bcl associated death protein


Summary: A pro-apoptotic protein and member of the Bcl-2 protein family that is regulated by PHOSPHORYLATION. Unphosphorylated Bad protein inhibits the activity of BCL-XL PROTEIN.

Top Publications

  1. Tripathy D, Grammas P. Acetaminophen protects brain endothelial cells against oxidative stress. Microvasc Res. 2009;77:289-96 pubmed publisher
  2. Han B, Ren Y, Guan L, Wei W, Hua F, Yang Y, et al. Sodium selenite induces apoptosis in acute promyelocytic leukemia-derived NB4 cells through mitochondria-dependent pathway. Oncol Res. 2009;17:373-81 pubmed
  3. Tsuda H, Ochiai K, Suzuki N, Otsuka K. Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells. J Periodontal Res. 2010;45:626-34 pubmed publisher
    ..These data suggest that butyrate induces apoptosis and autophagic cell death. ..
  4. Yang Y, Huang F, Ren Y, Xing L, Wu Y, Li Z, et al. The anticancer effects of sodium selenite and selenomethionine on human colorectal carcinoma cell lines in nude mice. Oncol Res. 2009;18:1-8 pubmed
    ..SSe and SeMet may be the selective, low-toxic anticancer agents to treat human colorectal carcinoma cancer...
  5. Frenzel A, Labi V, Chmelewskij W, Ploner C, Geley S, Fiegl H, et al. Suppression of B-cell lymphomagenesis by the BH3-only proteins Bmf and Bad. Blood. 2010;115:995-1005 pubmed publisher
    ..Loss of Bmf reduced the pressure to inactivate p53, whereas Bad deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc. ..
  6. Chaturvedi D, Cohen M, Taunton J, Patel T. The PKARIalpha subunit of protein kinase A modulates the activation of p90RSK1 and its function. J Biol Chem. 2009;284:23670-81 pubmed publisher
    ..Thus, in unstimulated cells, the increased phosphorylation and activation of RSK1 after silencing of PKARIalpha or depletion of D-AKAP1 are due to decreased association of PP2Ac in the RSK1 complex. ..
  7. Bhaskar P, Nogueira V, Patra K, Jeon S, Park Y, Robey R, et al. mTORC1 hyperactivity inhibits serum deprivation-induced apoptosis via increased hexokinase II and GLUT1 expression, sustained Mcl-1 expression, and glycogen synthase kinase 3beta inhibition. Mol Cell Biol. 2009;29:5136-47 pubmed publisher
    ..Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2(-/)(-) cells to serum deprivation-induced apoptosis. ..
  8. Wang S, Wang Y, Jiang J, Wang R, Li L, Qiu Z, et al. 15-HETE protects rat pulmonary arterial smooth muscle cells from apoptosis via the PI3K/Akt pathway. Prostaglandins Other Lipid Mediat. 2010;91:51-60 pubmed publisher
    ..These changes depended on the PI3K/Akt signaling pathway in PASMCs. Thus a signal transduction pathway was demonstrated which is necessary for the effects of 15-HETE in protection PASMCs from apoptosis. ..
  9. Lahiry L, Saha B, Chakraborty J, Adhikary A, Mohanty S, Hossain D, et al. Theaflavins target Fas/caspase-8 and Akt/pBad pathways to induce apoptosis in p53-mutated human breast cancer cells. Carcinogenesis. 2010;31:259-68 pubmed publisher

More Information


  1. Abramovich D, Irusta G, Parborell F, Tesone M. Intrabursal injection of vascular endothelial growth factor trap in eCG-treated prepubertal rats inhibits proliferation and increases apoptosis of follicular cells involving the PI3K/AKT signaling pathway. Fertil Steril. 2010;93:1369-77 pubmed publisher
    ..The PI3K/AKT signaling pathway is one of the pathways involved in this mechanism. Therefore, VEGFA has a role as an antiapoptotic and proliferative factor in follicular cells from the rat ovary. ..
