Linear and nonlinear rheology of liberase-treated breast cancer tumors.

Extracellular matrix (ECM) rigidity has been shown to increase the invasive properties of breast cancer cells, promoting transformation and metastasis through mechanotransduction. Reducing ECM stiffness via enzymatic digestion could be a promising approach to slowing breast cancer development by de-differentiation of breast cancer cells to less aggressive phenotypes and enhancing the effectiveness of existing chemotherapeutics via improved drug penetrance throughout the tumor. In this study, we examine the effects of injectable liberase (a blend of collagenase and thermolysin enzymes) treatments on the linear and nonlinear rheology of allograft 4T1 mouse mammary tumors. We perform two sets of in vivo mouse studies, in which either one or multiple treatment injections occur before the tumors are harvested for rheological analysis. The treatment groups in each study consist of a buffer control, free liberase enzyme in buffer, a thermoresponsive copolymer called LiquoGel (LQG) in buffer, and a combined, localized injection of LQG and liberase. All tumor samples exhibit gel-like linear rheological behavior with the elastic modulus significantly larger than the viscous modulus and both independent of frequency. Tumors that receive a single injection of localized liberase have significantly lower tumor volumes and lower tissue moduli at both the center and edge compared to buffer- and free liberase-injected control tumors, while tissue viscoelasticity remains relatively unaffected. Tumors injected multiple times with LQG and liberase also have lower tissue volumes but possess higher tissue moduli and lower viscoelasticities compared to the other treatment groups. We propose that a mechanotransductive mechanism could cause the formation of smaller but stiffer tumors after repeated, localized liberase injections. Large amplitude oscillatory shear (LAOS) experiments are also performed on tissues from the multiple injection study and the results are analyzed using MITlaos. LAOS analysis reveals that all 4T1 tumors from the multiple injection study exhibit nonlinear rheological behavior at high strains and strain rates. Examination of the Lissajous-Bowditch curves, Chebyshev coefficient ratios, elastic moduli, and dynamic viscosities demonstrate that the onset and type of nonlinear behavior is independent of treatment type and elastic modulus, suggesting that multiple liberase injections do not affect the nonlinear viscoelasticity of 4T1 tumors.

Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture.

Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.

Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture.

OBJECTIVE: Recent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to investigate the potential of human amniotic membrane (hAM) as a viable scaffold for the growth and proliferation of pluripotent-derived RPE cells. METHODS AND ANALYSIS: Three enzymatic hAM de-epithelialisation methods (thermolysin, trypsin-EDTA and dispase II) were assessed by histological analysis and optical coherence tomography (OCT). We generated RPE cells from a human embryonic stem cell (hESC) line subjected to spontaneous differentiation in feeder-free conditions. The hESC-derived RPE cells were seeded over denuded hAM at a density of 2.0×105 cells/cm2 and maintained in culture for up to 4 weeks. Immnofluorescence was carried out to evaluate the development of a confluent monolayer of RPE cells on the top of the hAM. Conditioned medium was collected to measure pigment epithelium-derived factor (PEDF) concentration by ELISA. RESULTS: Laminin α5 and collagen IV staining confirmed the efficiency of the de-epithelialisation process. In particular, thermolysin showed good retention of tissue integrity on OCT images and greater preservation of the hAM basement membrane. The hESC-derived RPE cells formed patches of pigmented cells interspersed along the denuded hAM, but failed to form a regular sheet of RPE cells. These cells expressed typical RPE markers, such as PMEL17 and RPE65, but they secreted low levels of PEDF. CONCLUSION: The biological variability of the hAM could influence the adhesion and the expansion of hESC-derived RPE cells. Further studies are required to verify whether a non-confluent monolayer might represent a limit to transplantation.

21†Denuded descemet's membrane as potential tool to support human embryonic stem cell derived retinal pigment epithelial cells culture.

