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71.
Frieden IJ 《Lymphatic research and biology》2003,1(4):313-316
Hemangiomas of infancy are very common tumors, but they are heterogeneous in their behavior. A small, but significant, subset causes medical complications or permanent disfigurement, but due to their heterogeneity, there is no appropriate "one size fits all" approach to management. In addition, the rapid evolution of this tumor over the first weeks-to-months of infancy renders even more difficult the task of predicting which infants will have medical complications or permanent disfigurement. This article outlines the clinical characteristics that help to stratify hemangiomas into those which are high risk, and likely to require either active treatment or closer scrutiny, and those which are low risk, and likely to behave in an innocuous manner. Five major factors are emphasized: the age of the child, the location of the hemangioma(s), the total number of hemangiomas present, the hemangioma subtype, and the presence and nature of dermal involvement. 相似文献
72.
Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm. 相似文献
73.
Kovács I Lasztóczi B Szárics E Héja L Sági G Kardos J 《Neurochemistry international》2003,43(2):101-112
Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain. 相似文献
74.
Carugo O Cemazar M Zahariev S Hudáky I Gáspári Z Perczel A Pongor S 《Protein engineering》2003,16(9):637-639
The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines is in a distorted trans conformation, the omega torsion angle ranges from 159 to -133 degrees, with an average value of 171 degrees. The constrained nature of the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal disulfides may be employed as a 'redox-activated' conformational switch. 相似文献
75.
76.
77.
Biodegradation of Aliphatic-Aromatic Copolyesters by Thermomonospora fusca and Other Thermophilic Compost Isolates
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Ilona Kleeberg Claudia Hetz Reiner Michael Kroppenstedt Rolf-Joachim Müller Wolf-Dieter Deckwer 《Applied microbiology》1998,64(5):1731-1735
Random aliphatic-aromatic copolyesters synthesized from 1,4-butanediol, adipic acid, and terephthalic acid (BTA) have excellent thermal and mechanical properties and are biodegradable by mixed cultures (e.g., in compost). Over 20 BTA-degrading strains were isolated by using compost as a microbial source. Among these microorganisms, thermophilic actinomycetes obviously play an outstanding role and appear to dominate the initial degradation step. Two actinomycete strains exhibited about 20-fold higher BTA degradation rates than usually observed in a common compost test. These isolates were identified as Thermomonospora fusca strains. They appeared to be particularly suitable for establishment of rapid degradation tests and were used in comparative studies on the biodegradation of various polyesters. 相似文献
78.
Sabine Kirner Philip E. Hammer D. Steven Hill Annett Altmann Ilona Fischer Laura J. Weislo Mike Lanahan Karl-Heinz van Pée James M. Ligon 《Journal of bacteriology》1998,180(7):1939-1943
Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.The antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] (PRN) is produced by many pseudomonads and has broad-spectrum antifungal activity (1, 5, 12–14, 17). PRN has been implicated as an important mechanism of biological control of fungal plant pathogens by several Pseudomonas strains (12–14), including P. fluorescens BL915, from which the prn genes were isolated (10).Tryptophan was identified as the precursor for PRN, based on the feeding of cultures with isotopically labeled and substituted tryptophan (2, 7, 8, 17, 25). Biosynthetic pathways were proposed as early as 1967 (7) and have been refined on the basis of tracer studies and the isolation of intermediates (Fig. (Fig.1)1) (2, 8, 17, 19, 23, 25). Recently, Hammer et al. (9) described the cloning and characterization of a 5.8-kb DNA region which encodes the PRN biosynthetic pathway. This DNA region confers the ability to produce PRN when expressed heterologously in Escherichia coli and contains four genes, prnABCD, each of which is required for PRN production. In the research described here, we used mutants in which each of the four genes was disrupted and strains which overexpress the individual genes to elucidate the function of each gene product in PRN biosynthesis. Open in a separate windowFIG. 1Biosynthetic pathways for PRN as proposed by van Pée et al. (23) (A) and by Chang et al. (2) (B). The reactions catalyzed by the PRN biosynthetic enzymes encoded by the prnABCD genes are indicated above the appropriate reaction arrows.