  2. Schallenberg M, Charalambous P, Thanos S. GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death. Exp Eye Res. 2009;89:665-77 pubmed publisher
    ..Like in vitro, GM-CSF counteracts the staurosporine-induced cell death in vivo and protects RGCs from axotomy-induced degeneration. Our data suggest that GM-CSF might be a novel therapeutic agent in neuropathic disease of the eye. ..
  3. Li Y, Wang Y, Taniguchi T, Kawakami T, Baba T, Ishibashi H, et al. Identification of stemonamide synthetic intermediates as a novel potent anticancer drug with an apoptosis-inducing ability. Int J Cancer. 2010;127:474-84 pubmed publisher
    ..These observations indicate that the chemicals and its related compounds may be effective for the treatment of tumors of endoderm-derived organs, particularly the pancreas. ..
  4. Montalvo V, Gery I. Constitutive pro-apoptotic molecules are differentially expressed in the fetal and adult mouse eye. Curr Eye Res. 2009;34:328-32 pubmed publisher
    ..Different mechanisms of apoptosis are prominent at the fetal and mature stages of the mouse eye. ..
  5. Gilchrist M, Henderson W, Morotti A, Johnson C, Nachman A, Schmitz F, et al. A key role for ATF3 in regulating mast cell survival and mediator release. Blood. 2010;115:4734-41 pubmed publisher
    ..This dual role of ATF3 provides insight into the complex interplay between MC development and its subsequent physiologic role. ..
  6. Huang C, Gu H, Zhang W, Herrmann J, Wang M. Testosterone-down-regulated Akt pathway during cardiac ischemia/reperfusion: a mechanism involving BAD, Bcl-2 and FOXO3a. J Surg Res. 2010;164:e1-11 pubmed publisher
  7. Brutus N, Hanley S, Ashraf Q, Mishra O, Delivoria Papadopoulos M. Effect of hyperoxia on serine phosphorylation of apoptotic proteins in mitochondrial membranes of the cerebral cortex of newborn piglets. Neurochem Res. 2009;34:1219-25 pubmed publisher
    ..We speculate that phosphorylation of Bcl-2 and Bcl-xl will result in loss of their anti-apoptotic potential by preventing their dimerization with Bax leading to activation of the caspase cascade of neuronal death. ..
  8. Oh J, Jang D, Kim H, Kang H, Chung C, Park W, et al. alpha3beta1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins. Exp Cell Res. 2009;315:3187-200 pubmed publisher
  9. Kurita Ochiai T, Ochiai K. Butyric acid induces apoptosis via oxidative stress in Jurkat T-cells. J Dent Res. 2010;89:689-94 pubmed publisher
    ..These results suggest that butyric acid is involved in mitochondrial-dysfunction- and endoplasmic reticulum stress-mediated apoptosis in human Jurkat T-cells via a ROS-dependent mechanism...
  10. Das A, Durrant D, Mitchell C, Mayton E, Hoke N, Salloum F, et al. Sildenafil increases chemotherapeutic efficacy of doxorubicin in prostate cancer and ameliorates cardiac dysfunction. Proc Natl Acad Sci U S A. 2010;107:18202-7 pubmed publisher
    ..In conclusion, these results provide provocative evidence that sildenafil is both a powerful sensitizer of DOX-induced killing of prostate cancer while providing concurrent cardioprotective benefit. ..
  11. Schuhmann D, Godoy P, Weiss C, Gerloff A, Singer M, Dooley S, et al. Interfering with interferon-? signalling in intestinal epithelial cells: selective inhibition of apoptosis-maintained secretion of anti-inflammatory interleukin-18 binding protein. Clin Exp Immunol. 2011;163:65-76 pubmed publisher
    ..Selective inhibition of IFN-? mediated apoptosis, while preserving its beneficial consequences on the ratio of IL-18/IL-18bp, could contribute to the integrity of the mucosal barrier in intestinal inflammation...
  12. Sanderson T, Kumar R, Murariu Dobrin A, Page A, Krause G, SULLIVAN J. Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia. Neurol Res. 2009;31:947-58 pubmed publisher
    ..The downstream components of insulin-induced Akt survival signaling after transient global brain ischemia remain to be identified. ..
