Benzenesulfonyl Fluoride Hydrochloride Research Articles

Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Protein Expression Signatures for Inhibition of Epidermal Growth Factor Receptor-mediated Signaling

Analysis of cellular signaling networks typically involves targeted measurements of phosphorylated protein intermediates. However, phosphoproteomic analyses usually require affinity enrichment of phosphopeptides and can be complicated by artifactual changes in phosphorylation caused by uncontrolled preanalytical variables, particularly in the analysis of tissue specimens. We asked whether changes in protein expression, which are more stable and easily analyzed, could reflect network stimulation and inhibition. We employed this approach to analyze stimulation and inhibition of the epidermal growth factor receptor (EGFR) by EGF and selective EGFR inhibitors. Shotgun analysis of proteomes from proliferating A431 cells, EGF-stimulated cells, and cells co-treated with the EGFR inhibitors cetuximab or gefitinib identified groups of differentially expressed proteins. Comparisons of these protein groups identified 13 proteins whose EGF-induced expression changes were reversed by both EGFR inhibitors. Targeted multiple reaction monitoring analysis verified differential expression of 12 of these proteins, which comprise a candidate EGFR inhibition signature. We then tested these 12 proteins by multiple reaction monitoring analysis in three other models: 1) a comparison of DiFi (EGFR inhibitor-sensitive) and HCT116 (EGFR-insensitive) cell lines, 2) in formalin-fixed, paraffin-embedded mouse xenograft DiFi and HCT116 tumors, and 3) in tissue biopsies from a patient with the gastric hyperproliferative disorder Ménétrier's disease who was treated with cetuximab. Of the proteins in the candidate signature, a core group, including c-Jun, Jagged-1, and Claudin 4, were decreased by EGFR inhibitors in all three models. Although the goal of these studies was not to validate a clinically useful EGFR inhibition signature, the results confirm the hypothesis that clinically used EGFR inhibitors generate characteristic protein expression changes. This work further outlines a prototypical approach to derive and test protein expression signatures for drug action on signaling networks.

Inhibition of Transcription Factor Specificity Protein 1 Alters the Gene Expression Profile of Keratinocytes Leading to Upregulation of Kallikrein-Related Peptidases and Thymic Stromal Lymphopoietin

Transcription factor specificity protein 1 (Sp1) is involved in diverse cellular functions. We recently found that Sp1 was significantly decreased in skin biopsies from patients with atopic dermatitis (AD) and had an even greater reduction in AD patients with a history of eczema herpeticum. In the current study, we sought to better understand the role of Sp1 in skin biological processes by using a small interfering RNA (siRNA) technique to knock down Sp1 gene expression in normal human keratinocytes (NHK) and investigated the genome-wide gene expression profiling of Sp1- silenced NHK. The gene arrays revealed that 53 genes had more than three-fold changes in expression in Sp1-silenced NHK as compared to scrambled siRNA silenced cells. Strikingly, six kallikrein-related peptidase genes, KLK5, KLK6, KLK7, KLK8, KLK10, and KLK12 were up-regulated in NHK following Sp1 silencing. Functionally, protease activity was significantly enhanced in Sp1-silenced keratinocytes as compared to scrambled siRNA silenced keratinocytes. Moreover, thymic stromal lymphopoietin (TSLP), an epithelial derived TH2 promoting cytokine, was induced in Sp1-silenced keratinocytes due to elevated kallikrein activity. These results indicate that Sp1 expression deficiency leads to abnormally increased kallikrein protease activity in keratinocytes and may contribute to TH2 immune responses in the skin by inducing TSLP.

