Low Concentrations Of Trypsin Research Articles

Improvement of protocol for detachment assays in B16F10 cells

B16F10 (B16) murine melanoma cells have the characteristic of strong adhesion to culture surfaces. The use of trypsin solutions in high doses to release this type of cell is not suitable. When B16F10 cells are released with high concentrations of trypsin they do not form a precipitate after centrifugation and consequently the cells will be lost when discarding the supernatant. To solve this problem, we established a solution with phosphate buffered saline (PBS) and ethylenediaminetetraacetic acid (EDTA) at low trypsin concentrations. Finally, we verified the efficiency of this solution on the quantity of cells recovered after centrifugation.

Establishment, characterization, and transfection potential of a new continuous fish cell line (CAM) derived from the muscle tissue of grass goldfish (Carassius auratus).

A new continuous fish cell line (CAM) has been successfully derived from the muscle tissues of grass goldfish, Carassius auratus. The primary cell cultures were initiated by incomplete trypsinization first and then explant culture in a Leibovitz-15 medium supplemented with 15% fetal bovine serum and 10% fish muscle extract. It was found that the CAM cells were very sensitive to trypsinization and needed to be sub-cultured at a low trypsin concentration of 0.0625% to be able to go through the crisis of spontaneous immortalization transformation, and afterward a total of five derivative cell strains were isolated from the original CAM cell line. This spontaneous immortalization transformation event was recorded successively at passages 44-47, beginning with a large-scale apoptosis and senescence and followed by mitosis arrest and re-activation, thus designated as cell strain CAM-44A, 44B, 45A, 44B, and 47A. Now both the CAM cell line and strains had been sub-cultured for more than 89 times and could be well cryopreserved in the growth medium containing 5% dimethylsulfoxide. Chromosome analysis and COI gene analysis had confirmed the grass goldfish origin of these CAM cells. Transfection potential analysis indicated that Lipofectamine LTX and Xfect were two suitable transfection reagents to be used in the gene delivery of CAM cells with a transfection efficiencies up to 11±6% and 8±3% in the CAM cell lines, respectively. Among the five cell strains, CAM-47A showed the highest transfection potential with a transfection efficiency up to 28 ± 5%. This work will provide a useful cell source for works on the cell-based artificial fish meat production and functional studies of fish myogenesis-related genes.

The effect of proteolytic enzymes and pH on GII.4 norovirus, during both interactions and non-interaction with Histo-Blood Group Antigens

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Histo-Blood Groups Antigens (HBGAs) have been described as attachment factors, promoting HuNoV infection. However, their role has not yet been elucidated. This study aims to evaluate the ability of HBGAs to protect HuNoVs against various factors naturally found in the human digestive system. The effects of acid pH and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs were studied, both during interactions and non-interaction with HBGAs. The results showed that GII.4 VLPs and GII.4 HuNoVs behaved differently following the treatments. GII.4 VLPs were disrupted at a pH of less than 2.0 and in the presence of proteolytic enzymes (1,500 units/mL pepsin, 100 mg/mL trypsin, and 100 mg/mL chymotrypsin). VLPs were also partially damaged by lower concentrations of trypsin and chymotrypsin (0.1 mg/mL). Conversely, the capsids of GII.4 HuNoVs were not compromised by such treatments, since their genomes were not accessible to RNase. HBGAs were found to offer GII.4 VLPs no protection against an acid pH or proteolytic enzymes.

Effective and Intact Cell Detachment from a Clinically Ubiquitous Culture Flask by Combining Ultrasonic Wave Exposure and Diluted Trypsin