  13. Yang J. Molecular modeling of human BAD, a pro-apoptotic Bcl-2 family member, integrating glycolysis and apoptosis. Protein Pept Lett. 2010;17:206-20 pubmed
    ..PKAc may prevent Bcl-xL from rebinding to BAD by phosphorylating human BAD at Ser118; whereas human BAD is phosphorylated on Ser75 in a BAD-Bcl-xL complex, resulting in the dissociation of BAD. ..
  14. Barba Barajas M, Hernandez Flores G, Lerma Diaz J, Ortiz Lazareno P, Dominguez Rodriguez J, Barba Barajas L, et al. Gossypol induced apoptosis of polymorphonuclear leukocytes and monocytes: involvement of mitochondrial pathway and reactive oxygen species. Immunopharmacol Immunotoxicol. 2009;31:320-30 pubmed publisher
    ..The findings in this report may be important to consider in light of possible gossypol use in clinical strategies for cancer treatment. ..
  15. Semedo P, Palasio C, Oliveira C, Feitoza C, Gonçalves G, Cenedeze M, et al. Early modulation of inflammation by mesenchymal stem cell after acute kidney injury. Int Immunopharmacol. 2009;9:677-82 pubmed publisher
    ..And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. ..
  16. Yu B, Sun X, Shen H, Gao F, Fan Y, Sun Z. Expression of the apoptosis-related genes BCL-2 and BAD in human breast carcinoma and their associated relationship with chemosensitivity. J Exp Clin Cancer Res. 2010;29:107 pubmed publisher
    ..The combined detection of BCL-2 and BAD may be markers for predicting the responses to anticancer drugs. ..
  17. Tripathy D, Grammas P. Acetaminophen inhibits neuronal inflammation and protects neurons from oxidative stress. J Neuroinflammation. 2009;6:10 pubmed publisher
  18. Kuo W, Chung L, Liu C, Wu S, Kuo C, Tsai F, et al. Effects of insulin replacement on cardiac apoptotic and survival pathways in streptozotocin-induced diabetic rats. Cell Biochem Funct. 2009;27:479-87 pubmed publisher
    ..The findings may provide possible diabetes-related apoptotic, hypertrophic, and survival pathways for potentially preventing cardiac abnormality in diabetes. ..
  19. Szanto A, Bognar Z, Szigeti A, Szabo A, Farkas L, Gallyas F. Critical role of bad phosphorylation by Akt in cytostatic resistance of human bladder cancer cells. Anticancer Res. 2009;29:159-64 pubmed
    ..Akt-mediated Bad phosphorylation plays an important role in preservation of mitochondrial membrane systems leading to paclitaxel resistance in T24 cells. ..
  20. Ahn S, Kim J, Hara M, Ren X, Lefkowitz R. {beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation. J Biol Chem. 2009;284:8855-65 pubmed publisher
    ..These findings establish a key role for beta-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved. ..
  21. Chen G, Zhou X, Florea S, Qian J, Cai W, Zhang Z, et al. Expression of active protein phosphatase 1 inhibitor-1 attenuates chronic beta-agonist-induced cardiac apoptosis. Basic Res Cardiol. 2010;105:573-81 pubmed publisher
    ..These findings suggest that mitochondrial signaling and ERK activation may be involved in the I-1c cardioprotective effects against apoptosis induced by prolonged beta-adrenergic stimulation. ..
  22. Aburai N, Yoshida M, Ohnishi M, Kimura K. Pisiferdiol and pisiferic acid isolated from Chamaecyparis pisifera activate protein phosphatase 2C in vitro and induce caspase-3/7-dependent apoptosis via dephosphorylation of Bad in HL60 cells. Phytomedicine. 2010;17:782-8 pubmed publisher
  23. Seow H, Yip W, Loh H, Ithnin H, Por P, Rohaizak M. Immunohistochemical detection of phospho-Akt, phospho-BAD, HER2 and oestrogen receptors alpha and beta in Malaysian breast cancer patients. Pathol Oncol Res. 2010;16:239-48 pubmed publisher
    ..This study does not support the simple model of linear HER2/PI3K/Akt pathway in breast cancer. ..
