34) groups according to the median IEQ/g or total IEQ values. Body weight and age were significantly different between the two groups. Being male or a positive history of pregnancy in females was factors favoring successful islet isolation. General condition assessments failed to estimate islet isolation results. Long anesthesia duration, which might have caused ischemic injury to the pancreas, negatively affected islet isolation results. Decapsulation, cannulation duration, and subsequent pancreas distension were significantly important in successful islet isolation. Inter-lot variability of Liberase was not observed because of screening processes performed before purchase. Isolation index and islet recovery rate correlated well with islet yields. CONCLUSIONS: Multivariate analysis using total IEQ and IEQ/g as outcome variables indicated that age older than 2, being male and moderate distension by Liberase injection are major determinants influencing successful islet isolation.

Import of preproteins into the chloroplast inner envelope membrane.

The chloroplast inner envelope membrane contains many integral proteins which differ in the number of alpha-helices that anchor the protein into the bilayer. For most of these proteins it is not known which pathway they engage to reach their final localisation within the membrane. In yeast mitochondria, two distinct sorting/insertion pathways have been described for integral inner membrane proteins, involving the Tim22 and Tim23 translocases. These routes involve on the one hand a conservative sorting, on the other hand a stop-transfer pathway. In this study we performed a systematic characterisation of the import behaviour of seven inner envelope proteins representing different numbers of predicted alpha-helices. We investigated their energy dependence, import rate, involvement of components of the chloroplast general import pathway and distribution between soluble and membrane fractions. Our results show the existence of at least two different families of inner envelope proteins that can be classified due to the occurrence of an intermediate processing form. Each of the proteins we investigated seems to use a stop-transfer pathway for insertion into the inner envelope.

An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation.

V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis--the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date.

Collagenase-assisted fat dissociation for autologous fat transfer.

BACKGROUND: The quality of fat for autologous transfer procedures has been a major focus of research in the past few years. The primary goal of these efforts is to improve the viability and longevity of the graft in human subjects. One possible factor in the permanence of theses transplants is the size of the adipose tissue grafts. OBJECTIVE: This study evaluated the effects of collagenase digestion on the viability of human adipose tissue. MATERIALS AND METHODS: Samples of fat were obtained from subjects undergoing tumescent liposuction. The tissue was digested in a variety of concentrations of collagenase using optimized methods of processing. The digested fat was also subjected to mock injections through small bore needles. RESULTS: Eight subjects completed the study. The viability of the fat using the optimized methods of collagenase digestion was consistently higher than 79%. During the mock injection trials, the viability of fat was improved from approximately 17% to 84% by collagenase digestion. CONCLUSIONS: Our results show increased viability of human adipose tissue when digested by collagenase. These techniques can be applied to human autologous lipoaugmentation procedures in an effort to improve longevity of the transplanted tissue.

Immunofluorometric activity-based probe analysis of active KLK6 in biological fluids.

Immunoassay measurements of human kallikrein-related peptidases (KLKs) such as prostate-specific antigen (KLK3) are of great value as diagnostic indices of cancer. Despite extensive knowledge of the abundance of immunoreactive KLKs in normal and cancer-related settings, there is little information available about the proportion of immunoreactive KLK that represents active enzyme in such samples. Using KLK6 as a prototype enzyme, we have developed an assay using a serine proteinase-targeted activity-based probe coupled to antibody capture. By employing activity-based labeling, we were able to quantify the proportion of enzymatically active relative to total immunoreactive KLK6 in crude cerebrospinal fluid from routine analyses and ascites fluid from ovarian cancer patients, as well as in supernatants from cancer cell lines. Our approach allowed monitoring of pro-KLK6 conversion to its active enzyme species and demonstrated that up to 5% of immunoreactive KLK6 detected in clinical samples represents active enzyme. We suggest that this new activity-based probe assay will prove of value as a complement to routine KLK immunoassay measurements for validating KLKs as cancer biomarkers.