Group XV phospholipase A₂, a lysosomal phospholipase A₂

A phospholipase A₂ was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A₂ (LPLA₂) and subsequently designated group XV phospholipase A₂. LPLA₂ has 49% of amino acid sequence identity to lecithin-cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA₂ is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA₂^−/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA₂^−/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA₂ may play a primary role in phospholipid homeostasis, drug toxicity, and host defense.     

Recent research progress on soil microbial responses to drying–rewetting cycles

Climatic change, such as increases in extreme drought and rainfall events and changes in rainfall intensity and pattern, has been strongly influencing soil moisture. The climatic change impact is particularly common in arid, semi-arid and Mediterranean regions, which is causing dramatic changes in the intensity and frequency of soil drying–rewetting cycles. The soil drying–rewetting cycle is a natural phenomenon that the soil experiences drying, then wetting, and then drying and rewetting again and again. When a dry soil is being rewetted, the amount of soil microbial biomass and its activity can be sharply increasing in a short time period, and then a large amount of gaseous carbon (C) and nitrogen (N) erupts from the soil. The sudden release of gaseous C and N is caused by the stimulation of the soil microbes. Such a phenomenon is called “Birch effect”. The drying–rewetting cycles have direct and indirect effects on soil microbes, and soil microbial responses to the drying and rewetting events play an important role in the feedbacks of terrestrial ecosystems. From aspects of soil microbial biomass, microbial activities and microbial structure, we review recent advances on studies regarding microbial responses to soil drying–rewetting cycles. We interpret the microbial responses using five different types of mechanisms: (1) Microbial stress mechanism: when a soil becomes dry, microorganisms must accumulate compatible solutes such as carbohydrates and aminoacids so that the soil microbes can equilibrate with their environment in order to avoid dehydrating and being killed. When the soil is rewetted, soil microbes must dispose of those osmolytes rapidly by transforming them into carbon dioxide (CO₂), dissolved organic carbon (DOC) and nutrients in order to prevent water from being flowing into the cells. (2) Substrate supply mechanism: low soil moisture may result in the physical disruption of soil aggregates which leads to the exposure of new soil surfaces and of previously protected organic matter. When the soil is rewetted, its physical structure is further disrupted by swelling. The increased new soil surfaces and previously protected organic matter will improve the microorganism’s nutrient availability. (3) Soil hydrophobicity mechanism: soil hydrophobicity can cause the reduction of soil moisture and nutrient availability and inhibition of microbial decomposition of soil organic matter. Therefore, soil hydrophobicity is an important factor of explaining the activity of microorganism in drying and rewetting events. (4) Diffusive limitations mechanism: transportation of the soil microbe is limited in a dry soil. When soil moisture is increasing, soil microbial activity is enhanced along with the increased availability of substrate nutrients. (5) Predation mechanism: a moist soil is usually conducive to the increase of bacteria and fungi populations. In response, protozoa and nematodes also increase, leading to the fluctuation of the soil microbial community structure. On the basis of the literature review, we propose five important aspects to be considered in the future: (1) assessing soil microbes’ concrete adapting ways to the drying–rewetting cycles, (2) evaluating the microbial responses to the drying–rewetting cycles based on suitable indicators, (3) interpreting microbial responses to the drying–rewetting cycles by combining field investigation and laboratory controlling experiment, (4) investigating the microbial responses to the drying–rewetting cycles at different temporal and spatial scales.     

An acidic phospholipase A₂ with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A₂ (PLA₂s) can be found. Basic PLA₂s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA₂s tend to have a lower toxicity. A novel PLA₂, here named PnPLA₂, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA₂ is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA₂ had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA₂s from viperid venoms. PnPLA₂ displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA₂ showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA₂, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C₂C₁₂. This is the first report on a bactericidal protein of Porthidium nasutum venom.     

Transglutaminase inhibition: A therapy to protect cells from death in neurodegeneration?

