Amelioration of TNBS-induced colon inflammation in rats by phospholipase A2 inhibitor

The pathophysiology of inflammatory bowel disease (IBD) involves the production of diverse lipid mediators, namely eicosanoids, lysophospholipids, and platelet-activating factor, in which phospholipase A2 (PLA2) is the key enzyme. Accordingly, it has been postulated that control of lipid mediator production by inhibition of PLA2 would be useful for the treatment of IBD. This hypothesis was tested in the present study by examining the therapeutic effect of a novel extracellular PLA2 inhibitor (ExPLI), composed of carboxymethylcellulose-linked phosphatidylethanolamine (CMPE), on trinitrobenzenesulfonic acid-induced colitis. Intraperitoneal administration of CMPE suppressed the colitis as measured by mortality rate, intestinal permeability, plasma PLA2 activity, intestinal myeloperoxidase activity, and histological morphometry. Current therapeutic approaches for inflammatory conditions focus on the selective control of a lipid mediator(s) (e.g., prostaglandins or leukotrienes). The present study supports the concept that inclusive control of lipid mediator production by PLA2 inhibition is a plausible approach to the treatment of colitis and introduces the ExPLIs as a prototype of a novel NSAID for the treatment of intestinal inflammation.     

Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2

Phospholipases A2 (PLA2s) play a key role in inflammatory processes through production of precursors of eicosanoids and platelet-activating factor. Recently, we described the purification of a novel approximately 100-kDa cytosolic PLA2 (cPLA2) from human monoblast U937 cells that is activated by physiological (intracellular) concentrations of Ca2+ (Kramer, R. M., Roberts, E. F., Manetta, J., and Putnam, J. E. (1991) J. Biol. Chem. 266, 5268-5272). Here we report the isolation of the complementary DNA encoding human cPLA2 and confirm its identity by expression in bacteria and in hamster cells. The predicted 749-amino acid cPLA2 protein has no similarity to the well known secretory PLA2s, but contains a structural element homologous to the C2 region of protein kinase C. The molecular cloning of cPLA2 will allow further studies defining the structure, function, and regulation of this novel PLA2.     

APOE genotype and an ApoE-mimetic peptide modify the systemic and central nervous system inflammatory response

Human apolipoprotein E is the major apolipoprotein expressed in the brain and exists as three isoforms, designated E2, E3, and E4. Although evidence suggests that apolipoprotein E plays an important role in modifying systemic and brain inflammatory responses, there is little data investigating apoE isoform-specific effects in vivo. In this study, we compared the inflammatory responses of targeted-replacement mice expressing the human APOE3 and APOE4 genes after intravenous administration of lipopolysaccharide. Animals expressing the E4 allele had significantly greater systemic and brain elevations of the pro-inflammatory cytokines TNFalpha and IL-6 as compared with their APOE3 counterparts, suggesting an isoform-specific effect of the immunomodulatory properties of apoE. Furthermore, intravenous administration of a small apoE-mimetic peptide derived from the receptor-binding region of the apoE holoprotein (apoE-(133-149)) similarly suppressed both systemic and brain inflammatory responses in mice after lipopolysaccharide administration. These results suggest that apoE plays an isoform-specific role in mediating the systemic and brain inflammatory responses. Moreover, because exogenous administration of this apoE mimetic peptide is effective at suppressing both systemic and brain inflammation, it may represent a novel therapeutic strategy for diseases characterized by systemic or central nervous system inflammation, such as septic shock, multiple sclerosis, and traumatic brain injury.     

Trichinella spiralis induces de novo expression of group IVC phospholipase A2 in the intestinal epithelium

Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.     

Duration and intensity of NF-kappaB activity determine the severity of endotoxin-induced acute lung injury

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-kappaB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-kappaB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-kappaB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-kappaB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IkappaB kinase inhibitor (BMS-345541) to down-regulate NF-kappaB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-kappaB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-kappaB activation correlates with severity of lung injury, and interdiction in the NF-kappaB pathway is beneficial even after the onset of lung inflammation.     

Localized changes in the gp120 envelope glycoprotein confer resistance to human immunodeficiency virus entry inhibitors BMS-806 and #155

BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the "phenylalanine 43 cavity" and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs.     