Open in a separate window
Open in a separate windowa+, PRN detected; −, PRN not detected. Hohaus et al. (11) presented additional evidence of the chlorinating activity of the prnA gene product, specifically, the chlorination of l-tryptophan to form 7-CLT by cell extracts from P. fluorescens strains which expressed the prnA gene, but which did not contain any of the other prn genes. To clarify which isomer was produced, Hohaus et al. (11) extracted 7-CLT from the bacteria and oxidized it to the corresponding indole-3-pyruvic acid with amino acid oxidases. Since the isolated 7-CLT was degraded by l-amino acid oxidase, but not by d-amino acid oxidase (11), it must be in the l configuration. The deduced amino acid sequence for prnA contains a consensus NAD binding site (9), and, indeed, NADH is a required cofactor for the prnA gene product.Cultures of BL915ΔORF2 produced 7-CLT, but 7-chloro-d-tryptophan (11) and other PRN biosynthetic intermediates were not detected (Fig. (Fig.3).3). BL915ΔORF2 produced PRN when supplied with exogenous MDA or APRN, but not when supplied with 7-CT (Table (Table2).2). When prnB was expressed in strain BL915ΔORF1–4, exogenously supplied 7-CT was converted to MDA (Fig. (Fig.4).4). These results indicate that the prnB gene product catalyzes the rearrangement of the indole ring to a phenylpyrrole and the decarboxylation of 7-CLT to convert 7-CLT to MDA. While it is somewhat surprising that a single enzyme carries out both the ring rearrangement and decarboxylation, Chang et al. (2) postulated a mechanism for such a reaction on a single enzyme some 16 years ago. The prnB gene product also catalyzed the production of APP (Fig. (Fig.4),4), presumably by using tryptophan as a substrate. Open in a separate windowFIG. 4In vivo conversion of PRN biosynthetic intermediates by the products of single prn genes. Individual genes were expressed on plasmids in the host strain BL915ΔORF1–4, and biosynthetic intermediates were added to the culture medium as indicated. Culture extracts were separated by TLC on silica plates with toluene as the mobile phase. Metabolites were visualized with van Urk’s reagent. Arrows indicate the positions of APP (dark green), MDA (olive green), APRN (reddish brown), and PRN (purple).MDA accumulated in cultures of BL915ΔORF3, but APP, APRN, and PRN were not detected (Fig. (Fig.3).3). BL915ΔORF3 was able to produce PRN when supplied with APRN in the culture medium, but not when supplied with 7-CT or MDA (Table (Table2).2). Strain BL915ΔORF1–4 expressing prnC converted exogenously supplied MDA to APRN (Fig. (Fig.4).4). These data indicate that the prnC gene product catalyzes the chlorination of MDA to form APRN. Cell extracts of the P. fluorescens strain which overexpresses the prnC gene (but does not contain the other prn genes) can also catalyze the chlorination of MDA to form APRN (11).The prnC gene is homologous to the chl gene from Streptomyces aureofaciens, which encodes a chlorinating enzyme for tetracycline biosynthesis (3, 9). Like prnA, the prnC deduced amino acid sequence contains a consensus NAD binding region (9), and NADH is required for the chlorination of MDA (11). While both prnA and prnC encode halogenating enzymes, they show no homology to previously cloned haloperoxidases (9) or to each other. Furthermore, in contrast to haloperoxidases (16), the two NADH-dependent halogenating enzymes in the PRN biosynthesis pathway are substrate specific (i.e., the tryptophan halogenase does not catalyze the