Loss of Catalase-1 (Cat-1) results in decreased conidial viability enhanced by exposure to light in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Light is one of the most important factors inducing morphogenesis in Neurospora crassa. The reception of light triggers the generation of reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂). Catalase-1 (Cat-1) is one of three catalases known to detoxify H₂O₂ into water and oxygen. We reported that the photomorphogenetic characteristics of mutants in nucleoside diphosphate kinase-1 (NDK-1), a light signal transducer, are severely affected, and NDK-1 interacted with Cat-1 in a yeast two-hybrid assay. To disclose the function of Cat-1, we created a Cat-1 loss-of-function mutant (cat-1 ^RIP ) by the repeat induced point-mutation (RIPing) method. No Cat-1 activity was detected in the mutant strain. Forty guanines were replaced with adenines in the cat-1 gene of cat-1 ^RIP , which caused 30 amino acid substitutions. The mutant strain grew normally, but its conidia and mycelia were more sensitive to H₂O₂ than those of the wild type. The lack of Cat-1 activity also caused a significant reduction in the conidial germination rate. Furthermore, light enhanced this reduction in cat-1 ^RIP more than that in the wild type. Introduction of cat-1 into the mutant reversed all of these defective phenotypes. These results indicate that Cat-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Singlet oxygen is part of a hyperoxidant state generated during spore germination

We show that singlet oxygen is generated in asexual spores (conidia) from Neurospora crassa at the onset of germination. Oxidation of N. crassa catalase-1 (Cat-1) was previously shown to be caused by singlet oxygen (Lledías et al. J. Biol. Chem. 273, 1998). In germinating conidia, increased protein oxidation, decrease of total protein, Cat-1 oxidation and accumulation of cat-1 mRNA was detected. These changes were modulated in vivo by light intensity, an external clean source of singlet oxygen, and by carotene amount and content of coordinated double bonds. Conditions that stimulated singlet oxygen formation increased Cat-1 oxidation and accumulation of cat-1 mRNA. Germinating conidia from mutant strains altered in carotene synthesis showed increased levels of protein degradation, Cat-1 oxidation and accumulation of cat-1 mRNA. During germination Cat-1a was oxidized, oxidized Cat-1c-Cat-1e conformers disappeared and Cat-1a was synthesized de novo. Furthermore, spontaneous oxygen-dependent chemiluminescence increased as soon as conidia absorbed dissolved oxygen. Low-level chemiluminescence is due to photon emission from excited electrons in carbonyls and singlet oxygen as they return to their ground state. H2O2 added to conidia under Ar caused a peak of chemiluminescence and germination of 20% of conidia, suggesting that a hyperoxidant state suffices to start germination under anaerobic conditions. Taken together, these results show that singlet oxygen is part of a hyperoxidant state that develops at the start of germination of conidia, in consonance with our proposal that morphogenetic transitions occur as a response to a hyperoxidant state.     

Immunological detection of alkaline-diaminobenzidine-negativeperoxisomes of the nematode Caenorhabditis elegans purification and unique pH optima of peroxisomal catalase.

We purified catalase-2 of the nematode Caenorhabditis elegans and identified peroxisomes in this organism. The peroxisomes of C. elegans were not detectable by cytochemical staining using 3, 3'-diaminobenzidine, a commonly used method depending on the peroxidase activity of peroxisomal catalase at pH 9 in which genuine peroxidases are inactive. The cDNA sequences of C. elegans predict two catalases very similar to each other throughout the molecule, except for the short C-terminal sequence; catalase-2 (500 residues long) carries a peroxisomal targeting signal 1-like sequence (Ser-His-Ile), whereas catalase-1 does not. The catalase purified to near homogeneity from the homogenate of C. elegans cells consisted of a subunit of 57 kDa and was specifically recognized by anti-(catalase-2) serum but not by anti-(catalase-1) serum. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected catalase-2 inside vesicles judged to be peroxisomes using morphological criteria. The purified enzyme (220 kDa) was tetrameric, similar to many catalases from various sources, but exhibited unique pH optima for catalase (pH 6) and peroxidase (pH 4) activities; the latter value is unusually low and explains why the peroxidase activity was undetectable using the standard alkaline diaminobenzidine-staining method. These results indicate that catalase-2 is peroxisomal and verify that it can be used as a marker enzyme for C. elegans peroxisomes.     

