Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potentially useful anticancer agent with exquisite selectivity for cancer cells. Unfortunately, many cancers exhibit or acquire resistance to TRAIL. We report herein that TRAIL activates a TGF-ß-activated kinase 1→mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)/MKK6→p38 pathway in prostate cancer cells that transcriptionally upregulates expression of the antiapoptotic BCL-2 family member MCL-1. TRAIL alone triggered robust formation of the "death-inducing signaling complex", activation of the initiator caspase-8, and truncation of the BH3-only protein BID (tBID). Nevertheless, simultaneous disruption of the p38 MAPK pathway was required to suppress MCL-1 expression, thereby allowing tBID to activate the proapoptotic BCL-2 family member BAK and stimulate mitochondrial outer membrane permeabilization (MOMP). Release of the inhibitor-of-apoptosis antagonist, Smac/DIABLO, from the intermembrane space was sufficient to promote TRAIL-induced apoptosis, whereas release of cytochrome c and apoptosome function were dispensable. Even following MOMP, however, mitochondrial-generated reactive oxygen species activated a secondary signaling pathway, involving c-Jun N-terminal kinases, that likewise upregulated MCL-1 expression and partially rescued cells from death. Thus, stress kinases activated at distinct steps in the extrinsic pathway mediate TRAIL resistance through maintenance of MCL-1 expression. / text

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Regulation of the inflammatory response is essential for the successful resolution of inflammation, and restoration of normal tissue homeostasis. Eosinophils are granulocytic cells of the innate immune system historically considered to be primarily involved in the defence against parasitic infection. Eosinophils are also key effector cells in the allergic inflammatory response, initiation of which is associated with the recruitment and activation of eosinophils culminating in the release of their intracellular granule contents. Eosinophil granules contain a range of cytotoxic proteins (major basic protein, eosinophil cationic protein and eosinophil peroxidase) that act to destroy infectious and parasitic organisms. However, these cytotoxic proteins can also cause damage to surrounding host tissue cells. The resolution of the inflammatory response acts to limit the extent of eosinophil-mediated tissue damage. Programmed cell death (apoptosis) of eosinophils represents an important component of this resolution process, limiting release of granule contents and triggering efferocytosis (the removal of apoptotic cells by phagocytes). Apoptosis is initiated by the activation of intracellular caspases, a family of cysteine proteases. Caspase activation primarily occurs as a result of changes in the balance of intracellular pro- and anti-apoptotic Bcl-2 family proteins. Mcl-1, an anti-apoptotic Bcl-2 protein has been shown to play a pivotal role in the regulation of neutrophil apoptosis. Pharmacological down-regulation of Mcl-1 initiates apoptosis and promotes the resolution of neutrophil-dominant inflammation. The importance of Mcl-1 in the regulation of apoptosis was shown using cyclin-dependent kinase inhibitors (CDKis), where induction of neutrophil apoptosis by CDKis was due to down-regulation of intracellular Mcl-1. Apoptotic cells display distinct surface molecules known as ‘eat-me’ signals that identify them for phagocytosis by macrophages and other phagocytes. One key receptor involved in the removal of apoptotic cells from tissue is the receptor tyrosine kinase Mer, a member of the Tyro3/Axl/Mer (TAM) family, which recognises the ‘eat me’ signal phosphatidylserine expressed on apoptotic cells. In the absence of Mer expression, clearance of apoptotic cells is compromised delaying the resolution of neutrophil-dominant inflammation. However, the roles of Mcl-1 and Mer in eosinophil apoptosis and clearance, respectively, and the resolution of allergic inflammation are not known. Asthma is a chronic inflammatory lung disease characterised by shortness of breath, airway obstruction, wheeze, non-specific bronchial hyper-responsiveness, excessive airway mucus production and an eosinophil dominant inflammatory infiltrate. The persistent presence of eosinophils in the lung, in chronic asthma, is likely due to a combination of excessive eosinophil recruitment and activation together with impaired eosinophil apoptosis. Investigation into the underlying mechanisms of these processes in allergic airway disease is of critical importance, as blocking eosinophil recruitment and/or promoting eosinophil apoptosis could provide a therapeutic approach to reduce associated eosinophil-mediated tissue damage. Understanding the regulation of eosinophil apoptosis and phagocytic clearance may identify novel pharmacological targets to enhance the resolution of allergic inflammation. We hypothesise that Mcl-1 and Mer play vital roles in the successful resolution of allergic airway inflammation. To investigate this hypothesis, we have used pharmacological and genetic manipulation of intracellular eosinophil Mcl-1 levels, and