chlorination of MDA and vice versa) (11).APRN accumulated in cultures of BL915ΔORF4 (Fig. (Fig.3),3), and this mutant was not able to produce PRN when supplied with any of the known PRN biosynthetic intermediates. Strain BL915ΔORF1–4 expressing prnD converted exogenously supplied APRN to PRN (Fig. (Fig.4).4). These results indicate that the prnD gene product catalyzes the oxidation of the amino group of APRN to a nitro group forming PRN. In vitro experiments by Kirner and van Pée (15) had suggested that this reaction is catalyzed by a chloroperoxidase; however, gene disruption experiments demonstrated that chloroperoxidases are not involved in PRN biosynthesis in vivo (16). Instead, this oxidation is more likely to be catalyzed by a class IA oxygenase (20), as suggested by the homology of prnD with these enzymes (9).We have shown that each prn gene encodes a protein found in the wild-type BL915 strain and have demonstrated in vivo that these four gene products carry out four biochemical steps which convert l-tryptophan to PRN. None of the conversions were observed in strain BL915ΔORF1–4, from which the entire 5.8-kb prn gene region has been deleted (Fig. (Fig.4).4). The arrangement of the genes in the operon is identical to the sequence of reactions in the biosynthetic pathway proposed by van Pée et al. (23) (Fig. (Fig.11).Chang et al. (2) proposed an alternate biosynthetic scheme (Fig. (Fig.1B)1B) and reported the conversion of exogenously supplied APP to PRN in vivo. Similarly, Zhou et al. (25) reported the conversion of APP to APRN in a cell-free system. These workers concluded that APP is an intermediate in PRN biosynthesis and that ring rearrangement precedes chlorination (Fig. (Fig.1B).1B). In the present study, APP accumulated only in strains which overexpressed the prnB gene. Furthermore, APP was not detected in cultures of BL915ΔORF1, which contains functional prnBCD genes expressed from the native promoter, as would be expected if the ring rearrangement (catalyzed by the prnB gene product) occurs before the first chlorination step (catalyzed by the prnA gene product). Like Hamill et al. (8) and van Pée et al. (23), we demonstrated that exogenously supplied 7-CT is converted to PRN. These results, together with the finding that the gene product of prnA catalyzes the NADH-dependent chlorination of l-tryptophan to 7-CLT (11), support the biosynthetic pathway proposed by van Pée et al. (23) (Fig. (Fig.1A)1A) and suggest that APP is a side product or dead-end metabolite. Purification and kinetic characterization of the prnA and prnB gene products, including investigations of substrate specificity and regioselectivity, will further clarify the roles of 7-CLT and APP in the PRN biosynthetic pathway.If APP is indeed a dead-end metabolite, it would be advantageous to tightly regulate the amount of prnB gene product present in cells, thus minimizing the diversion of substrate into APP. The prnB gene begins with GTG (9), which is a two- to threefold-less-efficient initiation codon than ATG (18); however, the prnB open reading frame is apparently translationally coupled to the prnA open reading frame (9). Coupling increases translational efficiency and is thought to be a mechanism to ensure coordinate expression of the coupled genes (18). In PRN biosynthesis, translational coupling of prnA and prnB may be a mechanism to regulate the level of prnB gene product present in cells and minimize the diversion of tryptophan to APP. 相似文献
Bacterial strains and plasmids.
