mTORC2/AKT/HSF1/HuR constitute a feed-forward loop regulating Rictor expression and tumor growth in glioblastoma.

Overexpression of Rictor has been demonstrated to result in increased mechanistic target of rapamycin C2 (mTORC2) nucleation and activity leading to tumor growth and increased invasive characteristics in glioblastoma multiforme (GBM). However, the mechanisms regulating Rictor expression in these tumors is not clearly understood. In this report, we demonstrate that Rictor is regulated at the level of mRNA translation via heat-shock transcription factor 1 (HSF1)-induced HuR activity. HuR is shown to directly bind the 3' untranslated region of the Rictor transcript and enhance translational efficiency. Moreover, we demonstrate that mTORC2/AKT signaling activates HSF1 resulting in a feed-forward cascade in which continued mTORC2 activity is able to drive Rictor expression. RNAi-mediated blockade of AKT, HSF1 or HuR is sufficient to downregulate Rictor and inhibit GBM growth and invasive characteristics in vitro and suppress xenograft growth in mice. Modulation of AKT or HSF1 activity via the ectopic expression of mutant alleles support the ability of AKT to activate HSF1 and demonstrate continued HSF1/HuR/Rictor signaling in the context of AKT knockdown. We further show that constitutive overexpression of HuR is able to maintain Rictor expression under conditions of AKT or HSF1 loss. The expression of these components is also examined in patient GBM samples and correlative associations between the relative expression of these factors support the presence of these signaling relationships in GBM. These data support a role for a feed-forward loop mechanism by which mTORC2 activity stimulates Rictor translational efficiency via an AKT/HSF1/HuR signaling cascade resulting in enhanced mTORC2 activity in these tumors.

Expression of antisense hsp70 is a major determining factor in heat-induced cell death of P-19 carcinoma cells.

Overexpressed heat shock protein 70 (Hsp70) is known to be associated with thermoprotection in a number of cell lines and transgenic animals. We hypothesized that because overexpression of Hsp70 protects cells from lethal heat stress, inhibition of expression should make cells susceptible to heat stress. The model used for this study was a stably transfected P-19 carcinoma cell line expressing antisense hsp70 under the control of the hsp70b promoter. The results showed marked inhibition of Hsp70 expression after heat shock correlated with heat-induced cell death. Hsp90 and Hsc70 protein expression were not affected by the antisense construct. Unexpectedly, heme oxygenase (HO-1), another highly inducible heat shock protein, was not induced after heat shock in the antisense hsp70 cell line. Heat shock transcription factor-1 (HSF-1) was in a highly phosphorylated state in the antisense cell line before and after heat shock. This was in contrast to the untransfected control P-19 cells where HSF-1 was primarily highly phosphorylated after heat shock. A control cell line expressing only the vector, pMAMneo, without the antisense construct also showed partial loss of Hsp70 induction but not increased cell death after heat shock. The findings support the role of Hsp70 in thermoresistance.

Induction of cell death by L-alpha-aminoadipic acid exposure in cultured rat astrocytes: relationship to protein synthesis.

The excitotoxin, L-alpha-aminoadipic acid (L-AAA), kills primary astrocytes in the brain. The mechanism underlying the induction of cell death is not well understood although many possible mechanisms are theorized. Previous studies have reported that astrocytes die after prolonged exposure to L-AAA suggesting a delayed programmed cell death and apoptosis. In this study rat cortical astrocytes exposed to continuous 1 mM L-AAA exposure for 24-, 48-, or 72 hours demonstrated increased DNA laddering, a characteristic of apoptosis. Unexpectedly, this was not ameliorated by the presence of cycloheximide at 0.1 microg/ml medium. Because of our interest in cytoprotective heat shock proteins induced by excitoxic stress, we studied the effect of prolonged exposure of L-AAA on the synthesis of stress proteins and protein synthesis in rat cortical astrocytes. Protein synthesis as measured by [35S]-methionine labeling showed a marked and significant decrease in incorporation of radiolabel after 24 hours of exposure to L-AAA and prior to induction of significant cell death noted at 48- and 72 hours of L-AAA exposure. The inhibition of protein synthesis was partially reversible at 24 hours if cells were labeled in medium without L-AAA during the radiolabeling period. Heat shock or stress proteins, HSP70 and heme oxygenase-1 (HO-1), were analyzed after a 24 hour exposure to L-AAA and showed no significant induction of HSP70 or HO-1. The findings suggest that the prolonged inhibition of protein synthesis and associated lack of induction of HSP70 and HO-1 synthesis contributed to apoptotic cell death induced by the excitoxin L-AAA.