phagocyte Mer expression, to determine the role they play in the regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils, respectively. Human and mouse eosinophils were cultured, and rates of constitutive and CDKi-induced apoptosis were determined, to investigate eosinophil apoptosis in vitro. Mice expressing human Mcl-1 (hMcl-1) were used to determine the effect of over-expression of Mcl-1 on eosinophil viability in vitro. The effect of hMcl-1 on eosinophil viability and disease severity in vivo was determined using an ovalbumin-induced model of allergic airway inflammation, which mimicked the symptoms of human asthma. Apoptotic eosinophils were co-incubated with macrophages in vitro to investigate the capacity for phagocytosis by different macrophage populations. Apoptotic cell clearance was further investigated using a Mer-kinase-dead mouse, which lacked Mer expression, to determine the role of Mer-dependent phagocytosis on the process of resolution of inflammation in vivo. Over-expression of Mcl-1 in eosinophils significantly delayed both constitutive and CDKi-induced apoptosis in vitro. In vivo in the ovalbumin-induced model of allergic airway inflammation, over-expression of Mcl-1 resulted in a significantly increased number of eosinophils in the lung and delayed rate of resolution of allergic airway inflammation. Alveolar and bone marrow-derived macrophages exhibited Mer-dependent phagocytosis of eosinophils, which was significantly reduced by an inhibitor of Mer kinase activity (BMS777607) or lack of macrophage Mer expression. The absence of Mer expression resulted in a significant increase in the number of apoptotic eosinophils in the lung together with a delayed rate of resolution of allergic airway inflammation in vivo. Together this work has shown that delayed rates of eosinophil apoptosis and impaired phagocytic clearance both delayed the resolution of allergic airway inflammation. These data suggest that both Mcl-1 and Mer are pivotal for the successful regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils in asthma and may provide attractive novel therapeutic targets.

Apoptosis is an inherent suicide mechanism that cells invoke for a variety of reasons including embryo cavitation, tissue homeostasis, excessive DNA damage and aberrant oncogene activation. Apoptosis is regulated by a diverse set of proteins including, but not limited to, the Bcl-2 family. This family set is comprised of both pro-death and pro-survival proteins whose relative expression, localization and/or modifications regulate the balance between life and death for each cell. The keystones to this system are the proapoptotic proteins Bax and Bak, which are regulated by their conformation and localization. However, the exact mechanisms by which Bax and Bak become activated remains to be resolved. Similarly, research focusing on the cancer cell's ability to deregulate apoptosis by preventing the activation of Bax or Bak will provide further insight into the development of targeted therapies for cancer that will hopefully contribute to the cure of this formidable disease. Src, the classic oncogenic kinase, has been found to deregulate Bax activation in response to the detachment of a cell from its substratum support thereby preventing anoikis, the Bax-dependent apoptotic response involved in the impairment of metastatic dissemination of cancer. Our findings indicate that Src deregulates this response by altering the relative expression of Bcl-2 family members Mcl-1 and Bim through the PI3-K/Akt and Erk1/2 pathways. However, Src retains its ability to prevent anoikis even in the setting of Akt and Erk1/2 signaling inhibition. Further evaluation of the role of Src in this process revealed that Bif-1, a protein known to associate with and activate Bax, could be directly phosphorylated by Src which prevented the association of Bax with Bif-1 and impaired the anoikis response. In addition, our studies have also found that Bax activation in response to treatment with type I and II histone deacetylase inhibitors is dependent on the expression of the tumor suppressor p53. Acetylation of p53 at carboxy-terminal lysine residues enhances its transcriptional activity associated with cell cycle arrest and apoptosis. Here, we demonstrate that p53 acetylation at K320/K373/K382 is also required for its transcription-independent functions in Bax activation, ROS production, and apoptosis in response to the histone deacetylase inhibitors (HDACi) SAHA and LAQ824. Knockout of p53 in HCT116 cells markedly reduced HDACi-induced apoptosis. Unexpectedly, expression of transactivation-deficient p53 variants sensitized p53-null cancer cells to HDACi-mediated Bax-dependent apoptosis, whereas knockdown of endogenous mutant p53 inhibited HDACi-induced apoptosis. Evaluation of the mechanisms controlling this response led to the discovery of a novel interaction between p53 and Ku70. The association between these two proteins was acetylation independent, but acetylation of p53 could prevent and disrupt the Ku70/Bax complex and enhance apoptosis. These results suggest a new mechanism of acetylated p53 transcription-independent regulation of apoptosis.