Bioengineering research and applications are supported by cell culture technologies that produce a large number of homogeneous cells. However, trypsin used in the general culture procedure for cell detachment decreases cell activity and culture efficiency. Furthermore, manually conducted culture procedures, especially pipetting after trypsin treatment, can induce inhomogeneous mechanical stress in cells, which may influence cellular functions. Alternate detachment methods using specialized culture devices without trypsin and/or manual pipetting have been reported. However, conventional trypsinization is still widely used. Diluted trypsin increases culture efficiency. Therefore, we developed a cell-detaching method using diluted trypsin and ultrasonic vibration for cell detachment from ubiquitous culture vessels. To demonstrate our concept, we used a T25 flask. Vibration of the culture surface was excited by ultrasonic waves propagated from an ultrasonic transducer placed under the flask. Using the proposed method, cells were completely detached by diluted trypsin, whereas 8.6% of cells remained on the flask with manual pipetting. The viability and proliferation of cells detached by the proposed method were higher than those of cells detached by the conventional method, owing to the low concentration of trypsin. Furthermore, glucose consumption after detachment showed no abnormality, eliminating possible oncogenesis. Two membrane proteins were quantified immediately after detachment and at 24 h of culture, and there were no differences between the detachment methods. Thus, we conclude that our proposed method improves culture efficiency without any adverse effects and ensures homogeneous mechanical stress on cells.

Core-Shell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum.

We describe the application of a fluorescently labeled water-soluble core-shell molecularly imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect trypsin. p-Aminobenzamidine (PAB), a competitive inhibitor of trypsin, was immobilized in the wells of a microtiter plate enabling the capture of trypsin in an oriented position, thus maintaining its native conformation. Fluorescent MIP nanoparticles, which bound selectively to trypsin, were used for quantification. The MIP was prepared by a multistep solid-phase synthesis approach on glass beads functionalized with PAB, orientating all trypsin molecules in the same way. The core-MIP was first synthesized, using a thermoresponsive polymer based on N-isopropylacrylamide, so as to enable its facile liberation from the immobilized template by a simple temperature change. The shell, mainly composed of allylamine to introduce primary amino groups for postconjugation of fluorescein isothiocyanate (FITC), was grafted in situ on the core-MIP, whose binding cavities were still bound and protected by the immobilized trypsin. The resulting core-shell MIP was endowed with a homogeneous population of high-affinity binding sites, all having the same orientation. The MIP has no or little cross-reactivity with other serine proteases and unrelated proteins. Our MIP-based FIA system was successfully applied to detect low trypsin concentrations spiked into nondiluted human serum with a low limit of quantification of 50 pM, which indicates the significant potential of this assay for analytical and biomedical diagnosis applications.

Functional liver tissue engineering by an adult mouse liver-derived neuro-glia antigen 2-expressing stem/progenitor population

Deaths due to end-stage liver diseases are increasingly registered annually in the world. Liver transplantation is the ultimate treatment for end-stage liver diseases to date, which has been hampered by a critical shortage of organs. The potential of decellularized liver scaffolds (DLS) derived from solid organs as a three-dimensional platform has been evolved as a promising approach in liver tissue engineering for translating functional liver organ replacements, but questions still exist regarding the optimal cell population for seeding in DLS and the preparation of the DLS themselves. The aim of our study was to utilize a sodium dodecyl sulfate decellularization procedure in combination with a low concentration of trypsin (0.005%)-ethylenediaminetetraacetic acid (0.002%) process to manufacture DLS from whole mouse livers and recellularized with hepatic stem/progenitors for use in liver tissue engineering and injured liver treatment. Results showed that the DLS generated with all the necessary microstructure and the extracellular components to support seeded hepatic stem/progenitor cell attachment, functional hepatic cell differentiation. Hepatic differentiation from stem/progenitor cells loaded by DLS was more efficient than that of the stem/progenitor cells in the two-dimensional cell culture model. In summary, the method of DLS loaded by hepatic stem/progenitor cells provided by this study was effective in maintaining DLS extracellular matrix to introduce seeded stem/progenitor cell differentiation, hepatic-like tissue formation and functional hepatic protein production in vitro that promoted functional recovery and survival in a mouse model of dimethylnitrosamine-induced liver cirrhosis after auxiliary heterotopic liver transplantation. Copyright © 2016 John Wiley & Sons, Ltd.

