Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma(-/-) cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Consequences of the combined loss of BOK and BAK or BOK and BAX

The multi-BCL-2 homology domain pro-apoptotic BCL-2 family members BAK and BAX have critical roles in apoptosis. They are essential for mitochondrial outer-membrane permeabilization, leading to the release of apoptogenic factors such as cytochrome-c, which promote activation of the caspase cascade and cellular demolition. The BOK protein has extensive amino-acid sequence similarity to BAK and BAX and is expressed in diverse cell types, particularly those of the female reproductive tissues. The BOK-deficient mice have no readily discernible abnormalities, and its function therefore remains unresolved. We hypothesized that BOK may exert functions that overlap with those of BAK and/or BAX and examined this by generating Bok−/−Bak−/− and Bok−/−Bax−/− mice. Combined loss of BOK and BAK did not elicit any noticeable defects, although it remains possible that BOK and BAK have critical roles in developmental cell death that overlap with those of BAX. In most tissues examined, loss of BOK did not exacerbate the abnormalities caused by loss of BAX, such as defects in spermatogenesis or the increase in neuronal populations in the brain and retina. Notably, however, old Bok−/−Bax−/− females had abnormally increased numbers of oocytes from different stages of development, indicating that BOK may have a pro-apoptotic function overlapping with that of BAX in age-related follicular atresia.

The mood-stabilizer lithium prevents hippocampal apoptosis and improves spatial memory in experimental meningitis.

Pneumococcal meningitis is associated with high morbidity and mortality rates. Brain damage caused by this disease is characterized by apoptosis in the hippocampal dentate gyrus, a morphological correlate of learning deficits in experimental paradigms. The mood stabilizer lithium has previously been found to attenuate brain damage in ischemic and inflammatory diseases of the brain. An infant rat model of pneumococcal meningitis was used to investigate the neuroprotective and neuroregenerative potential of lithium. To assess an effect on the acute disease, LiCl was administered starting five days prior to intracisternal infection with live Streptococcus pneumoniae. Clinical parameters were recorded, cerebrospinal fluid (CSF) was sampled, and the animals were sacrificed 42 hours after infection to harvest the brain and serum. Cryosections of the brains were stained for Nissl substance to quantify brain injury. Hippocampal gene expression of Bcl-2, Bax, p53, and BDNF was analyzed. Lithium concentrations were measured in serum and CSF. The effect of chronic lithium treatment on spatial memory function and cell survival in the dentate gyrus was evaluated in a Morris water maze and by quantification of BrdU incorporation after LiCl treatment during 3 weeks following infection. In the hippocampus, LiCl significantly reduced apoptosis and gene expression of Bax and p53 while it increased expression of Bcl-2. IL-10, MCP-1, and TNF were significantly increased in animals treated with LiCl compared to NaCl. Chronic LiCl treatment improved spatial memory in infected animals. The mood stabilizer lithium may thus be a therapeutic alternative to attenuate neurofunctional deficits as a result of pneumococcal meningitis.

Nitric oxide contributes to LPS/IFN-gamma-induced macrophage apoptosis via JNK and BCL-2 family regulation

Lipopolysaccharide (LPS) and interferon-gamma (IFN) activate macrophages and produce nitric oxide (NO) by initiating the expression of inducible Nitric Oxide Synthase (iNOS). Prolonged LPS/IFN-activation results in the death of macrophage-like RAW 264.7 cells and wild-type murine macrophages. This study was implemented to determine how NO contributes to LPS/IFN-induced macrophage death. The iNOS-specific inhibitor L-NIL protected RAW 264.7 cells from LPS/IFN-activated death, supporting a role for NO in the death of LPS/IFN-activated macrophages. A role for iNOS in cell death was confirmed in iNOS-/- macrophages which were resistant to LPS/IFN-induced death. Cell death was accompanied by nuclear condensation, caspase 3 activation, and PARP cleavage, all of which are hallmarks of apoptosis. The involvement of NO in modulating the stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) signal transduction pathway was examined as a possible mechanism of LPS/IFN-mediated apoptosis. Western analysis demonstrated that NO modifies the phosphorylation profile of JNK and promotes activation of JNK in the mitochondria in RAW 264.7 cells. Inhibition of JNK with sIRNA significantly reduced cell death in RAW 264.7 cells, indicating the participation of the JNK pathway in LPS/IFN-mediated death. JNK has been demonstrated to induce mitochondrial-mediated apoptosis through modulation of Bcl-2 family members. Therefore, the effect of NO on the balance between pro- and anti-apoptotic Bcl-2 family members was examined. In RAW 264.7 cells, Bim was upregulated and phosphorylated by LPS/IFN independently of NO. However, co-immunoprecipitation studies demonstrated that NO promotes the association of Bax with the BimL splice variant. Examination of Bax phosphorylation by metabolic labeling demonstrated that Bax is basally phosphorylated and becomes dephosphorylated upon LPS/IFN treatment. L-NIL inhibited the dephosphorylation of Bax, indicating that Bax dephosphorylation is NO-dependent. NO also mediated LPS/IFN-induced downregulation of Mcl-1, an anti-apoptotic Bcl-2 family member, as demonstrated by Western blotting for Mcl-1 protein expression. Thus, NO contributes to macrophage apoptosis via a JNK-mediated mechanism involving interaction between Bax and Bim, dephosphorylation of Bax, and downregulation of Mcl-1. ^

Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria

Cytochrome c release and the mitochondrial permeability transition (PT), including loss of the transmembrane potential (Δψ), play an important role in apoptosis. Using isolated mitochondria, we found that recombinant Bax and Bak, proapoptotic members of the Bcl-2 family, induced mitochondrial Δψ loss, swelling, and cytochrome c release. All of these changes were dependent on Ca2+ and were prevented by cyclosporin A (CsA) and bongkrekic acid, both of which close the PT pores (megachannels), indicating that Bax- and Bak-induced mitochondrial changes were mediated through the opening of these pores. Bax-induced mitochondrial changes were inhibited by recombinant Bcl-xL and transgene-derived Bcl-2, antiapoptotic members of the Bcl-2 family, as well as by oligomycin, suggesting a possible regulatory effect of F0F1-ATPase on Bax-induced mitochondrial changes. Proapoptotic Bax- and Bak-BH3 (Bcl-2 homology) peptides, but not a mutant BH3 peptide nor a mutant Bak lacking BH3, induced the mitochondrial changes, indicating an essential role of the BH3 region. A coimmunoprecipitation study revealed that Bax and Bak interacted with the voltage-dependent anion channel, which is a component of PT pores. Taken together, these findings suggest that proapoptotic Bcl-2 family proteins, including Bax and Bak, induce the mitochondrial PT and cytochrome c release by interacting with the PT pores.

Steatosis and liver cell apoptosis in chronic hepatitis C: A mechanism for increased liver injury

Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis Q however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl-2, Bd-x(L), Bax, and tumor necrosis factor alpha (TNF-alpha) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (r = 0.42; P < .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio (r = -0.32, P = .007; and r = 0.27, P = .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis (r = 0.26, P = .11; r = 0.06, P = .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis (r = 0.35, P = .047; r = 0.33, P = .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF-α mRNA levels and active caspase-3 (r = 0.54, P = .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis.

T cells, adhesion molecules and modulation of apoptosis in visceral leishmaniasis glomerulonephritis

Background: Immune complex deposition is the accepted mechanism of pathogenesis of VL glomerulopathy however other immune elements may participate. Further in the present study, no difference was seen between immunoglobulin and C3b deposit intensity in glomeruli between infected and non-infected dogs thus T cells, adhesion molecules and parameters of proliferation and apoptosis were analysed in dogs with naturally acquired VL from an endemic area. The dog is the most important domestic reservoir of the protozoa Leishmania (L.) chagasi that causes visceral leishmaniasis (VL). The similarity of VL manifestation in humans and dogs renders the study of canine VL nephropathy of interest with regard to human pathology. Methods: From 55 dogs with VL and 8 control non-infected dogs from an endemic area, kidney samples were analyzed by immunohistochemistry for immunoglobulin and C3b deposits, staining for CD4+ and CD8+ T cells, ICAM-1, P-selectin and quantified using morphometry. Besides proliferation marker Ki-67, apoptosis markers M30 and TUNEL staining, and related cytokines TNF-alpha, IL-1 alpha were searched and quantified. Results: We observed similar IgG, IgM and IgA and C3b deposit intensity in dogs with VL and non-infected control dogs. However we detected the Leishmania antigen in cells in glomeruli in 54, CD4+ T cells in the glomeruli of 44, and CD8+ T cells in 17 of a total of 55 dogs with VL. Leishmania antigen was absent and T cells were absent/scarse in eight non-infected control dogs. CD 4+ T cells predominate in proliferative patterns of glomerulonephritis, however the presence of CD4+ and CD8+ T cells were not different in intensity in different patterns of glomerulonephritis. The expression of ICAM-1 and P-selectin was significantly greater in the glomeruli of infected dogs than in control dogs. In all patterns of glomerulonephritis the expression of ICAM-1 ranged from minimum to moderately severe and P-selectin from absent to severe. In the control animals the expression of these molecules ranged from absent to medium intensity. It was not observed any correlation between severity of the disease and these markers. There was a correlation between the number of Leishmania antigen positive cells and CD4+ T cells, and between the number of CD4+ T cells and CD8+ T cells. In dogs presenting different histopathological patterns of glomerulonephritis, parameters of proliferation and apoptosis were studied. Ki-67, a proliferative marker, was not detected locally, but fewer apoptotic cells and lower TNF-alpha expression were seen in infected animals than in non-infected controls. Conclusion: Immunopathogenic mechanisms of VL glomerulonephritis are complex and data in the present study suggest no clear participation of immunoglobulin and C3b deposits in these dogs but the possible migration of CD4+ T cells into the glomeruli, participation of adhesion molecules, and diminished apoptosis of cells contributing to determine the proliferative pattern of glomerulonephritis in VL.