Colorectal cancer is the third leading cause of cancer-related mortality. Much progress has been achieved in combating this disease with surgical resection and chemotherapy in combination with targeted drugs. However, most metastatic patients develop drug resistance so new modalities of treatment are needed. Notch signaling plays a vital role in intestinal homeostasis, self-renewal, and cell fate decisions during post-development and is activated in colorectal adenocarcinomas. Under debate is its role in carcinomas and metastatic disease. In theory, blocking Notch activation using gamma-secretase inhibitors (GSIs) may show efficacy alone or in combination with chemotherapy in the treatment of colon cancer. In Chapter Three, we tested the capacity for GSIs to synergize with oxaliplatin in colon cancer cell lines and evaluated the underlying molecular mechanisms. GSI alone had no effect on colon cancer cell lines. Surprisingly, we show that GSIs blocked oxaliplatin-induced apoptosis through increased protein levels of the anti-apoptotic Bcl-2 proteins Mcl-1 and/or Bcl-xL. Restoration of apoptosis was achieved by blocking Mcl-1 and/or Bcl-xL with obatoclax (an anti-apoptotic Bcl-2 agonist) or siRNA. An unexpected result was the induction of cell death with the combination of GSI and obatoclax. In Chapter Four, we examined the mechanism of GSI + obatoclax-mediated cell death. We found that apoptosis played a minimal role. Rather, we identified blockage of cytoprotective autophagy played a causative role. Interestingly, we also saw autophagy induction in GSI-treated cells, which could explain the insensitivity of colon cancer cells to GSI. When autophagy was blocked in GSI-treated cells, cells became sensitive to GSI and cell death was elicited. The mechanism by which induction of autophagy occurs in GSI- treated cells is an area for further research. Overall, our work questions the validity of the use of GSIs in the treatment of colorectal cancers. We show that GSIs may block apoptosis and induce cytoprotective autophagy simultaneously, resulting in increased drug resistance and cellular survival. Whether these two cellular survival processes occurs in patients needs to be examined before GSIs can be utilized in a clinical setting. If so, these two hurdles must be overcome.