  24. Greenhough A, Wallam C, Hicks D, Moorghen M, Williams A, Paraskeva C. The proapoptotic BH3-only protein Bim is downregulated in a subset of colorectal cancers and is repressed by antiapoptotic COX-2/PGE(2) signalling in colorectal adenoma cells. Oncogene. 2010;29:3398-410 pubmed publisher
    ..These observations highlight the COX-2/PGE(2) pathway as an important negative regulator of Bim expression in colorectal tumours and suggest that Bim repression may be an important step during colorectal cancer tumorigenesis. ..
  25. Gao F, Li H, Chen Y, Yu X, Wang R, Chen X. Upregulation of PTEN involved in scorpion venom-induced apoptosis in a lymphoma cell line. Leuk Lymphoma. 2009;50:633-41 pubmed publisher
    ..The results indicate that key regulators in BmK venom-induced apoptosis are PTEN, acting through downregulation of the PI3K/Akt signal pathway, in Raji cells and p27 in Jurkat cells. ..
  26. Xiao Y, Yuan T, Yao W, Liao K. 3T3-L1 adipocyte apoptosis induced by thiazolidinediones is peroxisome proliferator-activated receptor-gamma-dependent and mediated by the caspase-3-dependent apoptotic pathway. FEBS J. 2010;277:687-96 pubmed publisher
    ..Thus, TZDs promote apoptosis in adipocytes through a PPARgamma-dependent pathway. This apoptosis is mediated by the inhibition of Akt-1, which decreases Bad phosphorylation and activates the mitochondrial apoptotic pathway. ..
  27. Calvo N, de Boland A, Gentili C. PTH inactivates the AKT survival pathway in the colonic cell line Caco-2. Biochim Biophys Acta. 2010;1803:343-51 pubmed publisher
    ..Overall, our data suggest that in Caco-2 cells, PP2A and the cAMP pathway act in concert to inactivate the AKT survival pathway in PTH-induced intestinal cell apoptosis. ..
  28. Banerjee R, Huang Y, McNamee J, Todorova M, Jean Claude B. The combi-targeting concept: selective targeting of the epidermal growth factor receptor- and Her2-expressing cancer cells by the complex combi-molecule RB24. J Pharmacol Exp Ther. 2010;334:9-20 pubmed publisher
  29. Chien C, Chang S, Lin K, Chiu C, Chang L, Lin S. Taiwan cobra cardiotoxin III inhibits Src kinase leading to apoptosis and cell cycle arrest of oral squamous cell carcinoma Ca9-22 cells. Toxicon. 2010;56:508-20 pubmed publisher
    ..Taken together, these results indicate that CTX III induces apoptosis and S-phase arrest in Ca9-22 cells via concomitant inactivation of the Src, EGFR, STAT3, STAT5, PI3 K(p110), and Akt signaling pathways. ..
  30. Wu J, Lin T, Wu K. Rosiglitazone and PPAR-gamma overexpression protect mitochondrial membrane potential and prevent apoptosis by upregulating anti-apoptotic Bcl-2 family proteins. J Cell Physiol. 2009;220:58-71 pubmed publisher
    ..These results indicate that ligand-activated PPAR-gamma protects N2-A cells against H-R damage by enhancing Bcl-2/Bcl-xl and maintaining p-Bad via preservation of p-Akt. ..
  31. Calvert J, Jha S, Gundewar S, Elrod J, Ramachandran A, Pattillo C, et al. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res. 2009;105:365-74 pubmed publisher
    ..These results reveal that the cardioprotective effects of H(2)S are mediated in large part by a combination of antioxidant and antiapoptotic signaling. ..