Proteolytic activity in latices of Asteraceae and Campanulaceae

In the order Asterales only two species are known to have proteolytic activity in their latices, first Taraxalisin from dandelion Taraxacum officinale Webb s.l., and second Parthenain from Guayule Parthenium argentatum L.. Both are characterized as serine endopeptidases [1]. Proteolytic enzymes isolated from plant latex have received special attention in the pharmaceutical industry and biotechnology due to their property of being active over wide range of temperature and pH. Nearly half of the commercially available enzymes are proteases, frequently used in food processing, tenderization of meat, brewing, cheese elaboration, bread manufacturing, leather and textile industries [1]. In this investigation the latex of 40 species of the Asteraceae family and 8 species of the Campanulaceae, which are not biochemical characterized before, were collected in the Botanical Garden Berlin. To determine proteolytic activity we used the fluorogenic substrate BODIPY FL- casein (Moleculare Probes, Inc., USA) [2]. To investigate the type of endopeptidases, the latex samples were pre-incubated with specific inhibitors for serine proteases (AEBSF (4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride), cysteine proteases (E64 (4-(2-Aminoethyl) benzenesulfonylfluoride hydrochloride)), aspartatic proteases (Pepstatin A) and metalloprotease (EDTA) and the remaining activity was determined. In both families highly active serine proteases were found.

Serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibits the rat embryo implantation in vivo and interferes with cell adhesion in vitro

Background This study was conducted to observe the in vivo effect of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) on embryo implantation in rats and its in vitro effect on cell adhesion. Study Design The anti-implantation efficacy of AEBSF in rats was determined by counting the number of visible implanted embryos on day 8 of pregnancy following intrauterine (5 mg and 10 mg AEBSF per horn) or tail vein (10 mg AEBSF per rat) administration on day 3 of pregnancy. The effects of AEBSF on cell adhesion were detected, respectively, by using the mouse blastocysts–endometrial cells or the human umbilical vein endothelial cells (HUVECs)–HeLa cells co-culture model. The alteration in protein secretion pattern of HUVECs and HeLa cells was detected by the proteome analysis. Results 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride showed an in vivo inhibitory effect on embryo implantation in rat. In vitro, AEBSF could disturb the growth of blastocysts on endometrial cells and inhibit the adhesion of HeLa cells on HUVECs. The treatment of AEBSF could alter the protein secretion pattern of co-cultured HUVEC–HeLa cells. Conclusion 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride might be a potential leading compound for novel contraceptives, and its inhibitory effect on implantation might result from the interference in extracellular matrix remodeling process.

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

Lack of Tyrosylprotein Sulfotransferase-2 Activity Results in Altered Sperm-Egg Interactions and Loss of ADAM3 and ADAM6 in Epididymal Sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.

A practical guide for the stabilization of acylghrelin in human blood collections

To better understand acylghrelin plasma stability, human synthetic acylghrelin was spiked into plasma and tracked by liquid chromatography tandem mass spectrometry. To investigate the best method for quantifying clinical plasma acylghrelin levels, pre- and postprandial human blood was collected from healthy volunteers (n=6) using various sample collections and treatments. Plasma ghrelin levels from human blood collections were analysed by enzyme-linked immunosorbant assay (ELISA). Acylghrelin's half-life in plasma was approximately 45 min with the formation of des-acylghrelin approaching 50% before the end of the 60-min incubation. Loss of acylghrelin inversely correlated with an increase in des-acylghrelin (P<0.008; r(2) =0.870). Plasma pretreated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or protease inhibitor cocktail without acidification resulted in no detectible acylghrelin losses. Acylghrelin measurements with AEBSF-treated blood were minimally 40% higher than sodium citrate/citric acid, K(2) EDTA, aprotinin/HCl and P800 collections. HCl addition to AEBSF-treated plasma did not provide enhanced acylghrelin stability and induced deacylation at and above the 100 mM final concentration. Pre- and postprandial ghrelin attenuation was investigated using aprotinin/HCl, AEBSF, protease inhibitor cocktail and no treatment for blood and plasma preparations. Fasting samples treated with AEBSF and protease inhibitor cocktail were approximately threefold higher than aprotinin/HCl and control treatments (P<0.03). Pre- and postprandial ghrelin attenuation was approximately twofold different (P<0.04) with significant counterintuitive trends in aprotinin/HCl and K(2) EDTA groups. Our data suggest that AEBSF addition to K(2) EDTA blood immediately after collection without plasma acidification, processing on ice and 14-day 70 °C storage is the best treatment for accurately quantifying acylghrelin in human plasma.

