New role for tRNA and its fragment purified from human urinary bladder carcinoma conditioned medium: Inhibition of endothelial cell growth

The growth of endothelial cells is necessary for angiogenesis, which in turn is required for later steps of tumor progression. In an attempt to purify new modulators of endothelial cell growth from the conditioned medium of human urinary bladder carcinoma cells, we isolated a small and stable oligonucleotide containing 10 to 16 bases. This oligonucleotide inhibited the growth of endothelial cells in vitro and was identified as a fragment of transfer RNA (tRNA). When unfractionated bovine tRNA was added to the cell culture, it specifically inhibited growth of endothelial cells, but not smooth muscle cells, bovine kidney cells, 3T3 fibroblasts, and several cancer cell lines. In contrast, ribosomal RNA, total yeast RNA, and single nucleosides from tRNA hydrolysate had no effect. These results demonstrate a new role for tRNA and its fragment as a selective endothelial cell inhibitor in vitro. J. Cell. Biochem. 76:109–117, 1999. © 1999 Wiley‐Liss, Inc.     

Synergism of leukemic blast growth factors in medium conditioned by human bladder carcinoma cell line 5637

Leukemic blast growth factors (LBGFs) are necessary for in vitro growth of clonogenic cells from patients with acute myeloblastic leukemia. As the human bladder carcinoma cell line 5637 had previously been reported to secrete abundant LBGFs into the culture supernatant, the LBGFs in 5637-conditioned medium (5637-CM) were characterized. Measurement of LBGFs was done using an in vitro leukemic blast colony assay in methylcellulose culture. LBGFs in 5637-CM were fractionated by anion exchange chromatography, and two peaks of activity were recovered. Pool B (high-salt eluent) and/or purified granulocyte colony-stimulating factor (G-CSF) were added to the clonogenic leukemic blast cell assays. It was found that pool B was more active than G-CSF in the majority of cases examined and that the two types of activity were synergistic in some cases.     

Effect of endothelial cell conditioned medium on the growth of human bone marrow fibroblasts

Both endothelial cells (EC) and fibroblasts, two discrete populations of hemopoietic stroma, are known to modulate the proliferation and differentiation of hemopoietic progenitors. Recent reports also demonstrated that EC stimulate the in vitro growth of fibroblasts via a soluble factor. This finding seems to support the hypothesis that EC may play a role in the pathogenesis of bone marrow fibrosis in myeloproliferative disorders (MD). We have studied the effects of the conditioned medium (CM) from human umbilical vein EC cultures, obtained in serum free conditions, on the growth of bone marrow fibroblasts from normal donors and from patients with MD. The results show that EC derived CM contains a factor which stimulates the proliferation of fibroblasts and that can act as an authentic growth factor by inducing "quiescent" fibroblasts to proliferate. Moreover, we found that this endothelial derived growth factor (EDGF) equally promotes the proliferation of both normal and pathological progenitors of bone marrow fibroblasts (CFU-F) by increasing both the number and the size of the colonies.     

小鼠骨髓 内皮细胞无血清条件培养液及其超滤组分对粒巨噬系祖细胞？…

本研究通过传代培养小鼠骨髓内皮细胞系细胞,惧无血清条件培养液（ｍＢＭＥＣ－ＣＭ）,将其作多级串联超滤,获得分子量大于１０、３￣１０、１￣３、０．５￣１ｋＤ和小于０．５ｋＤ超滤组分。进行粒巨噬系造血祖细胞集落形成试验。检测了它们的ｍＢＭＥＣ－ＣＭ和３￣１０组分对粒巨噬系祖细胞（ＣＦＵ－ＧＭ）生长未见明显影响,而分子量大于１０ｋＤ和０．５￣１ｋＤ组分促进ＣＦＵ－ＧＭ的增殖,分子量１￣３ｋＤ和小于０．５     

Stimulation of oxidative metabolism of granulocytes by recombinant granulocyte-macrophage-colony-stimulating-factor and a conditioned medium of a urinary bladder carcinoma cell line

Neutrophils (PMN) are the major host defence cells protecting the body against invasion by microorganisms. Products of oxidative metabolism mediate PMN microbicidal and tumoricidal activity, but the mechanisms by which these pathways become activated are not well understood. The colony stimulating factors (CSF) are known to stimulate proliferation and differentiation of committed bone marrow stem cells. These regulators may probably play an important role in non specific resistance to infections. We studied the oxidative metabolism of neutrophils after stimulation with recombinant GM-CSF (r.GM-CSF) and the concentrated conditioned medium of the UBC-5637 cell line (UBC-CM) showing CSF activity. It could be demonstrated that the r.GM-CSF, as well as the UBC-CM, induce an activation of the neutrophil respiratory burst without any cofactors such as f-MLP, PMA, or zymosan. In addition, we observed an increase of the response to those stimulants in the presence of either r.GM-CSF or UBC-CM. These effects were not endotoxin-induced, since stimulation persisted after addition of Polymyxin B, which is known to inhibit the action of endotoxins.     

