Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC₅₀ measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin''s cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.     

Serum and Lymphocytic Neurotrophins Profiles in Systemic Lupus Erythematosus: a Case-Control Study

Background

Neurotrophins play a central role in the development and maintenance of the nervous system. However, neurotrophins can also modulate B and T cell proliferation and activation, especially via autocrine loops. We hypothesized that both serum and lymphocytic neurotrophin levels may be deregulated in systemic Lupus erythematosus (SLE) and may reflect clinical symptoms of the disease.

Methods

Neurotrophins in the serum (ELISA tests) and lymphocytes (flow cytometry) were measured in 26 SLE patients and 26 control subjects. Th1 (interferon-γ) and Th2 (IL-10) profiles and serum concentration of BAFF were assessed by ELISA in the SLE and control subjects.

Findings

We have demonstrated that both NGF and BDNF serum levels are higher in SLE patients than healthy controls (p=0.003 and p<0.001), independently of Th1 or Th2 profiles. Enhanced serum NT-3 levels (p=0.003) were only found in severe lupus flares (i.e. SLEDAI ≥ 10) and significantly correlated with complement activation (decreased CH 50, Γ=-0.28, p=0.03). Furthermore, there was a negative correlation between serum NGF levels and the number of circulating T regulatory cells (Γ=0.48, p=0.01). In circulating B cells, production of both NGF and BDNF was greater in SLE patients than in healthy controls. In particular, the number of NGF-secreting B cells correlated with decreased complement levels (p=0.05). One month after SLE flare treatment, BDNF levels decreased; in contrast, NGF and NT-3 levels remained unchanged.

Conclusion

This study demonstrates that serum and B cell levels of both NGF and BDNF are increased in SLE, suggesting that the neurotrophin production pathway is deregulated in this disease. These results must be confirmed in a larger study with naive SLE patients, in order to avoid the potential confounding influence of prior immune-modulating treatments on neurotrophin levels.     

MCAK associates with the tips of polymerizing microtubules

MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.     

Kar3 interaction with Cik1 alters motor structure and function

Kar3, a kinesin-14 motor of Saccharomyces cerevisiae required for mitosis and karyogamy, reportedly interacts with Cik1, a nonmotor protein, via its central, predicted coiled coil. Despite this, neither Kar3 nor Cik1 homodimers have been observed in vivo. Here we show that Kar3 is a dimer in vitro by analytical ultracentrifugation. The motor domains appear as paired particles by rotary-shadow electron microscopy (EM) and circular dichroism (CD) spectroscopy of the nonmotor region shows characteristics of helical structure, typical of coiled coils. Remarkably, the Kar3/Cik1 nonmotor region shows greater helicity by CD analysis and rotary-shadow EM reveals a stalk joined to one large or two smaller particles. The highly helical Kar3/Cik1 nonmotor region and visible stalk indicate that dimerization with Cik1 causes structural changes in Kar3. The Cik1 and Kar3 stalk regions preferentially associate with one another rather than forming homodimers. Kar3/Cik1 moves on microtubules at 2-2.4 microm min(-1), 2-5-fold faster than Kar3, and destabilizes microtubules at the lagging ends. Thus, structural changes in Kar3 upon dimerization with Cik1 alter the motor velocity and likely regulate Kar3 activity in vivo.     

Hematological changes in Florida pompano (Trachinotus carolinus) supplemented with β-glucan and Pediococcus acidilactici synbiotic

Florida pompano (Trachinotus carolinus) are a species of growing interest for commercial aquaculture. Effective health monitoring is crucial to the successful growout of the species, and prophylactic and therapeutic use of chemicals and antibiotics has been the traditional strategy for promoting stock health. However, concerns about antimicrobial resistance, chemical residues in seafood products and the environment, and resultant immunosuppression have prompted the industry to identify alternative management strategies, including supplementation with prebiotics, probiotics, and combinations of both (synbiotics). The objectives of this study are to determine and compare hematological, plasma biochemical, and plasma protein electrophoresis data of synbiotic-supplemented (β-glucan and Pediococcus acidilactici) and non-supplemented Florida pompano. Reference intervals for blood analytes are provided for both groups and for subgroups (females, males, large, and small fish) where statistically significant results exist. There are no differences between the hematological and plasma biochemistry analytes between the supplemented and control groups, except for blood urea nitrogen and carbon dioxide, indicating a possible effect of synbiotic supplementation on gill function and osmoregulation. Sex-related and size-related differences are observed within each of the control and supplemented groups; however, biometric measurements do not strongly correlate with blood analytes. These data represent baseline hematological and plasma biochemical data in the Florida pompano and indicate the safety of synbiotic supplementation in this commercially important species. This study serves to further the commercialization of Florida pompano by providing blood analyte reference intervals for health monitoring in the aquaculture setting.     

Sensitivity to low-dose/low-LET ionizing radiation in mammalian cells harboring mutations in succinate dehydrogenase subunit C is governed by mitochondria-derived reactive oxygen species

It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O? metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O?(?U+2212)) and hydrogen peroxide (H?O?) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O??- and H?O?. When B9 cells were exposed to low-dose/low-LET radiation (5-50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5-50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O??- and H?O?. These results also support the hypothesis that mitochondrial O??- and H?O? originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.     

Synthesis and characterisation of a novel tubulin-directed DO3A-colchicine conjugate with potential theranostic features

Colchicine is a known tubulin binding agent enabling necrosis in tumors. A novel tubulin-directed DO3A-colchicine conjugate and its Gd(III) complex were prepared from N-deacetylcolchicine, coupling alkaloid and polyaza-alicyclic functions via a peptide coupling methodology. The longitudinal proton relaxivity of the Gd(III) complex in water at 4.7 T is 2.86 mM⁻¹ s⁻¹ and a similar efficacy as colchicine towards ovarian carcinoma cells in vitro.