Transglutaminases(TGs;E.C.2.3.2.13)are ubiquitous enzymes which catalyze post-translational modifications of proteins.TGs and TG-catalyzed post-translational modifications of proteins have been shown to be involved in the molecular mechanisms responsible for several human diseases.In particular,TG activity has been hypothesized to also be involved also in the molecular mechanisms responsible for human neurodegenerative diseases.In support of this hypothesis,Basso et al recently demonstrated that the TG inhibition protects against oxidative stress-induced neuronal death,suggesting that multiple TG isoforms participate in oxidative stress-induced cell death and that nonselective TG isoform inhibitors will be most effective in fighting oxidative death in neurological disorders.In this commentary,we discuss the possible molecular mechanisms by which TG activity could be involved in the pathogenesis of neurological diseases,with particular reference to neurodegenerative diseases,and the possible involvement of multiple TG isoforms expressed simultaneously in the nervous system in these diseases.Moreover,therapeutic strategies based on the use of selective or nonselective TG inhibitors for the amelioration of thesymptoms of patients with neurological diseases,characterized by aberrant TG activity,are also discussed.     

A [Lys⁴⁹]phospholipase A₂ from Protobothrops flavoviridis venom induces caspase-independent apoptotic cell death accompanied by rapid plasma-membrane rupture in human leukemia cells

Protobothrops flavoviridis venom contains plural phospholipase A(2) (PLA(2)) isozymes. A [Lys(49)]PLA(2) called BPII induced cell death in human leukemia cells. PLA2, an [Asp(49)]PLA(2) that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA(2) lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.     

Cryptosporidiosis and Cryptosporidium species in animals and humans: A thirty colour rainbow?

Parasites of the genus Cryptosporidium (Apicomplexa) cause cryptosporidiosis in humans and animals worldwide. The species names used for Cryptosporidium spp. are confusing for parasitologists and even more so for non-specialists. Here, 30 named species of the genus Cryptosporidium are reviewed and proposed as valid. Molecular and experimental evidence suggests that humans and cattle are the hosts for 14 and 13 out of 30 named species, respectively. Two, four and eight named species are considered of major, moderate and minor public health significance, respectively. There are at least nine named species that are shared between humans and cattle. The aim of this review is to outline available species information together with the most commonly used genetic markers enabling the identification of named Cryptosporidium spp. Currently, 28 of 30 named species can be identified using the complete or partial ssrRNA, serving as a retrospective ‘barcode’. Currently, the ssrRNA satisfies the implicit assumption that the reference databases used for comparison are sufficiently complete and applicable across the whole genus. However, due to unreliable annotation in public DNA repositories, the reference nucleotide entries and alignment of named Cryptosporidium spp. has been compiled. Despite its known limitations, ssrRNA remains the optimal marker for species identification.     

Group X secreted phospholipase A₂ specifically decreases sperm motility in mice

Different mammalian secreted phospholipases A(2) (sPLA(2) s) are expressed in male reproductive organs and/or in sperm cells but their cellular functions are still not fully characterized. Because several reports indicate a link between cellular lipids and sperm motility, we have investigated the effect of mouse group IIA, IID, IIE, V, and X sPLA(2) s on sperm motility. Among these enzymes, only mouse group X sPLA(2) (mGX sPLA(2) ) acts as a potent inhibitor of sperm motility that decreases track speed (VCL) and lateral displacement of the head (ALH) of both noncapacitated and capacitated sperm. The inhibitory effect of mGX sPLA(2) is dependent on its enzymatic activity because (i) both the proenzyme form of mGX sPLA(2) (pro-mGX) and the H48Q mutant of mGX sPLA(2) have very weak enzymatic activity and are unable to modulate sperm motility and (ii) LY329722, a specific inhibitor of sPLA(2) s, blocks the inhibitory effect of mGX sPLA(2) . Moreover, mGX sPLA(2) exerts a gradual potency on sperm subpopulations with different velocities, an effect which may be linked to the heterogeneity of lipid composition in these sperm subpopulations. Finally, we found that endogenous mGX sPLA(2) released during spontaneous acrosome reaction modulates sperm motility of capacitated sperm. Together, our results suggest a new role of sPLA(2) in sperm physiology where the sPLA2 selects a sperm subpopulation for fertilization based on its effect on sperm motility.     

