The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site

The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-κB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were confirmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK–RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen- and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE.     

Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways

The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro. Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment.     

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.     

Evidence that enteroglucagon (II) is identical with the C-terminal sequence (residues 33-69) of glicentin.

Enteroglucagon (II) was isolated from extracts of pig ileum mucosa by repeated gel filtrations, and its immunochemical and chromatographic characteristics were compared with those of a synthetic peptide corresponding to the 33-69 sequence of pig glicentin, before and after digestion with trypsin or trypsin followed by carboxypeptidase B, by using five region-specific assays covering most of the glicentin sequence. Enteroglucagon (II) and the synthetic peptide behave identically under three different conditions of chromatography as determined with all five assays (including a highly specific radioreceptor assay), and gave rise to similar fragments after enzyme digestion. It was therefore concluded that enteroglucagon (II) and the 33-69 sequence of glicentin are most probably identical.     

Development of inductively coupled plasma-mass spectrometry-based protease assays

Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr∼Asp-Lys(biotin) and Lu-DTPA-βAla-βAla-βAla-βAla-Gly-Ser-Ala-Tyr∼Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for α-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2 pM could be readily detected, superior to the detection limit of an assay using the α-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.     

The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK

The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type potassium channel that passes an A-type current and is localized to dendrites and cell bodies in the hippocampus. The sequence of Kv4.2 contains several consensus sites for ERK phosphorylation. In the present studies, we tested the hypothesis that Kv4.2 is an ERK substrate. We determined that the Kv4.2 C-terminal cytoplasmic domain is an effective ERK2 substrate, and that it is phosphorylated at three sites: Thr(602), Thr(607), and Ser(616). We used this information to develop antibodies that recognize Kv4.2 phosphorylated by ERK2. One of our phospho-site-selective antibodies was generated using a triply phosphorylated peptide as the antigen. We determined that this antibody recognizes ERK-phosphorylated Kv4.2 in COS-7 cells transfected with Kv4.2 and native ERK-phosphorylated Kv4.2 in the rat hippocampus. These observations indicate that Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERK may regulate potassium-channel function by direct phosphorylation of the pore-forming alpha subunit.     

The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.     

A comparison of human immunodeficiency virus type-1 protease inhibition activities by the aqueous and methanol extracts of Chinese medicinal herbs

The aqueous and methanol extracts of thirty-one herbs traditionally used as anti-fever remedies in China were screened for their in vitro inhibition on human immunodeficiency virus type-1 protease (HIV-1 PR). The activity of recombinant HIV-1 protease was determined by sequence-specific cleavage at the Tyr-Pro bond of the fluorogenic substrate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg) or by HPLC anaylsis of the cleavage products after incubation of the enzyme with a synthetic peptide substrate (Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide). Among the herbal extracts examined, the aqueous extracts of Prunella vulgaris and Scutellaria baicalensis and the methanol extracts of Woodwardia unigemmata, Paeonica suffruticosa and Spatholobus suberectus elicited significant inhibition (>90%) at a concentration of 200 microg/ml.     

Glyoxalase II in Saccharomyces cerevisiae: in situ kinetics using the 5,5'-dithiobis(2-nitrobenzoic acid) assay.

The determination of glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) activity is usually accomplished by monitoring the decrease of absorbance at 240 nm due to the hydrolysis of S-d-lactoylglutathione. However, it was not possible, using this assay, to detect any enzyme activity in situ, in Saccharomyces cerevisiae permeabilized cells. Glyoxalase II activity was then determined by following the formation of GSH at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid). Using this method we characterized the kinetics of glyoxalase II in situ using S-d-lactoylglutathione as substrate and compared the results with those obtained for cell-free extracts. The specific activity was found to be (4.08 +/- 0.12) x 10(-2) micromol min-1 mg-1 in permeabilized cells and (3.90 +/- 0.04) x 10(-2) micromol min1 mg-1 in cell-free extracts. Kinetic parameters were Km 0.36 +/- 0.09 mM and V (7.65 +/- 0.59) x 10(-4) mM min-1 for permeabilized cells and Km 0.15 +/- 0.10 mM and V (7.23 +/- 1.04) x 10(-4) mM min-1 for cell-free extracts. d-Lactate concentration was also determined and increased in a linear way with permeabilized cell concentration. gamma-Glutamyl transferase (EC 2.3.2.2), which also accepts S-d-lactoylglutathione as substrate and hence could interfere with glyoxalase II assays, was found to be absent in Saccharomyces cerevisiae permeabilized cells.     