INTRODUCTION: Recent clinical studies suggest that RPE-cell replacement therapy may preserve vision and restore retinal structure in retinal degenerative diseases. New developments enabled the differentiation of RPE cells from pluripotent stem cells. Scaffold-based methods are being tested in ongoing clinical trials for delivering these cells to the back of the eye. Borrowed materials from donor tissues can be used as cell supports in subretinal transplantation. These biological matrices resemble the extracellular matrix microenvironment of the native tissue. The Descemet's membrane (DM) is an example of high collagen-rich basement membrane (BM). The potential of this tissue in retinal repair remains to be uncovered. AIMS: To investigate human embryonic stem cell-retinal pigment epithelium (hESC-RPE) cells survival and behaviour on a decellularized DM, which may be of clinical relevance in retinal transplantation. MATERIALS: DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope and histology. hESC-RPE cells were seeded onto the endothelial-side surface of acellular DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene, protein expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. RESULTS: Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. hESC-RPE cell attachment 6 days post-seeding and proliferation rates over the acellular DM were similar to hESC-RPE cells cultured on tissue culture inserts.On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell graft showed the characteristic RPE morphology. The expression of typical RPE genes, proper protein localization and key growth factor secretion further confirmed the correct RPE phenotype. The viability of the cells was maintained for up to 4 weeks in culture. CONCLUSION: Acellular DM was shown to be capable of sustaining hESC-RPE cells growth, thus confirming to be potentially a valid alternative to the Bruch's membrane.Further in vivo studies will need to verify if this product can represent a feasible tool to deliver RPE cells in the back of the eye.Our study highlights the possibility of recycling unsuitable corneal tissues, which would otherwise be discarded by the eye banks for clinical application.

Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24.

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

An Internally Quenched Fluorescent Peptide Substrate for Protealysin.

Protealysin, a metalloprotease of Serratia proteamaculans, is the prototype of a subgroup of the M4 peptidase family. Protealysin-like proteases (PLPs) are widely spread in bacteria but also occur in fungi and certain archaea. The interest in PLPs is primarily due to their putative involvement in the bacterial pathogenesis in animals and plants. Studying PLPs requires an efficient quantitative assay for their activity; however, no such assay has been reported so far. Here, we used the autoprocessing site sequence of the protealysin precursor to construct an internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. Protealysin and thermolysin, the prototype of the M4 family, proved to hydrolyze only the Ser-Val bond of the substrate. The substrate exhibited a KM = 35 ± 4 µM and kcat = 21 ± 1 s-1 for protealysin as well as a KM = 33 ± 8 µM and kcat = 7 ± 1 s-1 for thermolysin at 37 °C. Comparison of the effect of different enzymes (thermolysin, trypsin, chymotrypsin, savinase, and pronase E) on the substrate has demonstrated that it is not strictly specific for protealysin; however, this enzyme has higher molar activity even compared to the closely related thermolysin. Thus, the proposed substrate can be advantageous for quantitative studies of protealysin as well as for activity assays of other M4 peptidases.

Extraction and identification by high resolution mass spectrometry of bioactive substances in different extracts obtained from pomegranate peel.

This work proposes a method to separate proteins and polyphenols in a food byproduct with high bioactive properties and demonstrate by high performance liquid chromatography tandem mass spectrometry its efficiency. Bioactive substances were extracted using high intensity focused ultrasounds. Resulting extract (SR) was submitted to a step for the purification of proteins and to obtain a protein isolate (PI). Both extracts (SR and PI) were digested using two different enzymes (alcalase and thermolysin). Antioxidant, hypocholesterolemic, and antihypertensive properties of hydrolysates were explored. High antioxidant capacity, mainly due to the presence of polyphenols, was observed in SR and its hydrolysates. Hydrolysates obtained from PI using the alcalase enzyme showed simultaneously a high capacity to inhibit cholesterol esterase and to reduce micellar cholesterol solubility. Hydrolysate obtained from PI using the thermolysin enzyme showed a high antihypertensive capacity. Peptides and polyphenols in hydrolysates were identified by RP-HPLC-ESI-Q-TOF. Hydrolysates obtained from PI showed a high amount of peptides and a negligible amount of polyphenols while polyphenols were mainly present in hydrolysates from SR.

Enzyme-Responsive Polymer Nanoparticles via Ring-Opening Metathesis Polymerization-Induced Self-Assembly.

Open-to-air aqueous-phase ring-opening metathesis polymerization-induced self-assembly (ROMPISA) is reported for forming well-defined peptide polymer nanoparticles at room temperature and with high solids concentrations (10 w/w%). For these materials, ROMPISA is shown to provide control over molecular weight with high conversion while open-to-air. Moreover, these peptide polymer nanoparticles can spontaneously rearrange into larger aggregate scaffolds in the presence of the proteolytic enzyme, thermolysin. This work demonstrates the robust nature of ROMPISA, highlighted here for the preparation of stimuli-responsive nanostructures in one pot, in air.

