Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

Characterisation of a plasma membrane-associated phospholipase A2 activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A₂ (EC 3.1.1.4; PLA₂) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA₂ activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca²⁺, with full enzyme activity at 10 μmol/L Ca²⁺. The Ca²⁺-dependent PLA₂ activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca²⁺-dependent PLA₂ activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA₂ activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA₂ in cold acclimation in plants.     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.     

Molybdate and sulfide inhibit H2 and increase formate production from glucose by Ruminococcus albus

H₂ production from glucose by Ruminococcus albus was almost completely inhibited by 10^–5 M molybdate only when sulfide was present in the growth medium. Inhibition was accompanied by a significant increase in the production of formate. Extracts of molybdate-sulfide-grown cells did not contain hydrogenase activity. Active enzyme in extracts of uninhibited cells was not inhibited by the molybdate-sulfide-containing growth medium. The results indicate that a complex formed from molybdate and sulfide prevents the formation of active hydrogenase and electrons otherwise used to form H₂ are used to reduce CO₂ to formate. Growth was significantly inhibited when molybdate was increased to 10^–4 M. Reversal of growth inhibition but not inhibition of H₂ production occurred between 10^–4 and 10^–3 M molybdate. H₂ production by R. bromei but not by R. flavefaciens, Butyrivibrio fibrisolvens, Veillonella alcalescens, Klebsiella pneumoniae and Escherichia coli was inhibited by molybdate and sulfide.     

Influence of cis-[Re2GABA2Cl4]Cl2 on the antioxidant defense system parameters of normal human blood

The effect of the rhenium complex cis-[Re₂GABA₂Cl₄]Cl₂ on the antioxidant parameters of normal human blood in vitro have been studied. The results suggest that the complex influences various enzymes in the cascade of reactions utilizing active oxygen metabolites. However, the manifestation of this activity varies over the studied concentration range of the complex in the preincubation medium (10^–12-10^–4 M), so the effects appear to be concentration-dependent. The largest differences in antioxidant parameters in comparison with control were observed for the concentrations 10^–8, 10^–5, and 10^–4 M. Thus, correlations between the peroxidation level, superoxide dismutase (SOD) activity, antioxidant factor (F), and indexes of resistance of erythrocytes for hemolysis (TR) were found.     

Differential effects of ibuprofen,indomethacin, and meclofenamate on prostaglandin endoperoxide H2 metabolism

Summary Previous studies have suggested the possibility that the non-steroidal antiflammatory drug (NSAID), ibuprofen, may inhibit thromboxane (TX) A₂ synthase activity in addition to inhibiting cyclooxygenae activity. Microsomal fractions isolated from the cat lung contain cyclooxygenase as well as prostacyclin (PGI₂) synthase, TX synthase, and a GSH-dependent prostaglandin (PG) E₂ isomerase activities. When [1-¹⁴C] PG endoperoxide H₂ (PGH₂) was used as substrate, ibuprofen, indomethacin, and meclofenamate exhibited differential effects on terminal enzyme activities. Ibuprofen, at concentrations up to 1 mM, had no effect on the activities of PGI₂ synthase, TXA₂ synthase of GSH-dependent PGE₂ isomerase, whereas indomethacin selectively inhibited PGI₂ synthase activity at 5 x 10^–4 M and 10^–3 M. Meclofenamate selectively inhibited TXA₂ synthase activity at 5 x 10^–4 M and 10^–3 M. At concentrations of 5 x 10^–3 M, this selectivity was not oberved, and indomethacin and meclofenamate decreased the formation of both 6-keto-PGF₁ and TXB₂. These data indicate that the choice of NSAID and the concentration employed may specifically alter PGH₂ metabolism. This action may affect the physiologic consequences of the exchange of PGH₂ between cells. The data further indicate that indomethacin has the potential for use as a tool to specifically attenuate PGI₂ synthase activity in vitro.     

Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A₂ (sPLA₂s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A₂ (hsPLA₂-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA₂-IID in Escherichia coli, the DNA-coding sequence for hsPLA₂-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA₂-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A₂. The refolded recombinant hsPLA₂-IID demonstrated Ca²⁺-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA₂-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.     