Tin-mesoporphyrin, a potent heme oxygenase inhibitor, for treatment of intracerebral hemorrhage: in vivo and in vitro studies.

Spontaneous intracerebral hemorrhage (ICH) is the stroke subtype with highest mortality and morbidity. ICH can also occur following traumatic brain injury and thrombolysis for ischemic stroke and myocardial infarction. Development of ICH-induced hemispheric edema can elevate intracranial pressure and cause death. In survivors, edema-related white matter injury can lead to life-long neurological deficits. At present, there are no scientifically proven treatments for ICH. Heme oxygenase products, particularly iron and bilirubin, can be toxic to cells. In cerebral ischemia models, metalloporphyrins that are potent heme oxygenase inhibitors, reduce edema and infarct size. Tin-mesoporphyrin (SnMP) is a neuroprotectant that has also been used clinically to treat hyperbilirubinemia. Presently, we tested the hypothesis that SnMP treatment would reduce edema development following experimental ICH. We produced hematomas in pentobarbital-anesthetized pigs (9-11 kg) by infusing autologous blood into the frontal white matter. To maximize tissue concentrations, SnMP (87.5 microM in DMSO) or DMSO (vehicle controls) was included in the infused blood. Pig brains were frozen in situ at 24 hrs. following ICH and hematoma and edema volumes were determined on coronal sections by computer-assisted image analysis. We also examined the effects of SnMP in vitro on ferritin iron release, the formation of iron-induced thiobarbituric acid reactive substances (TBARS) and initial clot formation and hemolysis. SnMP treatment significantly reduced intracerebral mass following ICH. This was due to significant decreases in hematoma (0.68+/-0.08 vs. 1.39+/-0.30 cc, vehicle controls p<0.025) and edema volumes (edema = 1. 16+/-0.33 vs. 1.77+/-0.31 cc, p<0.05). In vitro, SnMP did not stabilize ferritin iron against reductive release nor did it decrease iron-induced TBARS formation in brain homogenates. SnMP or DMSO added to pig blood did not alter clot weights. In conclusion, SnMP reduced intracerebral mass in an ICH model by decreasing both hematoma and edema volumes SnMP's mechanism of action is presently unknown but may involve its potent inhibition of heme oxygenase activity. SnMP's effect appears unrelated to ferritin iron release, antioxidant activity or initial clot formation. Since SnMP treatment could be brain protective following ICH, further investigations into neurological and neuropathological outcomes and as well as into its mechanism of action are warranted.

The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that "senses" extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT-PCR using four CaR-specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR-specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti-CaR antiserum. In addition, raising Ca2+o from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a "calcimimetic" CaR activator (R-467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca(+)-activated K+ channel having a unitary conductance of 84+/-4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.

Protective properties of tin- and manganese-centered porphyrins against hydrogen peroxide-mediated injury in rat astroglial cells.

Tin-mesoporphyrin (tin-mp), a potent inhibitor of heme oxygenase, and manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a potent superoxide dismutase mimetic, reduced H2O2 toxicity in cultures of transformed rat astroglial cells if added 30 min before, or at the same time as, H2O2. Reduced toxicity was not observed if treatment was delayed for 60 min, the time by which H2O2 was essentially eliminated from cultures. Coadministration of tin-mp and MnTMPyP did not increase protection over either compound administered individually. Tin-mp, but not MnTMPyP, was stable in culture. MnCl2 was not protective, suggesting that protection by MnTMPyP was not dependent on manganous ion, a by-product of MnTMPyP breakdown. Protection by tin-mp and MnTMPyP was not associated with metalloporphyrin-mediated induction of heme oxygenase-1 or with changes in heme oxygenase-2 on western blots. Whereas protective concentrations of tin-mp did not have superoxide dismutase-mimetic properties in vitro, protective concentrations of MnTMPyP partially inhibited heme oxygenase. The data support the hypothesis that heme oxygenase inhibition is protective against acute oxidative injury.

Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia.

BACKGROUND AND PURPOSE: The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. METHODS: Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. RESULTS: The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. CONCLUSIONS: Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.

Heme oxygenase in the experimental ALS mouse.