  32. Kaneko A, Satoh Y, Tokuda Y, Fujiyama C, Udo K, Uozumi J. Effects of adipocytes on the proliferation and differentiation of prostate cancer cells in a 3-D culture model. Int J Urol. 2010;17:369-76 pubmed publisher
    ..Adipocytes could modulate the proliferation and differentiation of prostate cancer cell lines. Activation of the PI3K pathway might be involved in the prostate cancer cell-adipocyte interaction. ..
  33. Win H, Acevedo Duncan M. Role of protein kinase C-iota in transformed non-malignant RWPE-1 cells and androgen-independent prostate carcinoma DU-145 cells. Cell Prolif. 2009;42:182-94 pubmed publisher
  34. Zhang F, Zhang T, Jiang T, Zhang R, Teng Z, Li C, et al. Wortmannin potentiates roscovitine-induced growth inhibition in human solid tumor cells by repressing PI3K/Akt pathway. Cancer Lett. 2009;286:232-9 pubmed publisher
    ..Taken together, these results provide evidence for the potential application of roscovitine/wormannin combination in clinical treatment for solid tumors. ..
  35. Stumpner J, Redel A, Kellermann A, Lotz C, Blomeyer C, Smul T, et al. Differential role of Pim-1 kinase in anesthetic-induced and ischemic preconditioning against myocardial infarction. Anesthesiology. 2009;111:1257-64 pubmed publisher
    ..These data suggest that Pim-1 kinase mediates at least in part desflurane-induced preconditioning and IPC against myocardial infarction in mice. ..
  36. Jain H, NOR J, Jackson T. Quantification of endothelial cell-targeted anti-Bcl-2 therapy and its suppression of tumor growth and vascularization. Mol Cancer Ther. 2009;8:2926-36 pubmed publisher
    ..These results underscore the potential of mathematical modeling to guide the development of novel antiangiogenic therapies and to direct drug design. ..
  37. McGaffin K, Zou B, McTiernan C, O Donnell C. Leptin attenuates cardiac apoptosis after chronic ischaemic injury. Cardiovasc Res. 2009;83:313-24 pubmed publisher
  38. Rashmi R, Bode B, Panesar N, King S, Rudloff J, Gartner M, et al. Siglec-9 and SHP-1 are differentially expressed in neonatal and adult neutrophils. Pediatr Res. 2009;66:266-71 pubmed publisher
    ..Our data suggest that age-specific interactions between Siglec-9 and SHP-1 may influence the altered inflammatory responsiveness and longevity of neonatal PMN. ..
  39. Chen B, Longtine M, Sadovsky Y, Nelson D. Hypoxia downregulates p53 but induces apoptosis and enhances expression of BAD in cultures of human syncytiotrophoblasts. Am J Physiol Cell Physiol. 2010;299:C968-76 pubmed publisher
  40. Rahmani M, Anderson A, Habibi J, Crabtree T, Mayo M, Harada H, et al. The BH3-only protein Bim plays a critical role in leukemia cell death triggered by concomitant inhibition of the PI3K/Akt and MEK/ERK1/2 pathways. Blood. 2009;114:4507-16 pubmed publisher
    ..Collectively, these findings suggest that Bim, and Mcl-1, but not Bad, integrate death signaling triggered by concomitant disruption of the PI3K/Akt and MEK1/2/ERK1/2 pathways in human leukemia cells. ..
  41. Quoyer J, Longuet C, Broca C, Linck N, Costes S, Varin E, et al. GLP-1 mediates antiapoptotic effect by phosphorylating Bad through a beta-arrestin 1-mediated ERK1/2 activation in pancreatic beta-cells. J Biol Chem. 2010;285:1989-2002 pubmed publisher
    ..Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands. ..
  42. Saito Y, Owaki T, Matsunaga T, Saze M, Miura S, Maeda M, et al. Apoptotic death of hematopoietic tumor cells through potentiated and sustained adhesion to fibronectin via VLA-4. J Biol Chem. 2010;285:7006-15 pubmed publisher
    ..The TNIIIA2-related matricryptic site of TN-C could contribute, once exposed, to preventing prolonged survival of hematopoietic malignant progenitors through potentiated and sustained activation of VLA-4. ..