Group 1B phospholipase A₂ deficiency protects against diet-induced hyperlipidemia in mice

Excessive absorption of products of dietary fat digestion leads to type 2 diabetes and other obesity-related disorders. Mice deficient in the group 1B phospholipase A₂ (Pla2g1b), a gut digestive enzyme, are protected against diet-induced obesity and type 2 diabetes without displaying dietary lipid malabsorption. This study tested the hypothesis that inhibition of Pla2g1b protects against diet-induced hyperlipidemia. Results showed that the Pla2g1b^−/− mice had decreased plasma triglyceride and cholesterol levels compared with Pla2g1b^+/+ mice subsequent to feeding a high-fat, high-carbohydrate (hypercaloric) diet. These differences were evident before differences in body weight gains were observed. Injection of Poloxamer 407 to inhibit lipolysis revealed decreased VLDL production in Pla2g1b^−/− mice. Supplementation with lysophosphatidylcholine, the product of Pla2g1b hydrolysis, restored VLDL production rates in Pla2g1b^−/− mice and further elevated VLDL production in Pla2g1b^+/+ mice. The Pla2g1b^−/− mice also displayed decreased postprandial lipidemia compared with Pla2g1b^+/+ mice. These results show that, in addition to dietary fatty acids, gut-derived lysophospholipids derived from Pla2g1b hydrolysis of dietary and biliary phospholipids also promote hepatic VLDL production. Thus, the inhibition of lysophospholipid absorption via Pla2g1b inactivation may prove beneficial against diet-induced hyperlipidemia in addition to the protection against obesity and diabetes.     

Regulatory RNAs and chromatin modification in dosage compensation: A continuous path from flies to humans?

Chromosomal sex determination is a widely distributed strategy in nature. In the most classic scenario, one sex is characterized by a homologue pair of sex chromosomes, while the other includes two morphologically and functionally distinct gonosomes. In mammalian diploid cells, the female is characterized by the presence of two identical X chromosomes, while the male features an XY pair, with the Y bearing the major genetic determinant of sex, i.e. the SRY gene. In other species, such as the fruitfly, sex is determined by the ratio of autosomes to X chromosomes. Regardless of the exact mechanism, however, all these animals would exhibit a sex-specific gene expression inequality, due to the different number of X chromosomes, a phenomenon inhibited by a series of genetic and epigenetic regulatory events described as "dosage compensation". Since adequate available data is currently restricted to worms, flies and mammals, while for other groups of animals, such as reptiles, fish and birds it is very limited, it is not yet clear whether this is an evolutionary conserved mechanism. However certain striking similarities have already been observed among evolutionary distant species, such as Drosophila melanogaster and Mus musculus. These mainly refer to a) the need for a counting mechanism, to determine the chromosomal content of the cell, i.e. the ratio of autosomes to gonosomes (a process well understood in flies, but still hypothesized in mammals), b) the implication of non-translated, sex-specific, regulatory RNAs (roX and Xist, respectively) as key elements in this process and the location of similar mediators in the Z chromosome of chicken c) the inclusion of a chromatin modification epigenetic final step, which ensures that gene expression remains stably regulated throughout the affected area of the gonosome. This review summarizes these points and proposes a possible role for comparative genetics, as they seem to constitute proof of maintained cell economy (by using the same basic regulatory elements in various different scenarios) throughout numerous centuries of evolutionary history.     

Crystal structure of Bn IV in complex with myristic acid: a Lys49 myotoxic phospholipase A₂ from Bothrops neuwiedi venom

The LYS49-PLA₂s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA₂ is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA₂ was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH₃(CH₂)₁₂COOH) and its overall structure was refined at 2.2 Å resolution. The Bn IV crystals belong to monoclinic space group P2₁ and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 Å. The biological assembly is a “conventional dimer” and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes.     