Purification and characterization of a tissue plasminogen activator-inhibitor complex from human umbilical vein endothelial cell conditioned medium

Tissue plasminogen activator-inhibitor complexes were purified from the conditioned medium of human umbilical vein endothelial cells by affinity chromatography followed by gel filtration. It was found that a single complex was isolated which can exist in two distinct interconvertible conformations. These may be separated by electrophoresis into a form with a 105,000 apparent molecular weight and a form with an 88,000 apparent molecular weight. The particular conformation which predominates may be altered by changing the pH at which preparations are incubated or by including dithiothreitol in incubation buffers. Plasminogen activator enzymatic activity may be partially recovered from purified complexes by incubation in the presence of fibrin. Incubation in 1.5 M NH4OH results in the dissociation of the complex into two major polypeptides of 67 and 40 kilodaltons (kDa). The 40-kDa protein was isolated by gel filtration high-pressure liquid chromatography. N-Terminal amino acid analysis of this protein revealed three distinct sequences. Two of these were nearly identical and matched the N-terminal sequence recently reported for the native plasminogen activator inhibitor from endothelial cells. The third sequence exactly matched an internal portion of the same protein. The results suggest that the internal sequence is located at the site where the inhibitor is cleaved by tissue plasminogen activator.     

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium

In human unbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA)_and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70°C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.     

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts

A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

The resistance of epithelia to vascularization: Proteinase and endothelial cell growth inhibitory activities in urinary bladder epithelium

The avascularity of epithelia may be attributed to the presence of an extractable, low-molecular-weight factor. This factor contains potent inhibitors of proteolytic enzymes, as well as a growth inhibitory activity directed against endothelial cells in vitro. It is extracted from the epithelium of bovine urinary bladders by 1 M NaCl. The extract is ultrafiltered through an Amicon XM-50 membrane, then concentrated and dialyzed into a 0.9% NaCl solution, using a UM-2 membrane. This ultrafiltrate, called the UM-2 retentate (UM-2R). contains approximately 6 μg protein/ g tissue. The UM-2R has a low content of uronic acid and is practically devoid of hydroxyproline. SDS-PAGE reveals that the UM-2R consists of six major proteins. The UM-2R contains a Trasylol-like proteinase inhibitor that expresses strong trypsin inhibitory activity. Comparisons between bladder and serum UM-2Rs and electrophoretic mobility assays indicate that this proteinase inhibitory activity is derived from the bladder epithelium and not from the serum. The UM-2R is cytotoxic to cultured endothelial cells. Cultures of other cell types (normal and neoplastic) are not affected. The bladder-derived proteinase and endothelial cell growth inhibitory activities may protect epithelia from vascular invasion.     

Enhancement of erythroid progenitor cell growth in medium conditioned by a human cancer cell line, KONT

Erythroid-potentiating activity (EPA) was detected in culture medium conditioned by a human cancer cell line (KONT) that produces colony-stimulating activity (CSA), using erythroid colony formation in vitro. EPA in the medium conditioned by the KONT cells (KONT-CM) was markedly heat stable. After treating KONT-CM at 80 degrees C for 30 min, 30% EPA remained, while CSA was completely inactivated. Both EPA and CSA appeared in approximately the same fractions of the gel filtration, indicating a molecular weight of approximately 30,000 daltons. EPA bound partially to Concanavalin-A Sepharose, whereas CSA almost did not bind. Our results indicate that EPA can be separated from CSA based on heat stability and binding to Concanavalin-A Sepharose.     

Tumor-induced endothelial cell activation: role of vascular endothelial growth factor

Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl₂ significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl₂, both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects. vascular endothelial growth factor receptor 2; MG63-conditioned medium     

Purification and characterization of an endothelial cell growth factor from serum-free culture medium of human diploid fibroblast cells

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).     

The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other

In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.     

Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation

An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.     

A cytotoxic monoclonal antibody produced from a human urinary bladder carcinoma cell line (TSGH-8301)

A mouse monoclonal antibody, designated 1G3.10, directed against a human urinary bladder cancer cell line TSGH-8301 was generated using the conventional hybridoma technique. It was of the IgG3 subclass with a restricted specificity to bladder cancer and several epithelial cancers, as detected on a panel of various cell lines and tissues by radioimmunoassay and indirect immunofluorescence methods. It also exhibited a strong binding activity to human group A red blood cells, as demonstrated in hemagglutination and absorption tests. Radioimmunoprecipitation and thin-layer chromatography revealed that the antigens recognized by the antibody are a mixture of glycopeptides (M.W. 30, 54, and 108 KD) and glycolipid (retardation factor 0.67-0.83). From a fluorescence activated cell sorter (FACS) study, direct killing of tumor cells by the antibody at a concentration of more than 10 micrograms/ml was observed. This antibody could also mediate both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in vitro when tested in a 4-hr 51Cr-release test. These results suggest that the monoclonal antibody 1G3.10 may provide an agent for further immunodiagnosis and the treatment of human bladder cancer.     