Presenilin 1 promotes trypsin-induced neuroprotection via the PAR2/ERK signaling pathway. Effects of presenilin 1 FAD mutations

Mutants of presenilin 1 (PS1) increase neuronal cell death causing autosomal-dominant familial Alzheimer's disease (FAD). Recent literature shows that treatment of neuronal cultures with low concentrations of trypsin, a member of the serine family of proteases, protects neurons from toxic insults by binding to the proteinase-activated receptor 2 and stimulating survival kinase extracellular signal–regulated kinase (ERK 1/2). Other studies show that PS1 is necessary for the neuroprotective activity of specific neurotrophic factors, such as brain-derived neurotrophic factor, against excitotoxicity and oxidative stress. Here, we show that treatment of mouse cortical neuronal cultures with trypsin activates ERK1/2 and protects neurons against glutamate excitoxicity. The trypsin-dependent ERK activation and neuroprotection requires both alleles of PS1 because neither PS1 knockout nor PS1 hemizygous neuronal cultures can use exogenous trypsin to activate ERK1/2 or increase neuronal survival. The protective effect of PS1 does not depend on its γ-secretase activity because inhibitors of γ-secretase have no effect on trypsin-mediated neuroprotection. Importantly, cortical neuronal cultures either heterozygous or homozygous for PS1 FAD mutants are unable to use trypsin to activate ERK1/2 and rescue neurons from excitotoxicity, indicating that FAD mutants inhibit trypsin-dependent neuroprotection in an autosomal-dominant manner. Furthermore, our data support the theory that PS FAD mutants increase neurodegeneration by inhibiting the ability of neurons to use cellular factors as protective agents against toxic insults.

Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes.

Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1μM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [(3)H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10μM for SFLLRN or SLIGRL, 0.1U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [(3)H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed concept of bimodal PAR function in cardiomyocytes remains to be defined, particularly in vivo where hemodynamic and other regulatory factors may counteract or mask the direct cellular actions described here.

Proteolytic processing of Bacillus thuringiensis Vip3A proteins by two Spodoptera species

Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolytic activation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein.

The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß–ß–a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

Extraction and purification of tumorigenic Schwann cells in vitro from neurofibromatosis type 2 tissues

Objective To establish a simple method to extract and purify tumorigenic Schwann cells (TSCs) from neurofibromatosis type 2 (NF2) tumor tissues in vitro, in order to improve the successful rate of primary culture and obtain a large number of TSCs from NF2 patients for further study. Methods Six fresh tumor tissues from patients with NF2 were obtained from 2009 to 2010 in our hospital. TSCs were extracted after collagenase digestion, followed by cell purification through rapid digestion with low trypsin concentration, differential detachment and clone screening. Morphological changes of TSCs were observed under inverted microscope and cultured TSCs were identified immunocytochemically by S ⁃ 100 protein staining. Results TSCs were successfully cultured from 3 NF2 specimens. A large number of bipolar spindle or triangular⁃shaped cells could be seen on the third day of primary culture and 4 to 5 clones were formed after the third passage in each successfully cultured specimen. Purified TSCs expressed S ⁃ 100 protein detected by immunocytochemistry with the rate of positive cells of above 95%, and their cell activity was fine and were able to be passaged stably in the subculture. Conclusion It is a successful culture method to extract and purify TSCs, combining primary culture with rapid trypsin digestion with low concentration and differential detachment. After clone screening, the purified and energetic TSCs can be obtained and applied to clinical study for NF2 pathogenesis and treatment. Fibroblasts (FBs) can be removed effectively as well. DOI：10.3969/j.issn.1672-6731.2011.01.017

ENaC Channels in Oocytes from Xenopus laevis and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na⁺ currents (INa_(amil)). We observed a marked reduction in INa_(amil) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa_(amil) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa_(amil) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

DNA Binding by the ETS Protein TEL (ETV6) Is Regulated by Autoinhibition and Self-association

The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (K(d) = 2.8 x 10(-8) M). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by approximately 10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two alpha-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.

ENaC at the Cutting Edge: Regulation of Epithelial Sodium Channels by Proteases

Epithelial Na+ channels facilitate the transport of Na+ across high resistance epithelia. Proteolytic cleavage has an important role in regulating the activity of these channels by increasing their open probability. Specific proteases have been shown to activate epithelial Na+ channels by cleaving channel subunits at defined sites within their extracellular domains. This minireview addresses the mechanisms by which proteases activate this channel and the question of why proteolysis has evolved as a mechanism of channel activation.