Effects of Aflatoxin B(1) and Fumonisin B(1) on the Viability and Induction of Apoptosis in Rat Primary Hepatocytes

The present study evaluated the effect of aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)) either alone, or in association, on rat primary hepatocyte cultures. Cell viability was assessed by flow cytometry after propidium iodine intercalation. DNA fragmentation and apoptosis were assessed by agarose gel electrophoresis and acridine orange and ethidium bromide staining. At the concentrations of AFB(1) and FB(1) used, the toxins did not decrease cell viability, but did induce apoptosis in a concentration and time-dependent manner.

Ameloblast apoptosis and IGF-1 receptor expression in the continuously erupting rat incisor model

Enamel-producing cells (ameloblasts) pass through several phenotypic and functional stages during enamel formation. In the transition between secretory and maturation stages, about one quarter of the ameloblasts suddenly undergo apoptosis. We have studied this phenomenon using the continuously erupting rat incisor model. A special feature of this model is that all stages of ameloblast differentiation are presented within a single longitudinal section of the developing tooth. This permits investigation of the temporal sequence of gene and growth factor receptor expression during ameloblast differentiation and apoptosis. We describe the light and electron microscopic morphology of ameloblast apoptosis and the pattern of insulin-like growth factor-1 receptor expression by ameloblasts in the continuously erupting rat incisor model. In the developing rat incisor, ameloblast apoptosis is associated with downregulated expression of the insulin-like growth factor-1 receptor. These data are consistent with the hypothesis that ameloblasts are hard wired for apoptosis and that insulin-like growth factor-1 receptor expression is required to block the default apoptotic pathway. Possible mechanisms of insulin-like growth factor-1 inhibition of ameloblast apoptosis are presented. The rat incisor model may be useful in studies of physiological apoptosis as it presents apoptosis in a predictable pattern in adult tissues.

Serum from children with polyarticular juvenile idiopathic arthritis (pJIA) inhibits differentiation, mineralization and may increase apoptosis of human osteoblasts ""in vitro""

We examined the effects of polyarticular juvenile idiopathic arthritis (pJIA) serum on proliferation, differentiation, mineralization, and apoptosis of human osteoblast cells (hOb) in culture. The hOb were cultured with 10% serum from active pJIA and healthy controls (CT) and were tested for DNA synthesis, alkaline phosphatase (AP) activity, osteocalcin (OC) secretion, calcium levels, caspase 3 activity, and DNA fragmentation. None of the patients had used glucocorticoids for at least 1 month before the study, or any other drug that can affect bone mineral metabolism. Human inflammatory cytokine levels (IL-6, IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70) were measured in pJIA and CT sera. Low levels of AP activity was observed in pJIA cultures compared with CT cultures (67.16 +/- 53.35 vs 100.11 +/- 50.64 mu mol p-nitrophenol/h(-1) mg(-1) protein, P=0.008). There was also a significant decrease in OC secretion (9.23 +/- 5.63 vs 12.82 +/- 7.02 ng/mg protein, P=0.012) and calcium levels (0.475 +/- 0.197 vs 0.717 +/- 0.366 mmol/l, P=0.05) in pJIA hOb cultures. No difference was observed in cell proliferation (323.56 +/- 108.23 vs 328.91 +/- 88.03 dpm/mg protein, P=0.788). Osteoblasts cultured with JIA sera showed lower levels of DNA and increased fragmentation than osteoblasts cultured with CT sera. pJIA sera showed higher IL-6 values than CT (21.44 +/- 9.31 vs 3.58 +/- 2.38 pg/ml, P<0.001), but no difference was observed related to IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70 between pJIA and controls. This study suggests that serum from children with pJIA inhibits differentiation, mineralization and may increase apoptosis of hOb cultures, and inflammatory cytokines such as IL-6 might be a mechanism in this find. These results may represent an alternative therapeutic target for prevention and treatment of bone loss in JIA.