Alzheimer's Aß1-40 peptide degradation by thermolysin: evidence of inhibition by a C-terminal Aß product.

The interaction of the amyloid-ß peptide (Aß) with thermolysin (TLN) was investigated by X-ray crystallography. Structural models of the complexes of TLN with several Aß fragments show that, despite the numerous possible cleavage sites of the Aß sequence, the C-terminal product of Ala30-Ile31 cleavage does not dissociate, thus inhibiting the enzyme. The high similarity between the TLN structural motif and neprilysin (NEP), the most extensively studied peptidase associated with Aß clearance, suggests that NEP should be more efficient against Aß polymorphs where Ala30-Ile31 is inaccessible, which is in agreement with studies in living mice that point to the limited role of NEP in degrading soluble Aß and its higher ability to degrade insoluble and/or oligomeric Aß forms, producing only the Aß10-37 intermediate.

Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex.

BACKGROUND: The aim of this study was to examine the effectiveness of Tumor Dissociation Enzyme (TDE) on the viability of follicles after digestion of fresh and cryopreserved ovarian cortex fragments (OCFs). METHODS: Fresh and thawed OCF from 14 patients (29 ± 6 years), sized 20 to 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; and Group 4, fresh OCF digested with Liberase TM. Evaluation of follicle viability was performed under light microscope after staining with Neutral red. For visualization of viable and dead cells under a confocal laser scanning microscope, the follicles were stained with Calcein AM and ethidium homodimer-1. RESULTS: The results showed that the number of retrieved follicles was significantly higher (990 vs 487; P < 0.01) in the TDE-treatment group compared to the Liberase TM-group. The presence of intense neutral red stained follicles was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (70.3% ± +/- 6.22 vs 53,1% ± 2.03 and 94.2% ± 6.6 vs 79.1% ± 2.1; P < 0.01). The percentage of Calcein AM stained follicles of class V1 was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (95.97% ± 7.8 vs 87.87% ± 2.4; 97.1% ± 6.8 vs 91.3% ± 2.3; P < 0.01). CONCLUSION: The enzymatic digestion of ovarian cortex with TDE provides recovery of a higher number of healthy preantral follicles in contrast to earlier described Liberase TM procedure.

A Novel Antihypertensive Peptide Identified in Thermolysin-Digested Rice Bran.

SCOPE: Hypertension is a risk factor for arteriosclerosis. In this study, we investigate the antihypertensive effect of protease-digested rice bran in a spontaneously hypertension rat (SHR) model. We also purify a novel antihypertensive peptide from the digest. METHODS AND RESULTS: Thermolysin-digested rice bran (TRB) is administered to SHRs for 4 weeks, and systolic blood pressure (SBP) was measured weekly using the tail-cuff method. TRB shows an antihypertensive effect in a dose-dependent manner. TRB also reduces angiotensin I-converting enzyme (ACE) activity in lung tissue and serum troponin I levels. TRB is fractionated by HPLC and ACE-inhibitory activity in the HPLC fractions is measured. Peptides LRA and YY are identified from the two fractions with the strongest ACE-inhibitory activity. Amino acid sequence of these peptides are found in a vicilin-like seed storage protein, and identified in rice bran protein using the peptide mass fingerprint method. We confirm that LRA and YY are cleaved by thermolysin digestion of a model synthetic peptide. Orally administered LRA (0.25 mg kg-1 ) or YY (0.5 mg kg-1 ) lowers the SBP of SHRs at 4 h after administration. CONCLUSION: We identify a novel, orally active antihypertensive peptide, LRA from the digest of rice bran protein.

Peptide Brush Polymers and Nanoparticles with Enzyme-Regulated Structure and Charge for Inducing or Evading Macrophage Cell Uptake.

