Expression of group XIIA phospholipase A2 in human digestive organs.

Cellular distribution of group XIIA phospholipase A2 (GXIIA PLA2) was studied in human digestive organs by immunohistochemistry. GXIIA PLA2 protein was detected in epithelial cells of normal gastrointestinal tract, gallbladder and pancreatic acinar cells. The GXIIA PLA2 protein was evenly distributed in the cytoplasm in contrast to secretory granular distribution of GIB PLA2 and GIIA PLA2 in pancreatic acinar cells and small intestinal Paneth cells respectively. Epithelial cells of intestinal glands in Crohn's disease and ulcerative colitis expressed abundant GXIIA PLA2 , whereas inflammatory cells were devoid of the enzyme protein. Tumour cells in colonic adenomas and carcinomas and pancreatic ductogenic carcinomas expressed GXIIA PLA2 protein at varying intensity levels. The putative functions of GXIIA PLA2 remain to be investigated and its role in healthy and diseased digestive organs can only be speculated on at present.

Identification of novel phospholipase A2 group IX members in metazoans.

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA2 pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA2 sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA2s while metazoan representatives are similar with GIX PLA2 of the marine snail Conus magus. Members of GXIV and GIX PLA2s show modest overall sequence similarity (21-35%) but considerable motif conservation within the putative Ca(2+)-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA2s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA2 homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA2 deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA2s, two of which correspond to those localized in GXIV PLA2s. Phylogenetic analysis demonstrates that metazoan GIX PLA2s cluster separate from the bacterial and fungal GXIV PLA2s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA2 pfam06951. Duplicate PLA2 pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA2s and support the idea of the common evolutionary origin of GXIV and GIX PLA2 pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.

1-Cysteine peroxiredoxin: A dual-function enzyme with peroxidase and acidic Ca2+-independent phospholipase A2 activities.

Peroxiredoxins (Prx) are enzymes that catalyze the reduction of hydrogen peroxide and alkyl hydroperoxides. Prxs are ubiquitous enzymes with representatives found in Bacteria, Archaea and Eukarya. Many 1-cysteine peroxiredoxins (1-CysPrx) are dual-function enzyme with both peroxidase and acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)) activities. The functions proposed for 1-CysPrx/aiPLA(2) include the protection of cell membrane phospholipids against oxidative damage (peroxidation) and the metabolism (hydrolysis) of phospholipids, such as those of lung surfactant. The peroxidase active site motif PVCTTE of 1-CysPrx contains the conserved catalytic cysteine residue, and the esterase (lipase) motif GXSXG of the enzyme contains the conserved catalytic serine residue. In addition to the classic lipase motif GXSXG, various 1-CysPrx/aiPLA(2)s have closely related variant putative lipase motifs containing the catalytic serine residue. The PLA(2) moieties are prevalent and highly homologous in vertebrate and bacterial 1-CysPrx/aiPLA(2)s that is consistent with a high degree evolutional conservation of the enzyme.

Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators.

Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA(2) [mouse (m)GX] is one of the most highly expressed PLA(2) in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA(2)s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA(2) inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA(2)alpha and M-type sPLA(2) receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA(2) mitogenic effects. Together, our results indicate that group X sPLA(2) may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.

Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A(2), sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Group IIA phospholipase A as a prognostic marker in prostate cancer: relevance to clinicopathological variables and disease-specific mortality.

Group IIA Phospholipase A(2) (PLA2-IIA), a key enzyme in arachidonic acid and eicosanoid metabolism, participates in a variety of inflammatory processes but possibly also plays a role in tumor progression in vivo. Our aim was to determine the mRNA and protein expression of PLA2-IIA during prostate cancer progression in localized and metastatic prostate tumors. We evaluated the prognostic significance of PLA2-IIA expression in biochemical recurrence, clinical recurrence and disease-specific survival after surgical treatment. The expression of PLA2-IIA was examined by immunohistochemistry and chromogenic in situ hybridization in tissue microarrays of radical prostatectomy specimens and advanced/metastatic carcinomas. The expression data were analyzed in conjunction with clinical follow-up information and clinicopathological variables. The mRNA and protein expression of PLA2-IIA was significantly increased in Gleason pattern grade 2-4 carcinomas compared with benign prostate (p-values 0.042-0.001). In metastases, the expression was significantly lower than in local cancers (p=0.001). The PLA2-IIA expression correlated positively with Ki-67 and alpha-methylacyl CoA racemase (AMACR) expression. The prognostic evaluation revealed decreased PLA2-IIA protein expression among patients who had died of prostate cancer. In conclusion, PLA2-IIA expression is increased in carcinoma when compared with benign prostate. However, metastatic carcinoma showed decreased expression of PLA2-IIA when compared with primary carcinomas. PLA2-IIA may serve as a marker for highly proliferating, possibly poorly differentiated prostate carcinomas. The protein expression of PLA2-IIA may be diminished in patients who consequently die of prostate cancer.

Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels.

Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

Automated pattern ranking in differential display data analysis.

Gene expression analysis by differential display (DD) is limited by the labor-intensive visual evaluation of the electrophoretic data traces. We describe a flexible method for computer-assisted ranking of expression patterns in data from DD experiments. The method is based on a pairwise alignment and comparison of the quantitative trace data with respect to specific expression patterns defined by the investigator. The observed patterns are ranked according to a score value that identifies the most potential findings to be confirmed visually instead of the vast amount of original results. This two-step approach, enabled by the efficient computer algorithm for gene expression pattern comparison, will increase the percentage of true-positive findings chosen for the tedious downstream processing, while minimizing the cost and labor involved in large scale DD data analysis.

mRNA differential display of gene expression in colonic carcinoma.

We analyzed changes in gene expression in human colonic carcinoma by fluorescent mRNA differential display. RNA isolated from two samples of normal colon and four cases of colonic adenocarcinoma were amplified with a 15 x 32 set of primers resulting in 2880 cDNAs analyzed with an automated sequencer. Electrophoretic patterns implying constitutive gene expression as well as upregulated and downregulated expression in carcinomas were identified. Forty such cDNA fragments were purified by a novel fluorescent polyacrylamide gel electrophoresis (PAGE)-based method and identified by cyclic sequencing. Most genes showing differential expression were upregulated in colonic carcinoma. Upregulated genes included those for various ribosomal and mitochondrial proteins, heat shock proteins, nucleolar RNA-helicase and phosphoserine aminotransferase. Downregulated genes included histone H3.3. In conclusion, genes associated with vital cellular functions such as transcription, protein synthesis and mitochondrial metabolism were upregulated in colonic carcinoma. Fluorescent mRNA differential display can be applied to the identification of novel cancer-related genes for diagnostic, prognostic and therapeutic purposes.

Cloning of a novel phospholipase A2 from the cnidarian Adamsia carciniopados.

PLA2 catalytic activity was detected in homogenised tissues, including tentacles and acontia (structures for preying and defence, respectively), of the sea anemone Adamsia carciniopados. Nested reverse transcription polymerase chain reaction (RT PCR) with degenerate primers and rapid amplification of cDNA ends (RACE) were used to clone a novel phospholipase A2 from Adamsia carciniopados (AcPLA2). AcPLA2 contains a putative prepropeptide of 37 residues, ending with a basic doublet followed by a mature protein of 119 amino acids, including 12 cysteines. AcPLA2 displays only 30-42% similarity with other known secretory PLA2s (sPLA2). C-terminal extension, typical of groups II and X PLA2s, is absent. Predicted molecular weight and pI of the mature protein are 13.5 kDa and 9.1, respectively. Structural features and phylogenetic analysis set AcPLA2 apart from the known sPLA2s and define this molecule in the ancient metazoan phylum Cnidaria as a member of a new class of sPLA2s.

Catalytic activity of phospholipase A2 in blood plasma and pancreatic tissue perfusion in early experimental acute pancreatitis.

BACKGROUND: To investigate the sequence of changes in the catalytic activity of phospholipase A2 in plasma and pancreatic tissue perfusion and oxygenation in mild and severe acute pancreatitis in pigs. METHODS: Twenty-four pigs were randomized into the groups of severe acute pancreatitis, mild acute pancreatitis, and controls. The pancreatic duct of eight anesthetized and mechanically ventilated pigs was cannulated, and taurocholic acid was infused into the pancreatic duct to induce severe acute pancreatitis. Eight animals received intraductal saline and developed mild acute pancreatitis. Eight pigs were cannulated only and served as controls. RESULTS: Central hemodynamics, arterial blood gases, and acid-base balance were stable throughout the study period in all three groups. Pancreatic tissue oxygenation decreased in pigs with severe acute pancreatitis and increased in animals with mild acute pancreatitis. The catalytic activity of phospholipase A2 in plasma remained stable, and there was no difference between the groups. Similarly, C-reactive protein values remained within the normal range during the study period in all groups. CONCLUSION: Plasma phospholipase A2 levels do not react to the changes in pancreatic tissue perfusion in the early phase of mild and severe acute pancreatitis.