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

Summary Spleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens. Abbreviations used in this paper: TCC, transitional cell carcinoma of the urinary bladder; TAA, tumor-associated antigens; ELISA, enzyme-linked immunosorbent assay; NBCS, newborn calf serum; PBS, phosphate-buffered saline, pH 7.2; ALP, alkaline phosphatase; GDA, glutardialdehyde; BSA, bovine serum albumin; IF, indirect immunofluorescence; LCL, lymphoblastoid cell lines: B-lymphocytes transformed in vitro with Epstein-Barr virus     

AS2O3抑制人结肠癌生长的体内外实验研究

目的：通过研究三氧化二砷（AS2O3）对人结肠癌细胞株LS-174T在体内外实验中增殖情况的影响,探讨As2O3治疗结肠癌的机制。方法：1、试验分组：体外细胞培养及体内动物实验均分三组：对照组,As2O3低剂量组,As2O3高剂量组。2、MTT法测定细胞增殖数的情况。3、丫啶橙/溴乙啶（AO/EB）双荧光染色检测细胞凋亡率。4、免疫组织化学法测定结肠癌细胞和裸鼠实体肿瘤中HIF-1α和VEGF的表达。5、测量各组裸鼠实体肿瘤的重量及体积。结果：体外试验中,由MTT法可以看出,As2O3治疗组细胞生存率明显低于对照组（P〈0.01）,其中AS2O3高剂量组明显低于低剂量组。双荧光染色结果As2O3高剂量组细胞凋亡百分率明显高于低剂量组和对照组（P〈0.01）。免疫组化结果显示结肠癌细胞HIF-1α和VEGF在对照组的阳性表达率明显高于As2O3治疗组（P〈0.01）,低剂量组高于高剂量组（P〈0.05）。体内实验中,治疗组平均瘤重和瘤体积均较对照组明显减低,（P〈0.01）。HIF-1α及VEGF在裸鼠肿瘤组织中均有阳性表达,其中对照组较AS2O3治疗组高（P〈0.01）。结论：As2O3能够抑制体内外人结肠癌细胞的生长,并能明显抑制HIF-1α和VEGF的表达,从而抑制肿瘤的生长。     

Human Wharton's Jelly stem cell conditioned medium and cell‐free lysate inhibit human osteosarcoma and mammary carcinoma cell growth in vitro and in xenograft mice

Human Wharton's jelly stem cells (hWJSCs) were shown to inhibit the growth of human mammary carcinomas. It is not known whether cell‐free secretions or lysates of hWJSCs do the same on different cancers. They may be less controversial than cells to regulatory bodies for clinical application. We examined the influence of hWJSC conditioned medium (hWJSC‐CM) and cell‐free lysate (hWJSC‐CL) on two osteosarcoma cell lines (MG‐63, SKES‐1) in vitro and on human mammary carcinomas in immunodeficient mice. When exposed to hWJSC‐CL, increased vacuolations in MG‐63 and increased membrane fragmentation in SKES‐1 cells were observed, with greater cell death in SKES‐1. Exposure of SKES‐1 and MG‐63 cells to hWJSC‐CL showed significant decreases in cell proliferation of 46.48 ± 6.66% and 24.32 ± 5.67% respectively compared to controls. MG‐63 and SKES‐1 cells were annexin V‐FITC positive and SKES‐1 TUNEL positive following treatment with hWJSC‐CM and hWJSC‐CL. MG‐63 cells were positive and SKES‐1 cells negative for anti‐BECLIN‐1 and anti‐LC3B following treatment with hWJSC‐CM and hWJSC‐CL. RT‐PCR showed that the pro‐apoptotic BAX gene and the autophagy‐related ATG‐5 and BECLIN‐1 genes were up‐regulated while the anti‐apoptotic BCL2 and SURVIVIN genes were down‐regulated in MG‐63 and SKES‐1 cells treated with hWJSC‐CM and hWJSC‐CL. Injections of hWJSCs and hWJSC‐CM into mammary carcinomas in immunodeficient mice resulted in decreased tumor sizes and weights of 24.86 ± 6.05% to 37.03 ± 5.91% and 47.14 ± 7.36% to 55.09 ± 5.87% respectively at 6 weeks compared to controls. hWJSC‐CM and hWJSC‐CL inhibit mammary carcinoma and osteosarcoma cells via apoptosis and autophagy. J. Cell. Biochem. 114: 366–377, 2013. © 2012 Wiley Periodicals, Inc.