The bacterial strains and plasmids used in this study are described in Table Table1.1. Pseudomonas strains were cultured in Luria-Bertani medium at 28°C. Antibiotics, when used, were added at the following concentrations: tetracycline, 30 μg/ml; and kanamycin, 50 μg/ml. The expression vector pPEH14 consists of the Ptac promoter and rrnB ribosomal terminator from pKK223-3 (Pharmacia, Uppsala, Sweden) cloned into the BglII site of the broad-host-range plasmid pRK290 (4). Ptac is a strong constitutive promoter in Pseudomonas (unpublished data). The PRN biosynthetic genes are the coding regions described by Hammer et al. (9). Each coding region was cloned from the translation initiation codon to the stop codon by PCR with restriction sites added to the ends to facilitate cloning. For prnB, the native GTG initiation codon was changed to ATG. The clones were sequenced after PCR.TABLE 1
Bacterial strains and plasmids used in this studyP. fluorescens strain or plasmid | Characteristics | Source or reference |
---|---|---|
Strains | ||
BL915 | Wild type | 10 |
BL915ΔORF1 | Deletion in prnA of BL915, Prn−, Kmr | 9 |
BL915ΔORF2 | Deletion in prnB of BL915, Prn−, Kmr | 9 |
BL915ΔORF3 | Deletion in prnC of BL915, Prn−, Kmr | 9 |
BL915ΔORF4 | Deletion in prnD of BL915, Prn−, Kmr | 9 |
BL915ΔORF1–4 | Deletion in prnABCD of BL915, Prn−, Kmr | 9 |
Plasmids | ||
pPEH14(prnA) | pRK290 carrying Ptac functionally fused to the 1.6-kb prnA coding region | This study |
pPEH14(prnB) | pRK290 carrying Ptac functionally fused to the 1.1-kb prnB coding region | This study |
pPEH14(prnC) | pRK290 carrying Ptac functionally fused to the 1.7-kb prnC coding region | This study |
pPEH14(prnD) | pRK290 carrying Ptac functionally fused to the 1.1-kb prnD coding region | This study |
Chemical standards.
7-Cl-d,l-tryptophan (7-CT) was synthesized as described by van Pée et al. (24). Monodechloroaminopyrrolnitrin (MDA) was extracted from cultures of P. aureofaciens and verified as described by van Pée et al. (23). Aminopyrrolnitrin (APRN) was prepared from PRN by reduction with sodium dithionite (22). PRN was synthesized according to the method of Gosteli (6).Western analysis.
To produce antigen, each prn gene was subcloned into a pET3 vector and transformed into E. coli BL21(De3) (Novagen, Inc., Madison, Wis.). Inclusion bodies were purified from induced cultures with protocols from Novagen. Inclusion body protein (100 μg) was run on a preparative Laemmli polyacrylamide electrophoresis gel, blotted to nitrocellulose filters, and stained with Ponceau S. The major band was excised, solubilized in dimethyl sulfoxide, and used by Duncroft, Inc. (Lovettsville, Va.), to immunize goats and produce antiserum against each PRN protein.Cultures of P. fluorescens BL915 were grown for 48 h in Luria-Bertani medium with the appropriate antibiotics. The cells were pelleted and resuspended in a small volume of Tris-EDTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis were performed as described by Sambrook et al. (21). The primary antiserum (goat anti-PRN protein) was diluted 1/1,000, and the secondary antibody (rabbit anti-goat immunoglobulin G conjugated to peroxidase; Pierce, Rockford, Ill.) was diluted 1/3,000. Bands were visualized with an enhanced chemiluminescence kit (Amersham, Arlington Heights, Ill.). This Western analysis demonstrated that each antibody recognized a single protein band from wild-type BL915, and these bands were not present in BL915ΔORF1–4 (Fig. (Fig.2).2). The molecular weights of the recognized proteins were consistent with the sizes predicted from the gene sequences. Each prn gene was expressed on a plasmid in BL915ΔORF1–4. In each case, the protein product of the cloned gene reacted only with the expected antibody and was identical in size to the band detected by that antibody in wild-type BL915 (Fig. (Fig.2).2). Open in a separate windowFIG. 2Western blot analysis of the protein products of prn genes cloned from P. fluorescens BL915. Individual genes were expressed on plasmids in the host strain BL915ΔORF1–4. BL915 wild-type and BL915ΔORF1–4 controls are included on each blot. Blots A, B, C, and D were probed with antibodies raised against the products of prnA, prnB, prnC, and prnD, respectively. Arrows indicate the positions of the 60- and 42-kDa molecular mass markers.Intermediate analysis and feeding experiments.