Inhibition of Proprotein Convertase SKI-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone (dec-RRLL-cmk), inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Because SKI-1 is required for activation of SREBP and CREB (cAMP-response element-binding protein)/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5-3.0-fold including Phex, Dmp1, COL1A1, COL11A1, and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1, and fibronectin was reduced by both, whereas COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1-activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19-kDa caspase-3. Conversely, overexpression of SKI-1-activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells and possibly osteocytes depends upon the non-apoptotic actions of SKI-1.

Thrombin Activation of Protease-Activated Receptor-2 on Neutrophils Primes the Respiratory Burst.

AbstractAbstract 1119Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of protease-activated receptors (PARs). Thrombin is considered the prototypical protease to activate PARs through cleavage of the N-terminal exodomain of the receptor, thereby unmasking a tethered ligand which activates it. Neutrophils (PMNs) are reported to express PAR-2; however, little is known about the changes in PMN physiology following protease activation of PAR-2. In addition, proteomic analyses (two dimensional gel electrophoresis and mass spectroscopy (MALDI-TOF)) of injured patients (blunt trauma) who developed acute lung injury (ALI) and multiple organ failure (MOF) have identified a number of serine proteases which accumulate post-injury. Moreover, these identical proteases were also found via proteomics in stored, but not fresh, packed red blood cells, which are used to resuscitate these injured patients. We hypothesize that thrombin primes PMNs through activation of PAR-2. Methods: PMNs were isolated from whole blood drawn from healthy human donors using dextran sedimentation, ficoll-hypaque gradient centrifugation and hypotonic lysis of red blood cells. The proteins from whole cell lysates were separated by SDS-polyacrylamide electrophoresis, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to PAR-2. The PMNs were also incubated for 3–30 minutes at 37°C with thrombin [1-10 U/ml] followed by fMLP [1 μM] activation of the NADPH oxidase. Oxidase activity was measured by the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm. Results: PMNs express PAR-2 as detected by western blotting of whole cell lysates. Thrombin [1 U/ml] for 30 min, significantly primed the fMLP-activated respiratory burst: buffer control: 1.6 +/− 0.5 vs. 1 U/ml thrombin: 2.2 +/− 0.3* (*p<.05, n=5). Higher concentrations of thrombin or shorter incubation times did not induce PMN priming. Furthermore, pre-incubation with leupeptin [50 μM] and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) [250 μM], two effective protease inhibitors, abrogated the thrombin-induced PMN priming by 100±18%. We conclude that thrombin primes the PMN respiratory burst, which is dependent upon its protease activity. Because PMNs are the effector cell in ALI/MOF and priming agents have been linked to the development of clinical ALI and MOF, proteases that accumulate in the patient plasma post-injury or are transfused with stored PRBCs may be etiologic in post-injury ALI/MOF. Disclosures:No relevant conflicts of interest to declare.

An atypical proprotein convertase in Giardia lamblia differentiation

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.

Loop-Sheet Mechanism of Serpin Polymerization Tested by Reactive Center Loop Mutations

The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central β-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of α(1)-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8-P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama α(1)-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8-P6 region is only a small part of an extensive domain swap.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Inhibitory profile of latex-proteases in the genus Euphorbia

The latex of some plant families such as Apocynaceae, Asclepiadaceae, Asteraceae, Caricaceae, Convolvulaceae, Euphorbiaceae, and Moraceae are known to contain endopeptidases. Proteolytic enzymes from plant latex have received special attention in the pharmaceutical industry and biotechnology due to their property of being active over wide range of temperature and pH. Nearly half of the commercially available enzymes are proteases, frequently used in food processing, tenderization of meat, brewing, cheese elaboration, bread manufacturing, leather and textile industries [1]. In the genus Euphorbia (Euphorbiaceae) 64 different species are mentioned to have proteolytic activity in their latices [2]. In this investigation the latex of 23 species of the genus Euphorbia, which are not characterised before, were collected in the Botanical Garden Berlin. To determine proteolytic activity we used the fluorogenic substrate BODIPY FL- casein (Moleculare Probes, Inc., USA) [3]. All tested samples show proteolytic activity. To investigate the type of endopeptidases, the latex samples were pre-incubated with specific inhibitors for serine (AEBSF (4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride)-, cysteine(E64 (4-(2-Aminoethyl)benzenesulfonylfluoride hydrochloride)) -, aspartatic (Pepstatin A)- and metalloprotease (EDTA) and the remaining activity was determined. 18 plants were strongly inhibited only by serine specific inhibitor, one plant was not influenced by any inhibitor and 4 plants were influenced by at least two inhibitors and had still a remaining proteolytic activity.