Expression of genes related to apoptosis, cell cycle and signaling pathways are independent of TP53 status in urinary bladder cancer cells

Urinary bladder cancer is the fourth most common malignancy in the Western world. Transitional cell carcinoma (TCC) is the most common subtype, accounting for about 90% of all bladder cancers. The TP53 gene plays an essential role in the regulation of the cell cycle and apoptosis and therefore contributes to cellular transformation and malignancy; however, little is known about the differential gene expression patterns in human tumors that present with the wild-type or mutated TP53 gene. Therefore, because gene profiling can provide new insights into the molecular biology of bladder cancer, the present study aimed to compare the molecular profiles of bladder cancer cell lines with different TP53 alleles, including the wild type (RT4) and two mutants (5637, with mutations in codons 280 and 72; and T24, a TP53 allele encoding an in-frame deletion of tyrosine 126). Unsupervised hierarchical clustering and gene networks were constructed based on data generated by cDNA microarrays using mRNA from the three cell lines. Differentially expressed genes related to the cell cycle, cell division, cell death, and cell proliferation were observed in the three cell lines. However, the cDNA microarray data did not cluster cell lines based on their TP53 allele. The gene profiles of the RT4 cells were more similar to those of T24 than to those of the 5637 cells. While the deregulation of both the cell cycle and the apoptotic pathways was particularly related to TCC, these alterations were not associated with the TP53 status.

Pfaffia paniculata (Brazilian ginseng) roots decrease proliferation and increase apoptosis but do not affect cell communication in murine hepatocarcinogenesis

Pfaffia paniculata (Brazilian ginseng) roots and/or its extracts have shown anti-neoplastic, chemopreventive, and anti-angiogenic properties. The aim of this work was to investigate the chemopreventive mechanisms of this root in Mice Submitted to the infant model of hepatocarcinogenesis, evaluating the effects oil cellular proliferation, apoptosis. and intercellular communication. Fifteen-day-old BALB/c male mice were given, i.p., 10 mu g/g of the carcinogen N-nitrosodiethylamine (DEN). Animals were separated into three groups at weaning and were given different concentrations of powdered P. paniculata root (0%, 2%, or 10%) added to commercial food for 27 weeks. Control group (CT) was not exposed to the carcinogen and was given ration without the root. After euthanasia, the animals` liver and body weight were measured. Liver fragments were sampled to Study intercellular communication, molecular biology, and histopathological analysis. Cellular proliferation was evaluated by immunohistochemistry for PCNA, apoptosis was evaluated by apoptotic bodies count and alkaline cornet technique, and inter-cellular communication by diffusion of lucifer yellow dye, immunofluorescence, western blot and real-time PCR for connexins 26 and 32. Chronic treatment with powdered P. paniculata root reduced cellular proliferation and increased apoptosis in the 2%, group. Animals in the 10% group had an increase in apoptosis with chronic inflammatory process. Intercellular communication showed no alterations in any of the groups analyzed. These results Indicate that chemopreventive effects of P. paniculata are related to the control of cellular proliferation and apoptosis, but not to cell communication and/or connexin expression, and are directly Influenced by the root concentration. (C) 2009 Elsevier GmbH. All rights reserved.

Early pregnancy factor suppresses the infiltration of lymphocytes and macrophages in the spinal cord of rats during experimental autoimmune encephalomyelitis but has no effect on apoptosis

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vbeta8.2(+) cells. In the present study, T cell, macrophage (CD11b/c(+)) and B cell (CD45RA(+)) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1 x 100 mug nor 2 x 100 mug doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5(+) and CD11b/c(+) cells. Treatment with 2 x 100 mug/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5(+), CD11b/c(+) and CD45RA(+) cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10-14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5(+) T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking. (C) 2003 Elsevier B.V. All rights reserved.

Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.