Lysosomotropic agents activate the capacity for calcium dependent pinocytosis in starvedAmoeba proteus: evidence for a mechanism involving phospholipase A2

Summary Pinocytosis induced by Na⁺ was assayed by phase contrast microscopy in 8–12 days starvedAmoeba proteus. These cultures were inactive with respect to calcium-dependent Na⁺-induced pinocytosis, but treatment with amino acid methyl and ethyl esters increased their capacity for pinocytosis. Besides promoting pinocytosis these compounds also stimulated calcium-sensitive secretion of lysosomal enzymes from normal, 2–3 days starved, cells. Only uncharged 1-forms of the amino acid esters were effective. Also other lysosomotropic compounds including monodansylcadaverine, glycine-phenylalanine-2-naphthylamide, NH₄Cl, and the ionophores monensin and A23187 activated starved cells. The effect of these agents (except A23187) was inhibited by the drug dantrolene suggesting that activation is a consequence of release of Ca²⁺ from intracellular stores. Several of the lysosomotropic agents also lost their activating effect in the presence of phospholipase A₂ (PLA₂) inhibitors. To investigate whether or not PLA₂ activity in the cell culture could imitate the effect of the lysosomotropic agents, we incubated starved cells with snake venom PLA₂s. These enzymes caused rapid, dantrolene-sensitive activation of the cells. Measurement of endogenous PLA₂in normal cells revealed significant cellular activity but no significant secretion of the enzyme into the culture medium was observed. Together the studies with enzyme inhibitors and dantrolene suggest that the process by which lysosomotropic agents affect pinocytosis involves activation of PLA₂ and release of Ca²⁺ from intracellular stores.Abbreviations AnBOMe amino-n-butyric acid methylester - Et ethylester - GPN glycine-1-phenylalanine-2-naphthylamide - MDC monodansylcadaverine - MDTC monodansylthiacadaverine - Me methylester - pBPB p-bromo phenacylbromide - PLA₂ phospholipase A₂     

Purification and characterization of a cytosolic Ca<Superscript>2+</Superscript>-independent phospholipase A<Subscript>2</Subscript> from bovine brain

The Ca²⁺-independent phospholipase A₂ (iPLA₂) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA₂ from bovine brain. iPLA₂ was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA₂ activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on twodimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃), Triton X-100, iron, and Ca²⁺. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA₂, and adenosine triphosphate (ATP). The spot with the iPLA₂ activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA₂.     

Purification and characterization of a potent hemolytic toxin with phospholipase A2 activity from the venom of Indian Russell's viper

A potent toxin with phospholipase A₂ (PLA₂) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc) of 15.3 kDa molecular weight, and a lethal toxicity dose (LD₅₀ i.p.) of 0.1 mg/kg body weight, was the most toxic PLA₂ so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA₂ toxin (RVV-PFIIc) was shown to be different from other PLA₂s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc showed variable degree of homology (42.1–94.7%) with those of other RVV-PLA₂s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc, though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc, failed to inhibit their PLA₂ activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc, at least two other PLA₂ isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

A turn in the road: How studies on the pharmacology of glucosylceramide synthase inhibitors led to the identification of a lysosomal phospholipase A2 with ceramide transacylase activity

A series of inhibitors of glucosylceramide synthesis, the PDMP based family of compounds, has been developed as a tool for the study of sphingolipid biochemistry and biology. During the course of developing more active glucosylceramide synthase inhibitors, we identified a second site of inhibitory activity for PDMP and its structural homologues that accounted for the ability of the inhibitors to raise cell and tissue ceramide levels. This inhibitory activity was directed against a previously unknown pathway for ceramide metabolism, viz. the formation of 1-O-acylceramide. In this pathway the addition of a fatty acyl group to the primary hydroxyl of ceramide occurs through a transacylation with either phosphatidylethanolamine or phosphatidylcholine as a substrate. However, both in the absence and presence of ceramide, water serves as an acceptor for the fatty acid. Thus the enzyme may be considered to be a phospholipase A₂. The enzyme is unique in that it has an acidic pH optimum and is localized to lysosomes by cell fractionation. More recently, the 1-O-acylceramide synthase has been purified, sequenced, and cloned. This phospholipase A₂ was discovered to be structurally homologous to lecithin cholesterol acyltransferase (LCAT). However, this phospholipase A₂ does not recognize cholesterol and lacks the defined lipoprotein-binding domain present in LCAT. We now refer to this enzyme as lysosomal phospholipase A₂ (LPLA₂). Although acidic phospholipase A₂ activities have been previously identified, LPLA₂ appears to be the first lysosomal PLA₂ to have been sequenced. This new phospholipase A₂ lacks an obvious and proven biological function. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (Maracambóia) venom