Cellular uptake by macrophages and ensuing clearance by the mononuclear phagocyte system stands as a significant biological barrier for nanoparticle therapeutics. While there is a growing body of work investigating the design principles essential for imparting nanomaterials with long-circulating characteristics and macrophage evasion, there is still a widespread need for examining stimuli-responsive systems, particularly well-characterized soft materials, which differ in their physiochemical properties prior to and after an applied stimulus. In this work, we describe the synthesis and formulation of polymeric nanoparticles (NPs) and soluble homopolymers (Ps) encoded with multiple copies of a peptide substrate for proteases. We examined the macrophage cell uptake of these materials, which vary in their peptide charge and conjugation (via the N- or C-terminus). Following treatment with a model protease, thermolysin, the NPs and Ps undergo changes in their morphology and charge. After proteolysis, zwitterionic NPs showed significant cellular uptake, with the C-terminus NP displaying higher internalization than its N-terminus analogue. Enzyme-cleaved homopolymers generally avoided assembly and uptake, though at higher concentrations, enzyme-cleaved N-terminus homopolymers assembled into discrete cylindrical structures, whereas C-terminus homopolymers remained dispersed. Overall, these studies highlight that maintaining control over NP and polymer design parameters can lead to well-defined biological responses.

Protein-ligand complex structure from serial femtosecond crystallography using soaked thermolysin microcrystals and comparison with structures from synchrotron radiation.

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein-ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.

Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities.

A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.

Quantifying the Effects of Different Neutral Proteases on Human Islet Integrity.

Efficient islet release from the pancreas requires the combination of collagenase, neutral protease (cNP), or thermolysin (TL). Recently, it has been shown that clostripain (CP) may also contribute to efficient islet release from the human pancreas. The aim of this study was to evaluate the impact of these proteases on human islet integrity in a prospective approach. Islets were isolated from the pancreas of 10 brain-dead human organ donors. Purified islets were precultured for 3 to 4 d at 37 °C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 °C in Hank's balanced salt solution supplemented with cNP, TL, or CP. The protease concentrations were calculated for a pancreas of 100 g trimmed weight utilizing 120 dimethyl-casein units of cNP, 70,000 caseinase units of TL, or 200 benzoyl-l-arginine-ethyl-ester units of CP (1×). These activities were then increased both 5× and 10×. After subsequent 24-h culture in enzyme-free culture medium, treated islets were assessed and normalized to sham-treated controls. Compared with controls and CP, islet yield was significantly reduced by using the 5× activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1× activity of cNP, 5× activity of TL, or 10× activity of CP. Although mitochondrial function was significantly lowered by 1× cNP and 5× TL, CP did not affect mitochondria at any concentration. cNP- and TL-incubated islets significantly lost intracellular insulin already at 1× activity, while the 10× activity of CP had to be added to observe a similar effect. cNP and TL have a similar toxic potency regarding islet integrity. CP also induces adverse effects on islets, but the toxic threshold is generally higher. We hypothesize that CP can serve as supplementary protease to minimize cNP or TL activity for efficient pancreas digestion.

Apricot and other seed stones: amygdalin content and the potential to obtain antioxidant, angiotensin I converting enzyme inhibitor and hypocholesterolemic peptides.

Stones from olives and Prunus genus fruits are cheap and sustainable sources of proteins and could be potential sources of bioactive peptides. The main limitation to the use of these seeds is the presence of amygdalin. This work proposes to determine amygdalin in olive and Prunus seeds and in protein isolates obtained from them. Moreover, antioxidant, angiotensin I converting enzyme (ACE) inhibitor, and hypocholesterolemic properties will be evaluated in hydrolysates obtained from these seeds. Despite some seeds contained amygdalin, all protein isolates were free of this substance. Two different procedures to obtain bioactive peptides from protein isolates were examined: gastrointestinal digestion and processing with Alcalase, Flavourzyme or Thermolysin. Higher antioxidant, ACE inhibitor and hypocholesterolemic activities were observed when proteins were processed with Alcalase, Flavourzyme or Thermolysin. The highest antioxidant and ACE inhibitor capacities were observed for the Prunus genus seed hydrolysates while the highest capacity to reduce micellar cholesterol solubility was observed for the apricot and olive seed hydrolysates.

Matrix metalloproteinases as reagents for cell isolation.

Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase™. Samples isolated with MMPs and Liberase™ had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase™. However, ADSCs isolated by Liberase™ displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to<1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.

Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity.

Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca(2+)-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1µM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300µM Trp-Val or Trp-Ile. On the basis of Ca(2+)-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca(2+)-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6µM, respectively.