Heme oxygenase-1 (HO-1) is a stress protein inducible in some cells by oxidative stress. The status of heme oxygenase was investigated in a transgenic mouse model of amyotrophic lateral sclerosis (ALS) since oxidative mechanisms are postulated in neuronal injury. Three ALS mice [(SOD1-G93A)1Gur] and three controls [(SOD-1)2Gur] were obtained from The Jackson Laboratory. Behavioral differences suggestive of neurodegeneration in ALS mice developed at 4-5 months of age. All mice were killed at 7-8 months of age. Tissue vacuolation, cell loss, and the presence of GFAP+ cells were noted in the spinal cords of ALS mice. Spinal cord motor neurons in both control and ALS mice stained positive for heme oxygenase-2 (HO-2). While not precluding the presence of low levels of HO-1 neither immunohistochemical staining nor Western blot analysis provided evidence for significant HO-1 induction in degenerating spinal cord.

Transient induction of heme oxygenase after cortical stab wound injury.

Heme oxygenase (HO) exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-1 has been identified as a heat shock or stress protein and is inducible whereas HO-2 is largely refractory to induction. HO-2 is the predominant isoenzyme in normal brain and appears to have a predominantly neuronal distribution in cerebral cortex. Cortical stab wound injury resulted in HO-1 induction as determined by Western blot analysis. Immunohistochemical analysis suggested that induced HO-1 was largely restricted to reactive astrocytes and macrophage-like cells. Enhanced HO-1 immunoreactivity was observed in hypertrophied, GFAP+ reactive astrocytes near the wound margin as early as 12 h after injury. Very rarely were HO-1+ neurons observed and then only up to 6 h after stabbing. Maximal numbers of HO-1+ astrocytes were found 3 days after stabbing. Their numbers declined thereafter. By 5 days after stab injury few HO-1+ reactive astrocytes were observed although GFAP+ reactive astrocytes were still prominent near the wound margin. HO-1+ macrophage-like cells were initially observed between 1 and 3 days after injury and they persisted in the margin of the wound for at least 14 days. The proximity of HO-1+ cells to the wound margin suggests that factors associated with injury contribute to the regulation of HO-1 in injured cortex.

Evidence for different mechanisms of induction of HSP70i: a comparison of cultured rat cortical neurons with astrocytes.

This study is a follow-up of previous work which demonstrated that cultured cortical neurons did not synthesize HSP70i immediately after heat stress when compared with cultured cortical astrocytes. We have extended the period of observation for HSP70i induction of cultured cortical neurons and astrocytes up to 24 h after heat stress. Cultured rat cortical neurons derived from 16-day-old fetal rats respond differently to heat stress than cultured rat astrocytes derived from newborn rats. They showed a delayed HSP70i induction in the majority of cultured neurons and the response was heterogeneous and was absent in most smaller neurons. The delayed neuronal induction was accompanied by a prolonged activation of heat-shock transcription factor 1 (HSF-1) and prolonged transcription of HSP70i mRNA. In comparison astrocytes showed a marked early induction of HSP70i mRNA and protein. In addition the induction of HSP70i in astrocytes was followed by translocation of the protein into the nucleus, a finding which we failed to demonstrate in neurons. Immunostaining for HSP70i was more uniform in astrocytes than neurons. Many neurons did not stain for up to 24 h after heat shock in this study. Immunocytochemical staining of HSF-1 and 2 showed major differences between neurons and astrocytes. Astrocytes showed localization of HSF-1 to the nucleus before and after heat stress, while neurons showed HSF-1 localization to the cytoplasm and nucleus before and after heat stress. Finally HSF-2 was undetectable in neurons when compared with astrocytes by Western immunoblot analysis. However, astrocytes and neurons revealed weak immunostaining of HSF-2 in the cytoplasm and nucleus. The staining in the neurons was likely secondary to cross-reactivity to an unidentified protein. We conclude that HSP70i expression after heat shock is delayed in rat cortical neurons when compared with rat cortical astrocytes. In addition most small neurons did not synthesize HSP70i after heat shock. This difference in induction of HSP70i may be secondary to localization and activation of HSF-1 but not HSF-2. Neuronal susceptibility to injury may be related to the delayed induction of HSP70i and also the possible failure of newly synthesized HSP70i to translocate into the nucleus.

Localization of heme oxygenase in rat retina: effect of light adaptation.

Heme oxygenase-2 isozyme is the predominant form of heme oxygenase in rat brain by western blot analysis. Heme oxygenase-1 isozyme is not induced by light adaptation in rat retina by western blot analysis. Immunocytochemistry localizes heme oxygenase-2 in three areas of the retina: the retinal pigment epithelium, inner segment and external nuclear layers of the rat retina. Ganglion cells and cell bodies of the internal nuclear layer of the retina and Müller cells were largely unstained for heme oxygenase-2. The localization of heme oxygenase-2 in the retina implies that its function is not associated with phototransduction. Also, light adaptation does not appear to induce heme oxygenase-1, a measure of oxidative injury.