The PLA₂ and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS—PAGE showed that the PLA₂ and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA₂ showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA₂ showed allosteric behavior, with maximal activity at pH 8.3 and 35–40°C. The C. d. cascavella PLA₂ required Ca²⁺ for activity, but was inhibited by Cu²⁺ and Zn²⁺ and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA₂ by acting as allosteric inhibitors.     

Inhibition of gastric acid secretion by a standardized aqueous extract of Cecropia glaziovii Sneth and underlying mechanism

Cecropia glazioui Sneth (Cecropiaceae) is used in folk medicine in tropical and subtropical Latin America as cardiotonic, diuretic, hypotensive, anti-inflammatory and anti-asthmatic. The hypotensive/antihypertensive activity of the plant aqueous extract (AE) and isolated butanolic fraction (BuF) has been confirmed and putatively related to calcium channels blockade in vascular smooth musculature [Lapa, A.J., Lima-Landman, M.T.R., Cysneiros, R.M, Borges, A.C.R., Souccar, C., Barreta, I.P., Lima, T.C.M., 1999. The Brazilian folk medicine program to validate medicinal plants – a topic in new antihypertensive drug research. In: Hostettman, K., Gupta, M.P., Marston, A. (Eds.), Proceedings Volume, IOCD/CYTED Symposium, Panamá City, Panamá, 23–26 February 1997. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Harwood Academic Publishers, Amsterdam, pp. 185–196; Lima-Landman, M.T., Borges, A.C., Cysneiros, R.M., De Lima, T.C., Souccar, C., Lapa, A.J., 2007. Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism. Phytomedicine 14, 314–320]. Bronchodilation and antidepressant-like activities of both AE and BuF have been also shown [Delarcina, S., Lima-Landman, M.T., Souccar, C., Cysneiros, R.M., Tanae, M.M., Lapa, A.J., 2007. Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles. Phytomedicine 14, 328–332; Rocha, F.F., Lima-Landman, M.T., Souccar, C., Tanae, M.M., De Lima, T.C., Lapa, A.J., 2007. Antidepressant-like effect of Cecropia glazioui Sneth and its constituents – in vivo and in vitro characterization of the underlying mechanism. Phytomedicine 14, 396–402]. This study reports the antiulcer and antisecretory gastric acid activities of the plant AE, its BuF and isolated compounds with the possible mechanism involved. Both AE and BuF were assayed on gastric acid secretion of pylorus-ligated mice, on acute models of gastric mucosal lesions, and on rabbit gastric H⁺, K⁺-ATPase preparations. Intraduodenal injection of AE or BuF (0.5–2.0 g/kg, i.d) produced a dose-related decrease of the basal gastric acid secretion in 4-h pylorus-ligated mice. At 1.0 g/kg, BuF decreased the volume (28%) and total acidity (33%) of the basal acid secretion, and reversed the histamine (2.5 mg/kg, s.c.)- or bethanecol (1.0 mg/kg, s.c.)-induced acid secretion to basal values, indicating inhibition of the gastric proton pump. Pretreatment of mice with the BuF (0.05–0.5 g/kg, p.o.) protected against gastric mucosal lesions induced by 75% ethanol, indomethacin (30 mg/kg, s.c.) or restraint at 4 °C. BuF also decreased the gastric H⁺, K⁺-ATPase activity in vitro proportionately to the concentration (IC₅₀=58.8 μg/ml). The compounds isolated from BuF, consisting mainly of cathechins, procyanidins and flavonoids [Tanae, M.M., Lima-Landman, M.T.R., De Lima, T.C.M., Souccar, C., Lapa, A.J., 2007. Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antacid secretion and antidepressant-like activities. Phytomedicine 14, 309–313], inhibited the in vitro gastric H⁺, K⁺-ATPase activity at equieffective concentrations to that of BuF. The results indicate that C. glazioui constituents inhibit the gastric proton pump; this effect may account for the effective antisecretory and antiulcer activities of the standardized plant extract.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^–9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