Enzymatic synthesis of chiral intermediates for Omapatrilat, an antihypertensive drug

Biocatalytic processes were used to prepare chiral intermediates required for the synthesis of Omapatrilat 1 by three different routes. The synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 3 to L-6-hydroxynorleucine 2 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of the ketoacid 3, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 4] with D-amino acid oxidase from porcine kidney or Trigonopsis variabilis followed by reductive amination to convert the mixture completely to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess (e.e.). The enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 5) was demonstrated using phenylalanine dehydrogenase (PDH) from T. intermedius. Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris. Using PDH from E. coli or P. pastoris, the enzymatic process was scale-up to prepare kg quantity of allysine ethylene acetal 5. The reaction yields of >94% and e.e. of >98% were obtained for allysine ethylene acetal 5. An enzymatic process was developed for the synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4 [[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid [BMS-199541-01]. The enzymatic oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[[(phenyl-methoxy)carbonyl] L-homocysteinyl] L-lysine)-1,1-disulphide [BMS-201391-01] to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase (LAT) from Sphingomonas paucimobilis SC 16113 was demonstrated. This enzyme was overexpressed in E. coli and a process was developed using the recombinant enzyme.     

Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response.

Serum levels of phospholipase A2 (PLA2) activity have been shown to be elevated in cases of septic shock and rheumatoid arthritis. The cellular origin of serum PLA2, however, is not known. In this report, we demonstrate that human group II PLA2 expression and secretion are induced in hepatoma cells (HepG2) following treatment with interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-1 (IL-1). Of the three cytokines, IL-6 is the most potent. Significant synergy is observed between IL-6 and IL-1 and between IL-6 and TNF, but not between IL-1 and TNF. PLA2 induction does not occur in human YT cells, which are known to have receptors for both IL-1 and IL-6, indicating that the regulatory mechanism involved is cell type-specific. The results of RNA blot analysis indicate that the PLA2 gene is regulated in HepG2 cells at the pretranslational level. Induction of PLA2 synthesis in HepG2 cells in response to these cytokines resembles the induction of the acute phase plasma proteins which are synthesized in cultured hepatocytes and hepatoma cells following exposure to the same cytokines and in liver in response to inflammation and infection. In addition, a putative IL-6-responsive element, which is homologous to a similar element found in several acute phase genes, is present in the 5'-promoter-proximal region of the PLA2 gene. These results suggest that serum PLA2 is synthesized in and secreted from liver cells in response to inflammatory stimuli, mediated primarily by IL-6, and therefore should be classified as an acute phase protein.     

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N(2)-

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine epsilon-aminotransferase.     

The mechanisms of action of interleukin-1 on rabbit intestinal epithelial cells

Interleukin-1 (IL-1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL-1's action, cells were treated with IL-1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2-activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL-1 caused concentration- and time-dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX-2 and PGE2. The IL-induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX-2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.     

The role of phosphatidylcholine and deoxycholic acid in inflammation

Aims

Phosphatidylcholine with deoxycholic acid (PC/DA) is widely used to reduce localized fat deposits with mild adverse effects. We previously demonstrated that PC induces lipolysis with mild PMN infiltration, while DA induces adipose tissue damage. Therefore, the aim of this study was to extend our understanding of the pro-inflammatory responses of PC, DA, and PC/DA.

Main methods

We evaluated the level of edema and polymononuclear (PMN) infiltration by histopathological examination. Myeloperoxidase (MPO) activity was analyzed using an MPO activity assay kit. Levels of inflammatory cytokines (IL-1β and IL-6) and PGE2 were measured by ELISA.

Key findings

A low and high dose of PC failed to induce an inflammatory response, whereas DA led to an intense inflammatory response in a dose dependent manner. Combined PC/DA treatment resulted in a mild inflammatory response that was notably less severe than higher DA. Together, these results demonstrated that DA plays a role in inflammation caused by combined PC/DA. Histopathological examination and measurement of MPO activity indicated that DA was the primary cause of edema and PMN infiltration. Further, increased levels of cytokines (IL-1β and IL-6) and PGE2 demonstrated that DA might directly induce inflammation, whereas PC alone has no effect on inflammation.