To determine which biosynthetic intermediates were produced by the prn gene deletion mutants, 2-day-old cultures were extracted with an equal volume of ethyl acetate. The organic phase was dried under vacuum, and the residue was dissolved in a small volume of methanol. Thin-layer chromatography (TLC) was performed on silica-coated plates with toluene or hexane-ethyl acetate (2:1) as the mobile phase. PRN, APRN, MDA, and aminophenylpyrrole (APP) were visualized with van Urk’s reagent as described previously (22).To further clarify which biosynthetic step was blocked in each deletion mutant, intermediate feeding experiments were conducted. Cultures (10 ml) were incubated at 28°C for 48 h. Biosynthetic intermediates were dissolved in a small volume of methanol and added to 4 ml of culture at the following final concentrations: 7-CT, 2.5 μg/ml; MDA, 25 μg/ml; APRN, 12.5 μg/ml. The cultures were incubated for an additional 4 h at 28°C and then extracted with ethyl acetate and analyzed by TLC and liquid chromatography-mass spectrometry as described above.MDA, APRN, and PRN were not detected in cultures of BL915ΔORF1 (Fig. (Fig.3),3), indicating that this mutant is blocked at an early step in PRN biosynthesis. BL915ΔORF1 was able to produce PRN when 7-CT, MDA, or APRN was supplied exogenously (Table (Table2).2). When prnA was expressed in the absence of other prn genes (i.e., in BL915ΔORF1–4), 7-chloro-l-tryptophan (7-CLT) accumulated. The identity of 7-CLT was verified by comparison of results of high-performance liquid chromatography and mass spectra with chemically synthesized 7-CT. These results indicate that the prnA gene product catalyzes the chlorination of l-tryptophan. Open in a separate windowFIG. 3Accumulation of PRN biosynthetic intermediates in P. fluorescens BL915 and prn gene deletion mutants derived from it. Extracts from 2-day-old cultures were separated by TLC on silica plates with hexane-ethyl acetate (2:1 [vol/vol]) as the mobile phase. Metabolites were visualized with van Urk’s reagent. Arrows indicate the positions of MDA (olive green), APRN (reddish brown), and PRN (purple).TABLE 2
Production of PRN by deletion mutants when supplied with biosynthetic intermediates in the growth mediumStrain | Result with intermediate added to culturesa
| ||
---|---|---|---|
7-CT | MDA | APRN | |
BL915ΔORF1 | + | + | + |
BL915ΔORF2 | − | + | + |
BL915ΔORF3 | − | − | + |
BL915ΔORF4 | − | − | − |
79.
Kelly A. Shipkowski Alexia J. Taylor Elizabeth A. Thompson Ellen E. Glista-Baker Brian C. Sayers Zachary J. Messenger Rebecca N. Bauer Ilona Jaspers James C. Bonner 《PloS one》2015,10(6)
Background
Multi-walled carbon nanotubes (MWCNTs) represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1β via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2) cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1β release in vitro and in vivo during allergic inflammation.Methods
THP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM) allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.Results
Priming of THP-1 macrophages with LPS increased pro-IL-1β and subsequent exposure to MWCNTs induced IL-1β secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1β secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1β. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1β in bronchoalveolar lavage fluid (BALF) and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1β in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1β mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs.Conclusions
These data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1β by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis. 相似文献80.
Julian Weghuber Anna M. Lipp Jacqueline Stadlbauer Michael C. Aichinger Verena Ruprecht Alois Sonnleitner Gerhard J. Schütz Tamás Henics 《Cell biology international》2010,34(11):1109-1112
The cationic antimicrobial immunomodulatory peptide, KLK (KLKL5KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK–membrane interactions that lead to these alterations require the ability of the peptide to move into an α‐helical conformation. We show that KLK induces an increase of the intracellular Ca2+ concentration in human T24 cells. The effect of KLK is buffer‐sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine‐to‐proline‐substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca2+ level of T24 cells. We show that, although KLK slightly stimulates Ca2+ influx into the cell, the bulk increase of Ca2+ levels is due to KLK‐induced depletion of intracellular Ca2+ stores. Finally, we demonstrate a KLK‐induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells. 相似文献