The non-venom insect phospholipases A2

Phospholipases A(2) (PLA(2)s) are responsible for releasing the fatty acid moiety from the sn-2 position of phospholipids. These enzymes are virtually ubiquitous proteins known from all major biological taxa. Various PLA(2)s act in a wide array of biological processes, including digestion of dietary lipids, cellular homeostasis, intra- and intercellular signaling, host defense and at least a few ecological interactions. PLA(2) activities have been recorded in a small number of insect species, which can be taken to represent the broad group, Insecta. Within insects, PLA(2)s act in functions expected from the background on these enzymes. So far, we know PLA(2)s act in lipid digestion, cellular host defense signaling, reproduction and in organismal-level metabolism. Additional PLA(2) actions are certain to emerge. This is the first article devoted to assembling the known information on insect PLA(2)s. I review the scant information available on the biological actions of PLA(2)s in insects, relate new findings on insect pathogens that disrupt insect immune functions by inhibiting PLA(2)s and mention the few reports of sequence information on insect PLA(2)s. Finally, I offer a brief prospectus on future research into insect PLA(2)s. There are two overarching points in this paper. One, there remains a great deal to learn about insect PLA(2)s and two, some of the findings on insect PLA(2)s will have meaningful practical significance.     

Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2

In an effort to identify the anticoagulant region of venom phospholipases A2, we have systematically compared the amino acid sequences of strong, weak and non-anticoagulant phospholipases. The comparison disclosed several significant substitutions in the region between residues 54 and 77 (homology numbers). This proposed anticoagulant region is positively charged in strong, but negatively charged in weak and non-anticoagulant phospholipases. The microenvironment of a tryptophan residue falls within the proposed region, accounting for the differential characteristics of intrinsic fluorescence changes observed at 335 nm after the binding of phospholipid vesicles to strong and weak anticoagulants. Four lysine residues are located in specific positions in the "anticoagulant" region of strong anticoagulants, and should form a cationic surface, based on analogy with the available crystallographic structures. The chemical modification of lysine, arginine, tyrosine, and tryptophan residues and carboxylate groups, performed by other investigators, not only provides added support for the predicted site, but also confirms the essentiality of the positive charges in the site. This region may participate in the formation of a specific preferential hydrolytic complex leading to the strong anticoagulant effect. The anticoagulant region is distinct and separate from the predicted neurotoxic and myotoxic sites, and is located on the opposite surface of the phospholipase molecule.     

The stimulatory effect of angiotensin II on Na-ATPase activity involves sequential activation of phospholipases and sustained PKC activity

Angiotensin II (Ang II) stimulates the proximal tubule Na⁺-ATPase through the AT₁ receptor/phosphoinositide phospholipase Cβ (PI-PLCβ)/protein kinase C (PKC) pathway. However, this pathway alone does not explain the sustained effect of Ang II on Na⁺-ATPase activity for 30 min. The aim of the present work was to elucidate the molecular mechanisms involved in the sustained effect of Ang II on Na⁺-ATPase activity. Ang II induced fast and correlated activation of Na⁺-ATPase and PKC activities with the maximal effect (115%) observed at 1 min and sustained for 30 min, indicating a pivotal role of PKC in the modulation of Na⁺-ATPase by Ang II. We observed that the sustained activation of PKC by Ang II depended on the sequential activation of phospholipase D and Ca²⁺-insensitive phospholipase A₂, forming phosphatidic acid and lysophosphatidic acid, respectively. The results indicate that PKC could be the final target and an integrator molecule of different signaling pathways triggered by Ang II, which could explain the sustained activation of Na⁺-ATPase by Ang II.     

In vitro study of antimicrobial activity of lactoferrins from various sources

Comparative antimicrobial activity of lactoferrins from various sources (native lactoferrin from Laprot, human hololactoferrin, recombinant human lactoferrin isolated from the cultural medium of permissive cell culture transfected using pseudoadenovirus nanostructure with the human lactoferrin gene, and native bovine lactoferrin) was studied to prove the possibility of their use for development of antimicrobial drugs. It was shown that all the substances were active against the Bacillus standard strains. The antibacterial activity was almost independent of the degree of saturation the lactoferrin molecules with Fe3+. The native human lactoferrin was more active than hololactoferrin against Candida when evaluated by the minimum inhibitory concentration (MIC). Fe(3+)-Non aturated recombinant human lactoferrin demonstrated the antimicrobial activity (by MIC) similar to that of the native human lactoferrin. The results showed that native and recombinant human lactoferrins might be used for the development of intravenous and intracavitary dosage forms, while the native bovine lactoferrin could be useful in development of oral drugs.     

The effect of chronic chlorpromazine administration on monoamine levels in various regions of rat brain

The neuroleptic drug, chlorpromazine (CPZ) has been shown to exert its antipsychotic effect by blocking post synaptic dopamine receptors. However, its effect on steady state levels of monoamines is still in discrepancy. In the present study, CPZ (4 mg/kg body weight) was administered intraperitoneally to adult Wistar rats chronically for 75 days and the levels of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) were assayed in various brain regions by high performance liquid chromatography (HPLC). After the experimental period body and brain weights were not statistically different from controls. NE and 5-HT levels were increased only in hippocampus by 15% (p<0.01) and 16% (p<0.01) respectively. DA levels were consistently increased in cortex by 39% (p<0.001), striatum-accumbens by 18% (p<0.01), hippocampus by 27% (p<0.01), hypothalamus by 34% (p<0.001), cerebellum by 36% (p<0.001) and brainstem by 40% (p<0.001) in CPZ treated rats compared to controls. The results suggest that chronic CPZ administration increases DA levels in almost all regions of brain and reflect the ability of CPZ to preferentially interfere with synaptic transmission mediated by DA in brain. It also suggests that this increase in DA might be responsible for certain side effects seen in patients after chronic CPZ treatment.     