The plant growth regulator activity of the fungicide,epoxiconazole, on Galium aparine L. (cleavers)

The plant growth regulator activity of epoxiconazole, a new triazole fungicide, was investigated by time-course, dose-response and histology experiments with Galium aparine L. (cleavers). Seven days after treatment with 125g ai ha^–1 epoxiconazole (field rate), plant height was reduced by 43%. After seventeen days, leaflet area was reduced by 27% but leaflet fresh weight was not significantly influenced. This was partly because leaflet thickness had increased by 20% following epoxiconazole application. Chlorophyll concentrations were also increased on a unit area basis. Examination of leaflet anatomy showed that epoxiconazole elongated palisade, spongy mesophyll and upper epidermal cells. For example, 125g ai ha^–1 caused a 35% increase in the length of spongy mesophyll cells. Epoxiconazole also prevented cell separation as there were significantly more palisade and spongy mesophyll cells per unit area than in leaflets sprayed with water. Stem development was reduced and 125g ai ha^–1 inhibited the elongation of pith cells in stem tissue by 53%. However, the simultaneous application of gibberellin A₃ (GA₃) with epoxiconazole resulted in stem elongation similar to that of control plants. These observations are consistent with the expected effects following the inhibition of cytochrome P-450 dependent enzyme activity.Abbreviations GA₃ gibberellin A₃ - g ai ha^–1 grams of active ingredient per hectare - L ha^–1 litres per hectare - PPFD photosynthetic photon flux density - RH relative humidity - SE standard error     

Properties of receptors for neurotoxic phospholipases A2 in different tissues

A radioiodinated derivative of OS₂ (¹²⁵I–OS₂), a neurotoxic monochain phospholipase A₂ isolated from taipan venom, was previously found to bind to a specific brain membrane receptor with very high affinity.¹²⁵I–OS₂ is now used to identify the properties of neurotoxic phospholipase receptors in other tissues. Heart, skeletal muscle, kidney, lung, liver, pancreas, and smooth muscle membranes also contain high-affinity binding sites for toxic phospholipases A₂. In most tissues, two different types of receptor sites have been characterized for¹²⁵I–OS₂ with K_d1 and K_d2 values in the 1–5 pM and the 10–50 pM range respectively. Whereas all receptors are similar in the different tissues in terms of their affinity for¹²⁵I–OS₂, maximal binding site capacities were very different, varying from 1.4 pmol/mg of protein in brain to 0.01 pmol/mg of protein in pancreaas. In brain, heart, and skeletal muscle, receptor densities vary with in vivo development. Affinity labeling experiments have identified the subunit composition of OS₂ receptors and indicated that these receptors do not have identical structures in the different tissues. Binding competition studies with OS₂ and other toxic phospholipases showed tissue-dependent pharmacological profiles. All these results taken together suggest the existence of a family of receptors for neurotoxic phospholipases.The abbreviations used are PLA₂ phospholipase A₂ - DSS suberic acid bis-N-hydroxysuccinimide ester - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - OS₁ Oxyuranus scutellatus scutellatus toxin 1 - OS₂ Oxyuranus scutellatus scutellatus toxin 2 Special issue dedicated to Dr. Lawrence Austin.     

Nonantibiotic Properties of Tetracyclines: Structural Basis for Inhibition of Secretory Phospholipase A2

Secretory phospholipase A₂ is involved in inflammatory processes and was previously shown to be inhibited by lipophilic tetracyclines such as minocycline (minoTc) and doxycycline. Lipophilic tetracyclines might be a new lead compound for the design of specific inhibitors of secretory phospholipase A₂, which play a crucial role in inflammatory processes. Our X-ray crystal structure analysis at 1.65 Å resolution of the minoTc complex of phospholipase A₂ (PLA₂) of the Indian cobra (Naja naja naja) is the first example of nonantibiotic tetracycline interactions with a protein. MinoTc interferes with the conformation of the active-site Ca²⁺-binding loop, preventing Ca²⁺ binding, and shields the active site from substrate entrance, resulting in inhibition of the enzyme. MinoTc binding to PLA₂ is dominated by hydrophobic interactions quite different from antibiotic recognition of tetracyclines by proteins or the ribosome. The affinity of minoTc for PLA₂ was determined by surface plasmon resonance, resulting in a dissociation constant K_d = 1.8 × 10^− 4 M.