Glycosylphosphatidylinositol Anchor Analogues Sequester Cholesterol and Reduce Prion Formation

A hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP(C) where C is cellular) into an alternatively folded, disease-related isoform (PrP(Sc), where Sc is scrapie), the accumulation of which is associated with synapse degeneration and ultimately neuronal death. The formation of PrP(Sc) is dependent upon the presence of PrP(C) in specific, cholesterol-sensitive membrane microdomains, commonly called lipid rafts. PrP(C) is targeted to these lipid rafts because it is attached to membranes via a glycosylphosphatidylinositol anchor. Here, we show that treatment of prion-infected neuronal cell lines (ScN2a, ScGT1, or SMB cells) with synthetic glycosylphosphatidylinositol analogues, glucosamine-phosphatidylinositol (glucosamine-PI) or glucosamine 2-O-methyl inositol octadecyl phosphate, reduced the PrP(Sc) content of these cells in a dose-dependent manner. In addition, ScGT1 cells treated with glucosamine-PI did not transmit infection following intracerebral injection to mice. Treatment with glucosamine-PI increased the cholesterol content of ScGT1 cell membranes and reduced activation of cytoplasmic phospholipase A(2) (PLA(2)), consistent with the hypothesis that the composition of cell membranes affects key PLA(2)-dependent signaling pathways involved in PrP(Sc) formation. The effect of glucosamine-PI on PrP(Sc) formation was also reversed by the addition of platelet-activating factor. Glucosamine-PI caused the displacement of PrP(C) from lipid rafts and reduced expression of PrP(C) at the cell surface, putative sites for PrP(Sc) formation. We propose that treatment with glucosamine-PI modifies local micro-environments that control PrP(C) expression and activation of PLA(2) and subsequently inhibits PrP(Sc) formation.

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.

Okadaic acid induces DNA fragmentation via caspase‐3‐dependent and caspase‐3‐independent pathways in Chinese hamster ovary (CHO)‐K1 cells

DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.

Estrogen regulates transcription factors STAT-1 and NF-kappaB to promote inducible nitric oxide synthase and inflammatory responses.

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-kappaB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-kappaB p65 and modifies nuclear NF-kappaBp50. Here, we demonstrated that both nuclear STAT-1 and NF-kappaB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-kappaB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-kappaB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-kappaB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-gamma production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-kappaB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-kappaB small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-kappaB in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-kappaB proteins.

Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.

Proteolytic activity in the genus Euphorbia

Over 110 latices of different plants are known to contain at least one proteolytic enzyme. Proteolytic enzymes from plant latex have received special attention in the pharmaceutical industry and biotechnology due to their property of being active over wide range of temperature and pH. In the latex of Euphorbiaceae only proteolytic endopeptidases of the subclass serine proteases (EC 3.4.21) are mentioned in the literature [1]. For detecting proteolytic activity in the genus Euphorbia we collected latex of 30 different plants which are located in the Botanical Garden Berlin. To determine proteolytic activity we used the fluorogenic substrate BODIPY FL- casein (Moleculare Probes, Inc., USA) [2]. The change in fluorescence was compared to that produced by trypsin as standard; with a volume of 100µl of each trypsin concentration. The plants were divided into two groups; group I has its activity in the range between 5µg/ml and 2.5mg/ml trypsin (27 plants) and group II has a very strong proteolytic activity higher than 2.5mg/ml trypsin (3 plants). Proteolytic activity of latices in the genus Euphorbia could be expect as a chemotaxonomic characteristic because every tested sample was able to make a significant change in fluorescence compared to the control. To investigate if serine proteases are the active endopeptidases, the latex samples were pre-incubated with serine protease specific inhibitors (AEBSF (4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride) and Aprotinin) and the remaining activity was determined. 9 plants are not influenced by the inhibitors, 19 plants were influenced but had still a remaining proteolytic activity, and by 2 plants the residual activity was negligible.