Homogeneous assays for cellular proteases employing the platinum(II)-coproporphyrin label and time-resolved phosphorescence

Phosphorescent platinum(II) coproporphyrin label (PtCP) is evaluated for the detection of cellular proteases by time-resolved fluorescence in homogeneous format. An octameric peptide containing the recognition motif for the caspase-3 enzyme was dual labeled with a new maleimide derivative of PtCP and with the dark quencher dabcyl. Following photophysical characterization, the quenched substrate was employed in cleavage assays for caspase-3 using Jurkat and HL60 cell lines treated with proapoptotic stimuli performed on a commercial plate reader. Dose-response and time course assays for the drug camptothecin were obtained for comparison with conventional fluorometric detection.     

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.     

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .     

Activation of the mitogen-activated protein kinase ERK1 during meiotic progression of mouse pachytene spermatocytes

Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OA-stimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G(2)/M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G(2)/M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.     

Plant glycerol-3-phosphate-1-acyltransferase (GPAT): structure selectivity studies

Squash glycerol-3-phosphate-1-acyltransferase has been crystallized and the structure of the enzyme determined, at 1.9-A resolution, using multiple isomorphous replacement of the wild type and a series of individual cysteine mutants. Competitive in vitro substrate selectivity assays have been established that differentiate between selective and non-selective forms of the enzyme. Particular care was taken to use near-physiological concentrations of both substrates. Clear substrate selectivity can be demonstrated with the natural substrate acyl-acyl carrier protein but not with the substrate analogue acyl-CoA. The use of site-directed mutagenesis, coupled to three-dimensional structural determinations, should provide a rational basis for elucidating structural components important in determining the substrate selectivity of this enzyme.     

Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580–598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570–581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570–595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4α-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.     

Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

A luminescent method to individually measure the chymotrypsin-like, trypsin-like, or caspase-like activities of the proteasome in cultured cells was developed. Each assay uses a specific luminogenic peptide substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. Luminescence is generated in a coupled-enzyme format in which proteasome cleavage of the peptide conjugated substrate generates aminoluciferin, which is a substrate for luciferase. The homogeneous method eliminates the need to prepare individual cell extracts as samples. Luminogenic proteasome substrates and buffer formulations enabled development of a single reagent addition method with adequate sensitivity for 96- and 384-well plate formats. Proteasome trypsin-like specificity was enhanced by incorporating a mixture of protease inhibitors that significantly reduce nonspecific serum and cellular backgrounds. The assays were used to determine EC₅₀ values for the specific proteasome inhibitors epoxomicin and bortezomib for each of the catalytic sites using a variety of cancer lines. These cell-based proteasome assays are direct, simple, and sensitive, making them ideal for high-throughput screening.     

A kinetic approach for the study of protein phosphatase-catalyzed regulation of protein kinase activity

The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.     

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.     

Aldehyde-oxidizing enzymes in an adult moth: in vitro study of aldehyde metabolism in Heliothis virescens

The conversion of pheromonal aldehydes to carboxylic acids in vitro in tissue extracts of Heliothis virescens is catalyzed by both aldehyde dehydrogenase and aldehyde oxidase enzymes. The aldehyde-oxidizing activity in antennae, heads, legs, and hemolymph from male and female moths was examined by radiochromatographic and spectroscopic assays. First, the enzymatic activity was measured in the presence or absence of added NAD+ using either (Z)-9-tetradecenal or (Z)-11-hexadecenal as tritiated substrate. Second, substrate specificity was determined spectroscopically by (i) indirect measurement of the AO-released hydrogen peroxide through the coupled AO-horseradish peroxidase reaction and by (ii) direct measurement of the ALDH-produced NADH. Both aldehyde-oxidizing activities were associated with soluble enzymes in the antennal extracts, and these enzymes degraded pheromone and nonpheromonal aldehydes. Both AO and ALDH activities were present in male and female tissues. AO activity was exhibited primarily in the antennal extracts and to a lesser degree in the leg extracts. Moreover, ALDH activity was distributed in the antenna, head, and leg extracts. A vinyl ketone analog of (Z)-11-hexadecenal preferentially inhibited the ALDH activity over the AO activity.     

Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts.

Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.