Regulation and oxidation of two large monofunctional catalases

The two Neurospora crassa catalase genes cat-1 and cat-3 were shown to encode Cat-1 and Cat-3 large monofunctional catalases. cat-1 and cat-3 genes are regulated differentially during the asexual life cycle and under stress conditions. A stepwise increase in catalase activity occurs during conidiation. Conidia have 60 times more catalase activity than exponentially growing hyphae. Cat-1 activity was predominant in conidia, during germination and early exponential growth. It was induced during prestationary growth and by ethanol or heat shock. Cat-3 activity was predominant during late exponential growth and at the start of the conidiation process. It was induced under stress conditions, such as H(2)O(2), paraquat, cadmium, heat shock, uric acid, and nitrate treatment. In general, Cat-1 activity was associated with nongrowing cells and Cat-3 activity with growing cells. The Cat-3 N-terminus sequence indicates that this catalase is processed and presumably secreted. Paraquat caused modification and degradation of Cat-1. Under heat shock both Cat-1 and Cat-3 were modified and degraded and Cat-1 was resynthesized. Paraquat and heat shock effects were observed only in the presence of air and are probably related to in vivo generation of singlet oxygen. Purified Cat-3 was modified with a photosensitizing reaction in which singlet oxygen is produced.     

The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation

Cat-1/Git-1 is a multifunctional protein that acts as a GTPase-activating protein (GAP) for Arf GTPases, as well as serves as a scaffold for a number of different signaling proteins. Cat-1 is best known for its role in regulating cell shape and promoting cell migration. However, whether Cat-1 might also contribute to cellular transformation is currently unknown. Here we show that ～95% of cervical tumor samples examined overexpress Cat-1, suggesting that the up-regulation of Cat-1 expression is a frequent occurrence in this type of cancer. We demonstrate further that knocking down Cat-1 from NIH3T3 fibroblasts expressing an activated form of Cdc42 (Cdc42 F28L), or from the human cervical carcinoma (HeLa) cell line, inhibits the ability of these cells to form colonies in soft agar, an in vitro measure of tumorgenicity. The requirement for Cat-1 when assaying the anchorage-independent growth of transformed fibroblasts and HeLa cells is dependent on its ability to bind paxillin, while being negatively impacted by its Arf-GAP activity. Moreover, the co-expression of Cat-1 and an activated form of Arf6 in fibroblasts was sufficient to induce their transformation. These findings highlight novel roles for Cat-1 and its interactions with the Arf GTPases and paxillin in oncogenic transformation.     

Differential parameters between cytosolic 2‐Cys peroxiredoxins,PRDX1 and PRDX2

Peroxiredoxins are thiol‐dependent peroxidases that function in peroxide detoxification and H₂O₂ induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H₂O₂ and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H₂O₂ than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H₂O₂ with rate constants of ca 2 × 10³ M^?1 s^?1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s^?1 for PRDX1, 0.2 s^?1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.     

The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species‐scavenging enzyme that shows promiscuity in thiol redox control

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (‐SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H₂O₂ reduction, a sulfenic acid (‐SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S‐S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx‐MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H₂O₂ stress, and its gene expression is clearly induced upon H₂O₂ challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.     

Production of catalases byComamonas spp. and resistance to oxidative stress

Bacterial isolates Comamonas terrigena N3H (from soil contaminated with crude oil) and C. testosteroni (isolated from the sludge of a wastewater treatment plant), exhibit much higher total catalase activity than the same species from laboratory collection cultures. Electrophoretic resolution of catalases revealed only one corresponding band in cell-free extracts of both C. testosteroni cultures. Isolates of C. terrigena N3H exhibited catalase-1 and catalase-2 activity, whereas in the collection culture C. terrigena ATCC 8461 only catalase-1 was detected. The environmental isolates exhibited much higher resistance to exogenous H2O2 (20, 40 mmol/L) than collection cultures, mainly in the middle and late exponential growth phases. The stepwise H2O2-adapted culture of C. terrigena N3H, which was more resistant to oxidative stress than the original isolate, exhibited an increase of catalase and peroxidase activity represented by catalase-1. Pretreatment of cells with 0.5 mmol/L H2O2 followed by an application of the oxidative agent in toxic concentrations (up to 40 mmol/L) increased the rate of cell survival in the original isolate, but not in the H2O2-adapted variant. The protection of bacteria caused by such pretreatment corresponded with stimulation of catalase activity in pretreated culture.     

Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: a role for NF-κB-STAT3-directed gene expression

Mitochondrial DNA depleted (ρ⁰) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ⁺ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ⁰ cells compared to ρ⁺ HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ⁺ HSF, but this response was substantially decreased in ρ⁰ HSF. Suppression of the IKK–NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2–STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ⁺ HSF. Inhibitory antibodies against IL6, the main activator of JAK2–STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ⁰ HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6–JAK2–STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.     