High-yielding enzymatic method for isolation and culture of microvascular endothelial cells from bovine retinal blood vessels

A simple and non-mechanical enzymatic method for the isolation and large-scale in vitro culture of bovine retinal endothelial cells (BRECs) is described. A sufficiently high yield of retinal microvessels was obtained from bovine eyes by brisk stirring of the retina with Minimum Essential Medium for 3 min, washing the pellet five times by repeated centrifugation at 300 × g and sieving in a single step. Pericyte contamination was eliminated by increasing the incubation period in an enzyme mixture and passaging the cells at a low concentration of trypsin for 1–2 min. The cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) with 10% fetal bovine serum. The cells were cobble-stone shaped and stained positive for von Willebrand Factor (vWF) and cluster differentiation factors, CD31 and CD146. These cells stained negative for α-smooth muscle actin (ASMA) and glial fibilary acidic protein (GFAP), forming characteristic capillary tube-like structures on matrigel. Here we optimized the method for the isolation of pure retinal endothelial cells without using complex procedures and sophisticated instruments. This method is simpler, more rapid and cost-effective, and also results in a higher yield of endothelial cells than other methods. Retinal endothelial cells cultured in this way will be used to study the pathogenesis of vascular eye diseases such as diabetic retinopathy and other neovascularization diseases. They can also serve as an excellent in vitro model system for drug analysis.

Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft

Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP) reportedly inhibits vascular smooth muscle cells (VSMCs) migration and proliferation. We hypothesized that adventitial administration of the PD-ECGF/TP gene will suppress intimal hyperplasia and prevent vein graft failure. The study used 68 female rabbits. Rabbit jugular vein was autogenously transplanted into carotid artery with a cuff anastomotic technique. To define vascular wall gene transfer efficiency, poloxamer hydrogel (20%) containing plasmid vector encoding the LacZ gene and different concentrations of trypsin (0%, 0.1%, 0.25%, and 0.5%, n = 5 for each group) was applied to the adventitia of the vein graft. Gene transfer efficiency was evaluated 7 days later by X-gal staining. An additional 48 rabbits received poloxamer hydrogel (20%) containing 0.25% trypsin and the human PD-ECGF/TP gene, LacZ gene, or saline. Intima thickness was evaluated at 2 and 8 weeks after grafting (n = 8 for each group at each time point). Transgene expression was examined by reverse transcriptase-polymerase chain reaction, immunoblotting assay, and immunohistochemical staining. Immunohistochemical staining was also used to determine VSMC proliferation, heme oxygenase-1 expression, and macrophage infiltration. Incorporation of trypsin into the poloxamer hydrogel significantly increased vessel wall gene transfer. Trypsin at 0.25% and 0.5% resulted in higher gene transfer at the same level without effecting intimal hyperplasia and inflammation; thus, trypsin at 0.25% concentration was used for subsequent experiments. Compared with the LacZ and saline groups, grafts receiving the PD-ECGF/TP gene significantly reduced intimal thickness at 2 and 8 weeks after treatment. The ratio of proliferative VSMC was lower in PD-ECGF/TP treated grafts. Histologic examination of the PD-ECGF/TP transgene grafts demonstrated high expression of heme oxygenase-1, which has been reported to inhibit VSMC proliferation, suggesting that heme oxygenase-1 may be important in the inhibition effect of PD-ECGF/TP on VSMC. No neoplastic or morphologic changes were found in the remote organs. A safe and highly efficient gene transfer method was developed by using poloxamer hydrogel and a low concentration of trypsin. Neointimal hyperplasia was significantly reduced by adventitial application of the PD-ECGF/TP gene to the vein graft. Our data suggest that adventitial delivery of the PD-ECGF/TP gene after grafting may be promising method for preventing vein graft failure.

Protease-activated Receptor-2 Increases Exocytosis via Multiple Signal Transduction Pathways in Pancreatic Duct Epithelial Cells

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.

Stoichiometry and Absolute Quantification of Proteins with Mass Spectrometry Using Fluorescent and Isotope-labeled Concatenated Peptide Standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.

The use of mild trypsinization conditions in the detachment of endothelial cells to promote subsequent endothelialization on synthetic surfaces

A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm 2 were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF™ for shear stresses up to 60 dyn/cm 2 applied for 4 h. Using 0.025% trypsin, initial cell spreading and cell surface α 5 β 1 integrins were increased relative to cells treated with 0.5% trypsin. After 1 h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.