Significance

These results indicate that DA rather than PC is responsible for inflammation, and that PC may not aggravate inflammatory responses induced by DA. Thus, the results of this study suggest that the adverse effects of PC/DA during localized fat treatment may be solely due to DA.     

P2X4 receptors mediate PGE2 release by tissue‐resident macrophages and initiate inflammatory pain

Prostaglandin E2 (PGE2) is a key mediator of inflammation and contributes to pain hypersensitivity by promoting sensory neurons hyperexcitability. PGE2 synthesis results from activation of a multi‐step enzymatic cascade that includes cyclooxygenases (COXs), the main targets of non‐steroidal anti‐inflammatory drugs (NSAIDs). Although NSAIDs are widely prescribed to reduce inflammatory symptoms such as swelling and pain, associated harmful side effects restrict their long‐term use. Therefore, finding new drugs that limit PG production represents an important therapeutic issue. In response to peripheral inflammatory challenges, mice lacking the ATP‐gated P2X4 channel (P2X4R) do not develop pain hypersensitivity and show a complete absence of inflammatory PGE2 in tissue exudates. In resting conditions, tissue‐resident macrophages constitutively express P2X4R. Stimulating P2X4R in macrophages triggers calcium influx and p38 MAPK phosphorylation, resulting in cytosolic PLA2 (cPLA2) activation and COX‐dependent release of PGE2. In naive animals, pain hypersensitivity was elicited by transfer into the paw of ATP‐primed macrophages from wild type, but not P2X4R‐deficient mice. Thus, P2X4Rs are specifically involved in inflammatory‐mediated PGE2 production and might therefore represent useful therapeutic targets.     

Prostaglandin production in human coronary artery endothelial cells is modulated differentially by selective phospholipase A(2) inhibitors

Atherosclerotic plaque formation is a dynamic process involving repeated injury and inflammation of the endothelium. We have demonstrated previously that thrombin and tryptase stimulation of human coronary artery endothelial cells (HCAEC) leads to increased phospholipase A(2) (PLA(2)) activity and generation of membrane phospholipid derived inflammatory metabolites, including eicosanoids and platelet activating factor. Thus, our hypothesis is that selective PLA(2) inhibitors have therapeutic potential as anti-inflammatory agents. Stimulation of confluent HCAEC monolayers with thrombin or tryptase resulted in a concentration and time-dependent increase in both prostaglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) production. Pretreatment with PX-18 to inhibit secretory PLA(2) or BEL to inhibit calcium-independent PLA(2) prior to thrombin or tryptase stimulation resulted in a significant inhibition of both PGI(2) and PGE(2) release. However, pretreatment with methyl arachidonyl fluorophosphonate (MAFP), a widely used inhibitor of cytosolic PLA(2) isoforms, resulted in a significant potentiation of both thrombin and tryptase stimulated PGI(2) and PGE(2) release as a consequence of increased free arachidonic acid production. We conclude that the use of selective PLA(2) inhibitors may be of therapeutic benefit in the development and progression of atherosclerosis, however, the development of such an agent requires rigorous screening.     

Calcium-independent phospholipase A(2) is required for human monocyte chemotaxis to monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A(2) (cPLA(2)) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA(2), calcium-independent PLA(2) (iPLA(2)) in comparison to cPLA(2). Pharmacological inhibitors of PLA(2) were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA(2) expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA(2) or cPLA(2) expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA(2)-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA(2)-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA(2)-deficient monocytes while having no effect on the iPLA(2)-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA(2) and cPLA(2) represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.     

Expression and function of phospholipase A(2) in brain

Phospholipase A(2) (PLA(2)) appears to play a fundamental role in cell injury in the central nervous system. We have investigated PLA(2) expression in the astrocytoma cell line 1231N1, and found that GIVA, GIVB, GIVC and GVI PLA(2) messages are expressed. PLA(2) activity is increased by inflammatory/injury stimuli such as interleukin-1beta and lipopolysaccharide in these cells but with very different time courses. The arachidonic acid liberated is converted to prostaglandin E(2), possibly by cyclooxygenase-2, which is induced by inflammatory stimuli. This cell system emerges as a model to study injury/inflammation-related activation of the new PLA(2) forms GIVB and GIVC.