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0–93 ⋅ 10^–6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10^–6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10^–6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1. Received: 9 May 1996 / Revision received: 24 February 1997 / Accepted: 8 March 1997     

Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum

Glucoamylase produced byScytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing 9.8% saccharide, pI of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C, either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed. The activity was activated by 1 mmol/L Mg²⁺ (27%), Zn²⁺ (21%), Ba²⁺ (8%) and Mn²⁺ (5%).K _m and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase activity was only slightly inhibited by glucose up to a 1 mol/L concentration.     

Increase of the expression and activity of ferredoxin-NADP<Superscript>+</Superscript> oxidoreductase in the cells adapted to low CO<Subscript>2</Subscript> in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> 6803

To investigate the effect of low CO₂ on the expression and activity of ferredoxin-NADP⁺ oxidoreductase (FNR) and this enzyme-mediated cyclic electron flow around photosystem I (cyclic PSI), the activity staining, immunoblotting and initial rate of P₇₀₀ ⁺ reduction were measured in high- or low-CO₂-grown (H or L)-cells of wild-type Synechocystis sp. strain PCC 6803 (WT) and its ΔndhB mutant (M55). Major results were depicted as follows. (1) The protein levels and activity of FNR were remarkably stimulated in L-cells of both WT and M55 relative to that in their H-cells. (2) The rate of cyclic PSI was significantly increased in L-cells of WT, not M55, when compared to that in respective H-cells. (3) N-ethylmaleimide, an inhibitor of FNR, partially inhibited the increase in the rate of cyclic PSI induced by low CO₂ in both WT and M55. These findings indicated that low CO₂ enhanced the expression and activity of FNR and the cyclic PSI mediated by FNR. The contribution of FNR to cyclic PSI is shortly discussed.     

Heterologous expression and characterization of endocellulase from the Japanese pine sawyer Monochamus saltuarius (MsGHF5)

In this study, the endocellulase gene from Monochamus saltuarius (MsGHF5) was transformed into Escherichia coli (RosettaBlue(DE3)pLysS strain), and induced by IPTG. The molecular weight of recombinant MsGHF5 (rMsGHF5) was 78 kDa and was expressed as a fusion protein with maltose binding protein in pMAL‐c2 expression vector. Native‐PAGE was conducted with 0.1% carboxymethyl cellulose as a substrate, and the zymogenic bands were observed. The Michaelis constant and maximum velocity of rMsGHF5 were 0.199 mg/mL and 0.034 μmol/min/mL, respectively. The optimal condition for rMsGHF5 occurred at pH 5 and 30°C. Fe²⁺ and Mn²⁺ stimulated the activity of rMsGHF5 by 167 and 114% respectively, whereas Cu²⁺, Hg²⁺ and Zn²⁺ inhibited its activity.     

Molecular and kinetic study of catalase-1, a durable large catalase of Neurospora crassa.

Catalase-1 (Cat-1), one of the two monofunctional catalases of Neurospora crassa, increases during asexual spore formation to constitute 0.6% of total protein in conidia. Cat-1 was purified 170-fold with a yield of 48% from conidiating cultures. Like most monofunctional catalases, Cat-1 is a homotetramer, resistant to inactivation by solvents, fully active over a pH range of 4-12, and inactivated by 3-amino-1,2,4-triazole. Unlike most monofunctional catalases, Cat-1 consists of 88 kDa monomers that are glycosylated with alpha-glucose and/or alpha-mannose, is unusually stable, and is not inactivated or inhibited by hydrogen peroxide. Cat-1 was more resistant than other catalases to heat inactivation and to high concentrations of salt and denaturants. Cat-1 exhibited unusual kinetics: at molar concentrations of hydrogen peroxide the apparent V was 10 times higher than at millimolar concentrations. Inactivation of Cat-1 activity with azide and hydroxylamine was according to first order kinetics, while cyanide at micromolar concentrations was a reversible competitive inhibitor.     