  43. Saldanha Gama R, Moraes J, Mariano Oliveira A, Coelho A, Walsh E, Marcinkiewicz C, et al. alpha(9)beta(1) integrin engagement inhibits neutrophil spontaneous apoptosis: involvement of Bcl-2 family members. Biochim Biophys Acta. 2010;1803:848-57 pubmed publisher
  44. Gao X, Deeb D, Hao J, Liu Y, Arbab A, Dulchavsky S, et al. Synthetic triterpenoids inhibit growth, induce apoptosis and suppress pro-survival Akt, mTOR and NF-{kappa}B signaling proteins in colorectal cancer cells. Anticancer Res. 2010;30:785-92 pubmed
    ..These studies provide rationale for clinical evaluation of CDDO-Me for the treatment of advanced chemotherapy refractory colorectal cancer. ..
  45. Jin L, Xiao C, Lu C, Xia M, Xing G, Xiong S, et al. Transcriptomic and proteomic approach to studying SNX-2112-induced K562 cells apoptosis and anti-leukemia activity in K562-NOD/SCID mice. FEBS Lett. 2009;583:1859-66 pubmed publisher
    ..Additionally, SNX-2112 prolonged the survival time of NOD/SCID mice inoculated with K562 tumor cells. Our results demonstrated the therapeutic potential of SNX-2112 against human CML. ..
  46. Weidinger C, Karger S, Krause K, Schierle K, Steinert F, Gimm O, et al. Distinct regulation of intrinsic apoptosis in benign and malignant thyroid tumours. Horm Metab Res. 2010;42:553-6 pubmed publisher
    ..Further knowledge on apoptosis-deregulation during thyroid carcinogenesis might confer diagnostic and therapeutic benefits in the management of thyroid cancer. ..
  47. Matheny R, Adamo M. Role of Akt isoforms in IGF-I-mediated signaling and survival in myoblasts. Biochem Biophys Res Commun. 2009;389:117-21 pubmed publisher
    ..Our results provide evidence for IGF-I-stimulated isoform-specific actions of Akt on molecules involved in promoting apoptosis. ..
  48. Karunakaran S, Ravindranath V. Activation of p38 MAPK in the substantia nigra leads to nuclear translocation of NF-kappaB in MPTP-treated mice: implication in Parkinson's disease. J Neurochem. 2009;109:1791-9 pubmed publisher
    ..Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression. ..
  49. Cho J, Sung J, Cho E, Won C, Lee H, Kim M, et al. Gingko biloba Extract (EGb 761) prevents ischemic brain injury by activation of the Akt signaling pathway. Am J Chin Med. 2009;37:547-55 pubmed
    ..In conclusion, this study suggests that EGb 761 prevents cell death due to brain injury and that EGb 761 protection is affected by preventing the injury-induce decrease of Akt phosphorylation. ..
  50. Samovski D, Kalderon B, Yehuda Shnaidman E, Bar Tana J. Gating of the mitochondrial permeability transition pore by long chain fatty acyl analogs in vivo. J Biol Chem. 2010;285:6879-90 pubmed publisher
    ..Low conductance PTP gating by LCFA/MEDICA may account for their thyromimetic calorigenic activity in vivo. ..
  51. Luo S, Rubinsztein D. Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL. Cell Death Differ. 2010;17:268-77 pubmed publisher
    ..The cleavage of Beclin 1 is a critical event whereby caspases inhibit autophagy, as a non-cleavable Beclin 1 mutant restored autophagy in cells overexpressing Bax. ..
  52. Takeuchi K, Ito F. EGF receptor in relation to tumor development: molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitors. FEBS J. 2010;277:316-26 pubmed publisher
    ..We also discuss tyrosine kinase inhibitor-induced apoptosis via c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase. ..
  53. Hida N, Awais M, Takeuchi M, Ueno N, Tashiro M, Takagi C, et al. High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases. PLoS ONE. 2009;4:e5868 pubmed publisher
    ..This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. ..