Alterations in the activity of phospholipases A2 in postmortem white matter from patients with multiple sclerosis

Activities toward arachidonyl-labelled phospholipase A₂ substrates were assayed in fractions of white matter and cerebral cortex from control subjects and in fractions of demyelinated plaque, normal-appearing white matter and cerebral cortex from subjects who died with multiple sclerosis. Membranous activity at pH 8.6 in the presence of Ca²⁺, characteristic of 14 kDa secretory phospholipase A₂, in either multiple sclerosis white matter or cortex did not differ from controls, whereas membranous activity at pH 4.5 in the absence of added Ca²⁺, characteristic of lysosomal enzymes was increased over controls in both plaque and normal-appearing white matter but not cerebral cortex. Activity in the cytosol fraction, at pH 8.6 in the presence of Ca²⁺ and glycerol characteristic of the cytosolic 85 kDa enzyme was decreased by greater than 50% in both white matter and cortex samples from multiple sclerosis subjects. Immuno-precipitation and-blotting confirmed that the deficient activity was largely attributable to the 85 kDa enzyme although the enzyme protein was not similarly reduced.Special issue dedicated to Dr. Leon S. Wolfe.     

不同碳源对悬浮培养玫瑰茄细胞主要基质消耗的影响

采用了蔗糖、葡萄糖、可溶性淀粉3种不同碳源,考察了在摇瓶培养过程中,这3种碳源对维持悬浮培养过程中玫瑰茄细胞的生长及主要基质消耗的影响。结果表明,蔗糖和葡萄糖在这些方面的表现基本相同,而可溶性淀粉由于植物细胞对其没有有效的水解利用手段,因而不能支持植物细胞的生长,从基质消耗水平上来看,也明显慢于上述两类碳源。     

A bactericidal homodimeric phospholipases A2 from Bungarus fasciatus venom

Group IIA secretory phospholipases A(2) (sPLA(2)-II) is generally known to display potent gram-positive bactericidal activity, while group IA sPLA(2) (sPLA(2)-I) reportedly is not. In this work, a novel sPLA(2)-I named BFPA was identified from Bungarus fasciatus venom, and its antimicrobial activity was studied as well. The amino acid sequence of the venomous protein precursor was 145-amino acid in length, and contained a predicted 27-amino acid signal peptide and a 118-amino acid mature protein. Unlike the well-known sPLA(2)-Is, which have 14 half-cysteines forming 7 intramolecular disulfide bridges, BFPA possesses 15 half-cysteines. The additional cysteine might contribute to the formation of an intermolecular disulfide bridge of the homodimeric protein. In the biological activities assays, BFPA displayed the activities of anticoagulation and bactericidal against Escherichia coli and Staphylococcus aureus. This study is the first report about gram-positive bactericidal activity of sPLA(2)-I.     

Juvenile hormone activity from various sources of termite castes and their fungus gardens

Summary Juvenile hormone (JH) activity is shown by extracts of the rectal contents of workers and soldiers, and anal exudate of queen of the termites,Microtermes sp.,Microcerotermes beesoni (Snyder),Odontotermes obesus (Rambur),O. qurdaspurensis (Holmgren) andO. assmuthi (Holmgren) as well as the fungus,Termitomyces sp. which is present in the termitaria. The possible significance of JH from various sources of termite castes and their fungus gardens is discussed.

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Zusammenfassung Es wurde festgestellt, dass bei den TermitenMicrotermes sp.,Microcerotermes beesoni (Snyder),Odontotermes obesus (Rambur),O. qurdaspurensis (Holmgren) andO. asmuthi (Holmgren). Extrakte aus dem Analexkret der Könign und aus dem Pilz des Termitenhaus (Termitomyces sp.) eine Juvenilhormonaktivität (JH) zeigen. Die wahrscheinliche Bedeutung dieserer Juvenilhormonaktivität aus verchredenen Termitenkasten und ihrer Pilzkultures wird diskutiert.

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Résumé L'activité de l'hormone juvénile (JH) est mise en évidence à partir d'extraits de contenu rectal d'ouvriers et de soldats et d'exsudat anal de reines de termites,Microtermes sp.,Microcerotermes beesoni (Snyder),Odontotermes obesus (Rambur),O. qurdaspurensis (Holmgren) etO. assmuthi (Holmgren), ainsi que du champignonTermitomyces sp. qui est présent dans la termitière. La signification possible de JH provenant de différentes sources, castes de termites ou champignons, est discutée.

     

Effect of phospholipases on Factor-VIII activity

Summary The effect of the enzymes phospholipases C and D on Factor VIII were investigated. Phospholipase D was found to activate the partially purified intact Factor-VIII molecule maximally at a final concentration of 0.6 U/ml. Neither the dissociated small molecular weight component nor the high molecular weight component were activated. Phospholipase C, on the other hand, inactivated both the intact and the dissociated Factor-VIII molecule. Phospholipase D, however, had no effect on the haemophilic cryoprecipitate or the partially purified haemophilic Factor VIII. The implications of these results for the genetic control of the Factor-VIII molecule are discussed. In this connection, haemophilia A is hypothesized to be caused by an X-linked enzyme effect that impairs phospholipid assembly of the Factor-VIII protein, whereas von Willebrand's disease might be due to a structural defect of the Factor-VIII protein.     

Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A(2) from Bothrops snake venoms

Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.