Recent progress in understanding Alzheimer's β-amyloid structures

The formation of amyloid fibrils, protofibrils and oligomers from the β-amyloid (Aβ) peptide represents a hallmark of Alzheimer's disease. Aβ-peptide-derived assemblies might be crucial for disease onset, but determining their atomic structures has proven to be a major challenge. Progress over the past 5 years has yielded substantial new data obtained with improved methodologies including electron cryo-microscopy and NMR. It is now possible to resolve the global fibril topology and the cross-β sheet organization within protofilaments, and to identify residues that are crucial for stabilizing secondary structural elements and peptide conformations within specific assemblies. These data have significantly enhanced our understanding of the mechanism of Aβ aggregation and have illuminated the possible relevance of specific conformers for neurodegenerative pathologies.     

Unexpected prion phenotypes in experimentally transfused animals: predictive models for humans?

ABSTRACT

The recently reevaluated high prevalence of healthy carriers (1/2,000 in UK) of variant Creutzfeldt-Jakob Disease (v-CJD), whose blood might be infectious, suggests that the evolution of this prion disease might not be under full control as expected. After experimental transfusion of macaques and conventional mice with blood derived from v-CJD exposed (human and animal) individuals, we confirmed in these both models the transmissibility of v-CJD, but we also observed unexpected neurological syndromes transmissible by transfusion: despite their prion etiology confirmed through transmission experiments, these original cases would escape classical prion diagnosis, notably in the absence of detectable abnormal PrP with current techniques. It is noteworthy that macaques developed an original, yet undescribed myelopathic syndrome associating demyelination and pseudo-necrotic lesions of spinal cord, brainstem and optical tract without affecting encephalon, which is rather evocative of spinal cord disease than prion disease in human medicine. These observations strongly suggest that the spectrum of human prion diseases may extend the current field restricted to the phenotypes associated to protease-resistant PrP, and may notably include spinal cord diseases.     

Neuroscience and education: from research to practice?

Cognitive neuroscience is making rapid strides in areas highly relevant to education. However, there is a gulf between current science and direct classroom applications. Most scientists would argue that filling the gulf is premature. Nevertheless, at present, teachers are at the receiving end of numerous 'brain-based learning' packages. Some of these contain alarming amounts of misinformation, yet such packages are being used in many schools. What, if anything, can neuroscientists do to help good neuroscience into education?     

Nitric Oxide：from a mysterious labile factor to the molecule of the Nobel Prize Recent progress in nitric oxide ressarch

NO is now known to be an important messenger molecule in biology.It regulates a variety of functions within cells and tissues including vasodilation,neurotransmission and immunological process.This review will focus on the nitric oxide synthase gene family and recent progress on molecular genetic analysis of NOS1,NOS2 and NOS3 genes.     

Quinoline-4-methyl esters as human nonpancreatic secretory phospholipase A₂ inhibitors

A series of novel fused heterocycle methyl esters were designed and synthesized as human nonpancreatic secretory phospholipase A? (hnps-PLA?) competitive inhibitors. Among the 22 synthesized compounds, 17 quinoline-4-methyl esters displayed hnps-PLA? inhibition activity in the in vitro bioassay. The IC?? value for the best compound 3o was 1.5 μM. The structure-inhibition-activity relationships of the compounds were studied using molecular docking.     

Regulation of macrophage differentiation and polarization by group IVC phospholipase A₂

Although the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbol ester-induced macrophage differentiation. We therefore examined the role of IVC-PLA(2) in macrophage differentiation using THP-1 cells. Two THP-1 cell lines stably expressing IVC-PLA(2)-specific shRNA were established. Differentiation and maturation into macrophages on treatment with phorbol ester were facilitated by knockdown of IVC-PLA(2) without the compensatory induction of mRNA expression for other group IV and VI PLA(2)s. Furthermore, the enhancement of macrophage differentiation by IVC-PLA(2)-knockdown were abolished by treatment with lysophosphatidylcholine, a metabolite of phospholipids generated by PLA(2)-mediated hydrolysis, indicating that PLA(2) activity is necessary for the inhibition of macrophage differentiation by IVC-PLA(2). Additionally, we found that the differentiation into classically activated M1 macrophage was superior in IVC-PLA(2)-knockdown cells, whereas the differentiation into alternatively activated M2 macrophage was suppressed by IVC-PLA(2)-knockdown. These findings suggest that IVC-PLA(2) is involved in regulations of macrophage differentiation and macrophage polarization.