Hypaphorine,an Indole Alkaloid Isolated from Caragana korshinskii Kom., Inhibites 3T3‐L1 Adipocyte Differentiation and Improves Insulin Sensitivity in Vitro

Obesity, a major health problem worldwide, is a complex multifactorial chronic disease that increases the risk for insulin resistance, type 2 diabetes, coronary heart disease, and hypertension. In this study, we assessed methods to isolate hypaphorine, a potent drug candidate for obesity and insulin resistance. Semi‐preparative reversed‐phase liquid chromatography (semi‐preparative RPLC) was established as a method to separate three compounds, adenosine, l ‐tryptophan, and hypaphorine, from the crude extracts of Caragana korshinskii Kom . Due to its specific chemical structure, the effect of hypaphorine on differentiation and dexamethasone (DXM) induced insulin resistance of 3T3‐L1 cells was investigated. The structures of the three compounds were confirmed by UV, ¹H‐NMR, and ¹³C‐NMR analysis and compared with published data. The activity results indicated that hypaphorine prevented the differentiation of 3T3‐L1 preadipocytes into adipocytes by down‐regulating hormone‐stimulated protein expression of peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPα), and their downstream targets, sterol regulatory element binding protein 1 c (SREBP1c) and fatty acid synthase (FAS). Hypaphorine also alleviated DXM‐induced insulin resistance in differentiated 3T3‐L1 adipocytes via increasing the phosphorylation level of Akt2, a key protein in the insulin signaling pathway. Taken together, we suggest that the method can be applied to large‐scale extraction and large‐quantity preparation of hypaphorine for treatment of obesity and insulin resistance.     

Comparison of amylolytic properties of Lactobacillus amylovorus and of Lactobacillus amylophilus

Partially purified amylases produced by Lactobacillus amylovorus and L. amylophilus were compared and they differed in several properties. The maximum amylase activity of L. amylovorus was higher than that of L. amylophilus. As estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the molecular mass of the enzymes was 140 kDa for L. amylovorus amylase and 100 kDa for L. amylophilus amylase. Maximum enzymatic activities were obtained when the strains were grown in the presence of CaCO₃, on maltose with L. amylovorus and on sucrose with L. amylophilus. Optimal activities were obtained at pH values between 5.0 and 6.0 for both amylases. The L. amylovorus amylase was stable at a higher temperature (50°C) than the L. amylophilus amylase (40°C). Of six substrates examined, greatest activity was obtained by both enzymes on soluble starch. Neither enzymes hydrolysed pullulan or - and \-cyclodextrins. With the exception of Hg²⁺, which partially inhibited both enzymes, various metal ions, such as 1 mm Ca²⁺ and Ba²⁺, stimulated L. amylophilus amylase activity whereas they inhibited L. amylovorus amylase activity. Correspondence to: J. Morlon-Guyot, ORSTOM     

Identification and partial characterization of two cysteine proteases from castor bean leaves (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.) activated by wounding and methyl jasmonate stress

Jasmonates are signaling molecules that play key roles in wound response and regulate the biosynthesis of many defensive proteins, including proteases. In this study, we investigate the effects of wounding and methyl jasmonate (MJ) application on the protein expression pattern of Ricinus communis L. leaves and on proteolytic activity. Gelatin zymography demonstrated that both MJ and mechanical wounding induce alterations in the proteolytic pattern of castor bean leaves (R. communis L.). Expression of two cysteine proteases (38 and 29 kDa) was induced by the treatments employed; however, MJ induced a higher protease level than mechanical wounding during the stress period (24, 48, and 72 h). The increase in protease activity mirrors the decline in soluble protein content and rubisco degradation that may indicate initiation of senescence in castor plants. The 29 kDa protease has an acidic optimal pH; whereas the 38 kDa protease has a neutral optimum activity. Both proteases were almost completely inhibited by E-64 and cystatin. The significant induction of these proteins by MJ suggests a possible role of cysteine proteases in leaf senescence as well as their involvement in regulating both the wound response and MJ in castor bean plants.     

Secretory expression and characterization of a highly Ca-activated thermostable L2 lipase

Thermostable lipases are important biocatalysts, showing many interesting properties with industrial applications. Previously, a thermophilic Bacillus sp. strain L2 that produces a thermostable lipase was isolated. In this study, the gene encoding for mature thermostable L2 lipase was cloned into a Pichia pastoris expression vector. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter, the recombinant L2 lipase was secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. After optimization the maximum recombinant lipase activity achieved in shake flasks was 125 U/ml. The recombinant 44.5 kDa L2 lipase was purified 1.8-fold using affinity chromatography with 63.2% yield and a specific activity of 458.1 U/mg. Its activity was maximal at 70 °C and pH 8.0. Lipase activity increased 5-fold in the presence of Ca²⁺. L2 lipase showed a preference for medium to long chain triacylglycerols (C₁₀–C₁₆), corn oil, olive oil, soybean oil, and palm oil. Stabilization at high temperature and alkaline pH as well as its broad substrate specificity offer great potential for application in various industries that require high temperature operations.