Neuroprotection against CA1 injury with metalloporphyrins.

The hippocampal slice was used to examine neuroprotection with metalloporphyrins, a class of drug which inhibits heme oxygenase and which has been found to be effective in the treatment of neonatal hyperbilirubinemia. Tin-protoporphyrin given during hypoxia significantly improved recovery of CA1 antidromic PS to a mean of 82 +/- 2% of initial amplitude, while unmedicated slices regained only 6 +/- 3% of initial amplitude. Tin-protoporphyrin also protected against fluid percussion injury with an EC50 of 10 microM when given after trauma. This protection extended to induction of long-term potentiation. Tin-mesoporphyrin and zinc-protoporphyrin protected against trauma with EC50's of 4 and 32 microM. Treatment with Sn-PP also protected against exposure to hydrogen peroxide, but not NMDA, AMPA, glycine or nitric oxide. These findings indicate that metalloporphyrins protect against CA1 neuronal injury through direct neural effects.

Pharmacological induction of heat shock protein 68 synthesis in cultured rat astrocytes.

The induction of the highly inducible 70-kDa heat shock protein (HSP 70) is associated with thermotolerance and survival from many other types of stress. This investigation studied the pharmacological induction of HSP 68 (HSP 68 is the rat homolog of human HSP 70) by 1,10-phenanthroline in cultured rat astrocytes under conditions that activated heat shock transcription factor-1 without inducing HSP 68 synthesis. Two conditions that activate heat shock transcription factor-1 and promote its binding to the heat shock element without subsequent transcription of HSP 68 mRNA, intracellular acidosis and exposure to salicylate, showed synthesis of HSP 68 when 1,10-phenanthroline was added to culture medium after the activation of heat shock transcription factor-1. 1,10-phenanthroline mimicked heat shock by inducing HSP 68 mRNA and protein under both conditions. 1,10-phenanthroline added alone to culture medium did not induce the synthesis of HSP 68 or activate heat shock transcription factor-1. These findings strongly suggest a multistep activation for HSP 68 synthesis and also demonstrate that the synthesis of HSP 68 can be pharmacologically regulated.

Differential localization of heme oxygenase and NADPH-diaphorase in spinal cord neurons.

Western blot analysis using several antibodies showed that rat spinal cord contained abundant immunostainable heme oxygenase-2 (HO-2) and barely detectable levels of heme oxygenase-1 (HO-1). Anti-HO-2 antibody stained large anterior horn motoneurones and numerous smaller neurons throughout spinal cord gray matter including the dorsal root entry zone. HO-2+ astrocytes were not evident in gray matter although their presence cannot be ruled out. The distribution of HO-2+ neurons was compared with the distribution of cells containing NADPH-diaphorase (NADPH-d) activity, a marker for nitric oxide synthase. NADPH-d activity was restricted to far fewer neurons, many of which were close to the central canal and dorsal root entry zone.

Differential expression of heme oxygenase-1 in cultured cortical neurons and astrocytes determined by the aid of a new heme oxygenase antibody. Response to oxidative stress.

Heme oxygenase exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-2 is made constitutively in many cell types whereas HO-1 is a stress protein inducible by heat, heavy metals, ultraviolet irradiation, and oxidative stress. Recombinant rat HO-1 was expressed in bacteria and antiserum designated HO-1713 was raised against the purified protein. HO-1713 detected recombinant rat HO-1 and recombinant rat HO-2. In rat tissues it detected HO-1 and a second, unidentified band designated HO-L (heme oxygenase-like immunoreactivity) which was not HO-2. Cultured rat cortical neurons and forebrain astrocytes were exposed to hydrogen peroxide (0.14-0.7 micromolar for 30 or 60 min). Neurons which contained little detectable HO-1 and which were sensitive to hydrogen peroxide at the high end of the dose curve failed to induce HO-1 by Western blot analysis. In contrast, cultured rat forebrain astrocytes which contained HO-1 under normal culture conditions and which were resistant to injury by hydrogen peroxide, increased their content of immunoreactive HO-1 by 7-fold within 3 h after exposure. Our results support a protective role for HO-1 in oxidative injury and suggest that the relative inability of neurons to increase HO-1 after oxidative stress may contribute to their selective vulnerability vis-a-vis astrocytes. They also suggest that differential expression of heme oxygenase in studies utilizing CNS cultures may alter normal cell physiology and cell survival.

Heat shock proteins in hypoxic-ischemic brain injury: a perspective.