Dichloroacetate (DCA) it a metabolic reprogramming agent that is used to target the unique metabolism of cancer cells, but is not always effective in colorectal cancer cells. In HCT116 cells, DCA was unable to induce apoptosis, but did decrease proliferation when compared to untreated cells. A decrease in full length Mcl-1 protein expression 7 hours following DCA treatment did not correspond with changes in mRNA production or changes in expression of inhibitory binding partners, but may be due to altered proteasomal degradation. Similar reduction in levels of a lower molecular weight Mcl-1 band occurred, which did not result from alternative splicing or from caspase-mediated cleavage. Mcl-1 showed primarily nuclear localization within the cell, and expression changes in full-length Mcl-1 were seen in nuclear lysate but not cytoplasmic lysate after 7 hours of DCA treatment. Changes in nuclear Mcl-1 expression did not correspond with cell cycle arrest or progression. These results suggest that proteasomal degradation of Mcl-1 may be altered following treatment with DCA, and this change may be associated with decreased proliferation, independent of cell cycle arrest. This may indicate a novel role of nuclear Mcl-1 in response of colorectal cancer to DCA exposure. / Final thesis for Leanne Delaney in partial fulfillment of requirements for the degree of Master of Science in Biomedical Sciences / NSERC

Melanoma is the most deadly form of skin cancers, with an incidence increasing more rapidly than any other malignant cancer in the past 40 years. Metastatic melanoma is resistant to conventional treatments, such as chemotherapy and radiation therapy. Our lab has previously demonstrated that Mcl-1 is a key contributor in protecting melanoma from therapy-induced cell death. RNAi therapeutics was employed as a novel way to silence the anti-apoptotic protein by using Mcl-1 mRNA sequence-specific siRNAs in vitro. In our hands, passive non-targeted delivery of RNAi therapy into melanoma tumours has been shown to be neither effective, nor selective in vitro and in vivo. Consequently, in this study, siRNA was linked to a delivery system which expressed a ligand specifically targeting melanoma cells. Previously shown, melanoma overexpresses the cell surface ganglioside GD3, thus it is my belief that the anti-GD3 R24 monoclonal antibody can function as a targeting molecule. The antibody was linked to coated cationic liposomes (CCLs) carrying siRNA molecules. Our first step was to confirm R24 ligation to CCLs. Untargeted CCLs showed insignificant values of antibody, whereas antibody-conjugated CCLs presented approximately 30 antibodies per liposome. I also confirmed that siRNA was internalized within CCLs using spectrometry, with an encapsulation efficiency of approximately 80%. Since liposomes need to be small to be effective in vitro and in vivo, CCLs were confirmed to be less than 100nm in diameter. In vitro studies using fluorescent microscopy demonstrated greater binding to melanoma cells with antibody-conjugated CCLs as compared to untargeted CCLs. In vivo experiments showed specific localization of targeted CCLs to induced subcutaneous mouse xenograft tumours. Western blotting demonstrated greater Mcl-1 knockdown using GD3-targeted CCLs. Taken together, these results suggest that anti-GD3 antibodies can serve as targeting molecules to deliver siRNA to melanoma cells and furthermore, GD3-targeted CCLs can promote siRNA-mediated gene silencing. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-09-02 10:29:37.944

蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶，CK2 作用的受質眾多且廣泛表現在哺乳類動物細胞中，對於細胞週期的發展、轉錄作用以及抗細胞凋亡等機制扮演非常重要的角色。在神經系統中，CK2 已知可以保護神經細胞以抵抗外來的傷害，但是其分子層面的機制目前尚未釐清。本篇論文的研究重點在於探討 CK2 保護作用可能參與的細胞分子機制。血清反應因子 SRF 是一種哺乳類動物細胞的轉錄因子，調控基因的轉錄作用來促進細胞的存活。Mcl-1 是抗細胞凋亡家族 Bcl-xL 家族蛋白成員之一，可以促進細胞的存活能力。先前研究指出，SRF 會受到 CK2 的磷酸化作用而增加本身的 DNA 結合能力。在其他研究也指出，Mcl-1 會受到 SRF 的調控。在本篇論文的第一部份，著重於 Mcl-1 的表現是否會受到 CK2 調控 SRF 的路徑所影響，實驗結果顯示，轉染野生型 CK2α 質體 DNA 可以增加海馬迴 CA1 腦區的 SRF 磷酸化，而轉染不活化的突變型 CK2αΑ156 質體 DNA 則會減少 SRF 的磷酸化。更進一步，轉染野生型 CK2α 會增加 Mcl-1 的 mRNA 及蛋白質表現，轉染突變型 CK2αΑ156 則減少 Mcl-1 的表現。此外，轉染突變型 SRF99A 也會減少 Mcl-1 的 mRNA 及蛋白質表現；而且在共同轉染實驗中，SRF99A 會拮抗野生型 CK2α 對促進的 Mcl-1 蛋白質表現的作用。 另一方面，DARPP-32 是一個在新紋狀體神經細胞中具有調控多巴胺訊息效力的訊息傳遞分子。先前研究指出，DARPP-32 具有抗細胞凋亡的功能，且發現在 DARPP-32 Ser102 氨基酸會受 CK2 的磷酸化作用。因此，本篇論文的第二部份主要是探討 CK2 的抗細胞凋亡能力是否是透過磷酸化 DARPP-32 來調控。實驗結果顯示，轉染野生型 CK2α 可以增加紋狀體 DARPP-32 的磷酸化，而轉染不活化的突變型 CK2αΑ156 則會減少 DARPP-32 的磷酸化。此外，轉染 CK2α 的小干擾 RNA (siRNA) 可以抑制內生性的 CK2 表現，同時也會減少 DARPP-32 的磷酸化以及抗細胞凋亡蛋白, Bcl-xL 的表現。綜合這些實驗結果，CK2α可以分別透過 SRF 或 DARPP-32 調控的訊息傳遞來促進 Mcl-1 或 Bcl-xL 的表現進而調控神經系統的抗細胞凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. In the nervous system, CK2 is shown to protect neurons against injury, but the cellular mechanisms are not well studies. In the present studies, we investigate which cellular mechanism might involve in the CK2 protection effects. The serum response factor (SRF) is a mammalian transcription factor which mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemin 1 (Mcl-1) is one of the anti-apoptotic Bcl-2 family members and is involved in promoting cell viability. Previous studied have revealed that the SRF phosphorylation by CK2 can enhance its DNA-binding activity. The regulation of Mcl-1 by SRF has also been reported in other studies. In the first part of the present studies, we investigate whether the Mcl-1 expression is regulated by CK2 through SRF mediated pathway. The results from wildtype CK2α plasmid DNA transfection revealed that the phosphorylated SRF were increased in hippocampus CA1 region, whereas transfection of the catalytically inactive CK2αA156 mutant plasmid DNA decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Moreover, transfection of the mutant SRF99A also decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A antagonized the upregulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments. In the other side, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Previous studies have revealed that DARPP-32 might involve in the anti-apoptosis and its Ser102 residue is phosphorylated by CK2. Therefore, in the second part of this study, we investigate whether one of the anti-apoptotic effects of CK2 is through DARPP-32 phosphorylation by CK2 in the present study. The results revealed that the phosphorylated DARPP-32 is increased in stratum by wildtype CK2α transfection and decreased by catalytically inactive CK2αA156 mutant transfection. Further, transfection of CK2α siRNA can inhibit endogenous CK2 expression and also decrease phosphorylation of DARPP-32 as well as the anti-apoptotic protein, Bcl-xL. These results together suggest that CK2α-mediated anti-apoptotic effects are partially through SRF mediated or DARPP-32 mediated signaling to regulate Mcl-1 or Bcl-xL expression, respectively.