There is much to suggest that the induction of heat shock protein synthesis is an important response to injury and stress in the brain. The role of heat shock proteins in neurological disease has been approached from two points-of-view. First, the induction and synthesis of specific proteins after brain cell injury provide a window through which insight on the regulation of gene expression in pathological tissue can be obtained. These studies have broad implications for understanding pathophysiological mechanisms of disease. Second, putative cell protective effects of heat shock proteins in brain tissue provide insight into biochemical mechanisms of selective neuronal vulnerability. These studies have extremely important clinical implications since cell sensitivity to injury can seemingly be modified. The role of heat shock proteins in hypoxic-ischemic brain injury is discussed forthwith.

Heme oxygenase is a heat shock protein and PEST protein in rat astroglial cells.

Cultured rat forebrain astrocytes contained significant amounts of immunostainable heme oxygenase-1 (HO-1) isozyme, whereas HO-1 was undetectable in spontaneously transformed rat astroglial cells (ATs). HO-1 was inducible in both cell types by heat shock and by submicromolar amounts of H2O2. Inhibition of RNA synthesis with actinomycin D or protein synthesis with cycloheximide resulted in the rapid loss of immunostainable heme oxygenase in astrocytes. Analysis of the primary structure of heme oxygenase suggests that it is a PEST protein, i.e., targeted for rapid turnover.

Characterization of the major 68 kDa heat shock protein in a rat transformed astroglial cell line.

The heat shock response in a transformed astrocyte line was compared with nontransformed astrocytes. The synthesis of HSP 68, the major inducible heat shock protein (HSP 68) was induced by a non-lethal 45 degrees C, 10 min heat shock. Although the incorporation of [35S]methionine into HSP 68 suggested that similar amounts of protein were being synthesized after heat shock, Western immunoblotting demonstrated striking differences in the HSP immunostaining between the two cell types. By one- and 'two-dimensional gel electrophoresis the major 68 kDa heat shock protein (HSP 68) was similar in both cell types. However, HSP 68 from heat shocked, transformed astrocytes did not immunostain with the monoclonal antibody, C-92, which is specific for the major inducible heat shock protein of HeLa cells. In contrast HSP 68 from heat shocked, nontransformed astrocytes immunostained quite well. A polyclonal antibody raised against the inducible 72 kDa heat shock protein of HeLa cells immunostained the HSP 68 from both astrocytes and transformed astrocytes. Analysis of the mRNA from the two cell types after heat shock revealed two bands of approximately 2.5 and 2.8 kb in astrocytes but only a single 2.5 kb band in the heat shocked transformed astroglia. These results suggest that structural differences in the HSP 68 may be present in the transformed astrocytes compared to the normal astrocytes.

Heat shock in cultured neurons and astrocytes: correlation of ultrastructure and heat shock protein synthesis.

Cultured cerebral cortical neurons and astrocytes were compared after a brief shock. Morphological findings were correlated with the synthesis of the 68 kD heat shock protein (HSP68). Heat shocked neurons demonstrated many severe morphological changes after exposure to temperatures of 43 degrees C for 15 min and 45 degrees C for 10 min. Nuclear membrane 'blebbing' with lysis of the membrane, chromatin clumping, and disappearance of the nucleolus were prominent after both conditions. Lysis of the cell membrane was noted in severely injured neurons; this was more prominent at the higher temperature. In addition, alterations to polyribosomes, Golgi apparatus, rough endoplasmic reticulum and mitochondria were noted in the cytoplasm of neurons after heat shock. In contrast, no significant changes were noted in either the nucleus or cytoplasm of heat shocked astrocytes. The severity of morphological changes in neurons directly correlated with the low level of induction of HSP68 in neurons. Neurons synthesized much less 68 kD heat shock protein than similarly heat shocked astrocytes. We conclude that cultured cerebral cortical neurons are more susceptible to injury after heat shock than heat resistant astrocytes and that one possible mechanism of injury is failure to synthesize adequate amounts of HSP68 after injury.

Regulation of heat shock protein synthesis in rat astrocytes.

Rat forebrain astrocytes synthesize heat shock proteins with molecular weights 97, 89, 70, 68, and 30-34 kilodaltons. The stress inducible 68-kDa heat shock protein (HSP-68) was vigorously expressed by astrocytes in culture after a 45 degrees C, 20 min heat shock. HSP-68 synthesis was poorly inducible by a second heat shock given 16 hr after the initial heat shock. Decreased [35S]methionine incorporation into HSP-68 correlated with low levels of HSP-68 mRNA present after the second heat shock. The data suggest that control of HSP-68 mRNA levels by transcriptional/posttranscriptional mechanisms is a major site for regulation of HSP-68 synthesis.