漢厚朴酚是從木蘭科植物中萃取之天然化合物，已知具有抗氧化、抗發炎及神經保護之生理活性功能。先前的研究證明漢厚朴酚可以保護多巴胺神經元對抗6-OHDA所引起的細胞傷害，並且可以減緩6-OHDA 動物模式由apomorphine所誘發的旋轉行為，但漢厚朴酚對於神經保護之分子機制的相關研究尚未釐清。蛋白激酶CK2是具有多功能的絲氨酸/蘇氨酸激酶，高度表現在大腦紋狀體中，先前的研究證實蛋白激酶CK2參與調節神經系統功能和具有神經保護之作用。先前研究也指出轉錄因子Nrf1（Nuclear factor E2-related factor 1）是蛋白激酶CK2下游磷酸化受質，會調控小鼠胚胎纖維細胞中蛋白酶體基因的表現。抗細胞凋亡蛋白Mcl-1 (myeloid cell leukemia 1) 屬於Bcl-2蛋白家族的成員之一，在細胞凋亡的過程中，其蛋白含量減少與細胞凋亡有密切關聯性，抑制Mcl-1蛋白的降解可以延遲細胞死亡。因此本論文主要探討漢厚朴酚的神經細胞保護機制是否透過CK2-Nrf1細胞訊息路徑調控蛋白酶體活性，進而減少Mcl-1的降解速率。實驗結果顯示，轉染CK2α-EGFP DNA質體會增加Nrf1磷酸化並抑制蛋白酶體活性，泛素化之Mcl-1蛋白含量亦伴隨增加；轉染CK2α siRNA則會降低Nrf1磷酸化並促進蛋白酶體活性，導致naive Mcl-1蛋白質含量減少24小時的漢厚朴酚後處理（post-treatment）可以部份恢復因轉染CK2α siRNA所造成之CK2蛋白、Phosphoserine蛋白和Mcl-1蛋白質含量減少，在設計縮短間隔5小時漢厚朴酚後處理（post-treatment）的實驗結果雖然仍無法有效恢復CK2蛋白含量，但對於Phosphoserine和Mcl-1蛋白含量以及蛋白酶體活性則具有部份恢復的功效。利用過氧化氫造成細胞氧化壓力環境下，實驗發現間隔3小時的漢厚朴酚後處理才能有效恢復細胞存活率，間隔5小時的漢厚朴酚後處理則無法恢復細胞存活率。在大白鼠紋狀體腦區給予漢厚朴酚微量注射則對pTH、TH和GAD蛋白質含量皆有促進增加的作用，乙醯化的Histone H3蛋白含量也有顯著增加。綜合以上結果，推測漢厚朴酚對細胞保護作用的其中一個機制是參與調控CK2-Nrf1路徑而抑制蛋白酶體活性，減少Mcl-1蛋白質降解速率和提升氧化壓力下之細胞存活能力；此外，從活體動物的實驗結果顯示漢厚朴酚亦可能參與調控多巴胺和γ-氨基丁酸神經細胞功能的機制之中。 / Honokiol is a natural compound, extracted from the Magnolia officinalis, and is known as its anti-oxidative, anti-inflammatory and neuroprotective effects. The previous study has been demonstrated that the honokiol can protect striatal dopamine neuron against 6-OHDA induced damage and reverse the apomorphine-induced rotational behavior in Parkinson’s disease model of rats. However, the cellular mechanisms for its neuroprotective effects are not fully investigated. Protein kinase Casine kinase 2 (CK2) is a serine/threonine kinase has a highly abundant expression in the striatum compared with other brain areas. Further, CK2 is shown to regulate many neuronal functions including neuroprotection. The nuclear factor E2-related factor 1 (Nrf1) has been identified as one of the substrate proteins for CK2 and is indicated to involve in the induction of proteasome subunits gene expressions in mouse embryonic fibroblasts. The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) is shown to play a critical initiation role during the apoptosis process due to its synthesis blockage and proteasome degradation. The present study is aimed to investigate whether one of protective effects of honokiol is through CK2-mediated Nrf1 signaling pathway to regulate the proteasome activity in the mouse N2a neuroblastoma cell line. In the current results, transfection of the CK2α-EGFP plasmid DNA increased Nrf1 phosphorylation accompanied with the decrease in the proteasome activity but increased the ubiquitinated Mcl-1 protein. Whereas, transfection of CK2α siRNA decreased Nrf1 phosphorylation leading to the increase in proteasome activity and Mcl-1 protein degradation. The 24 hr duration of honokiol post-treatment only slightly reversed the knock-down effect of CK2α siRNA on CK2α and Mcl-1 protein levels. However, 5 hr duration of honokiol post-treatment could partially reverse the Mcl-1 protein level and proteasome activity but no effect on CK2α protein levels. In the H2O2-induced oxidative stress condition, only 3 hr duration of honokiol post-treatment could protect cells against H2O2-induced cell death. In the experiments of in vivo rat animal model, local administration of honokiol was found to increase phospho-TH, naive TH, GAD as well as acetylated Histone H3 protein levels. These above results suggest one of the protective mechanisms of honokiol might be through CK2-mediated Nrf1 signaling to inhibit the proteasome activity. and to promote cell survival under oxidative stress. Beside these functions, honokiol might also involve in the regulation of nurophysiological functions of dopamine and GABA neurons.

No description available.