Interactions between quercetin and Warfarin for albumin binding: A new eye on food/drug interference

The interaction between quercetin, a popular antioxidant flavonoid, and human serum albumin (HSA) is investigated and characterized by means of induced circular dichroism and saturation transfer difference NMR. These techiques demonstrate the reversible binding of quercetin to the carrier protein, which is responsible for its dissolution in aqueous medium. Competition experiments with two classical probes for HSA binding sites, namely Ibuprofen and Warfarin (a common anticoagulant coumarin), demonstrate that quercetin has a primary binding site located in the subdomain IIA, where coumarins are hosted. The affinity for this site is large and we found that quercetin may effectively displace warfarin from HSA. This may have relevant consequences in rationalizing the interferences of common dietary compounds and food supplements to anticoagulant treatments. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Hypertension caused by prenatal testosterone excess in female sheep

Polycystic ovary syndrome (PCOS), a leading cause of infertility, affects approximately 10% of women of reproductive age. The etiology and pathophysiology of PCOS are poorly understood. PCOS is multifaceted and includes reproductive abnormalities and components of the metabolic syndrome such as insulin resistance, obesity, dyslipidemia, and hypertension. Exposure to excess testosterone (T) during the prenatal period may predispose individuals to PCOS phenotype. The goal of this study was to determine whether hypertension and dyslipidemia occur in a well-characterized model of PCOS produced by prenatal treatment of sheep with T. Radiotelemetry was used to measure blood pressure over a 24-h period in conscious, undisturbed animals. To normalize circulating estradiol levels across treatment, control (n = 4) and prenatal T-treated (100 mg T propionate im twice weekly from days 30 to 90 of fetal life, n = 4) 2-yr-old females were ovariectomized, instrumented with a radiotelemetry transmitter, and clamped with early follicular phase levels of estrogen using an implant. Six days later, a 24-h recording period commenced. Prenatal T-treated sheep were hypertensive compared with control sheep, and heart rate tended to be higher. T-treated sheep had hyperglycemia, insulin resistance, hypernatremia, and hyperchloremia, and both total and LDL cholesterol tended to be higher. Plasma aldosterone and epinephrine were significantly lower in T-treated sheep, whereas norepinephrine was unchanged. This first-ever use of radiotelemetric blood pressure recordings in sheep demonstrates that mild hypertension, a risk factor reported in some women with PCOS, is also a feature of the sheep model of PCOS produced by prenatal T treatment.     

Iron does not cause arrhythmias in the guinea pig model of transfusional iron overload

Cardiac events, including heart failure and arrhythmias, are the leading cause of death in patients with beta thalassemia. Although cardiac arrhythmias in humans are believed to result from iron overload, excluding confounding factors in the human population is difficult. The goal of the current study was to determine whether cardiac arrhythmias occurred in the guinea pig model of secondary iron overload. Electrocardiograms were recorded by using surgically implanted telemetry devices in guinea pigs loaded intraperitoneally with iron dextran (test animals) or dextran alone (controls). Loading occurred over approximately 6 wk. Electrocardiograms were recorded for 1 wk prior to loading, throughout loading, and for approximately 4 wk after loading was complete. Cardiac and liver iron concentrations were significantly increased in the iron-loaded animals compared with controls and were in the range of those reported for humans with thalassemia. Arrhythmias were rare in both iron-loaded and control guinea pigs. No life-threatening arrhythmias were detected in either group. These data suggest that iron alone may be insufficient to cause cardiac arrhythmias in the iron-loaded guinea pig model and that arrhythmias detected in human patients with iron overload may be the result of a complex interplay of factors.     

Mesenchymal differentiation propensity of a human embryonic stem cell line

Objectives: To characterize basal differentiation tendencies of a human embryonic stem (hES) cell line, KCL‐002. Materials and methods: In vitro specification and differentiation of hES cells were carried out using embryoid body (EB) cultures and tests of pluripotency and in vivo differentiation were performed by teratoma assays in SCID mice. Real‐time PCR, immunohistochemistry, flow cytometry and histological analyses were used to identify expression of genes and proteins associated with the ectodermal, endodermal and mesodermal germ layers. Results: Undifferentiated KCL‐002 cells expressed characteristic markers of pluripotent stem cells such as Nanog, Sox‐2, Oct‐4 and TRA 1‐60. When differentiated in vitro as EB cultures, expression of pluripotency, endodermal and ectodermal markers decreased rapidly. In contrast, mesodermal and mesenchymal markers such as VEGFR‐2, α‐actin and vimentin increased during EB differentiation as shown by qPCR, immunostaining and flow cytometric analyses. Teratoma formation in SCID mice demonstrated the potential to form all germ layers in vivo with a greater proportion of the tumours containing mesenchymal derivatives. Conclusions: The data presented suggest that the KCL‐002 hES cell line is pluripotent and harbours a bias in basal differentiation tendencies towards mesodermal and mesenchymal lineage cells. Characterizing innate differentiation propensities of hES cell lines is important for understanding heterogeneity between different cell lines and for further studies aimed at deriving specific lineages from hES cells.     

Clinical and molecular characterization of five patients with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inborn error of ketone body metabolism and causes episodic ketoacidosis. We report clinical and molecular analyses of 5 patients with SCOT deficiency. Patients GS07, GS13, and GS14 are homozygotes of S405P, L327P, and R468C, respectively. GS17 and GS18 are compound heterozygotes for S226N and A215V, and V404F and E273X, respectively. These mutations have not been reported previously. Missense mutations were further characterized by transient expression analysis of mutant cDNAs. Among 6 missense mutations, mutants L327P, R468C, and A215V retained some residual activities and their mutant proteins were detected in immunoblot analysis following expression at 37 °C. They were more stable at 30 °C than 37 °C, indicating their temperature sensitive character. The R468C mutant is a distinct temperature sensitive mutant which retained 12% and 51% of wild-type residual activities at 37 and 30 °C, respectively. The S226N mutant protein was detected but retained no residual activity. Effects of missense mutations were predicted from the tertiary structure of the SCOT molecule. Main effects of these mutations were destabilization of SCOT molecules, and some of them also affected catalytic activity. Among 5 patients, GS07 and GS18 had null mutations in both alleles and the other three patients retained some residual SCOT activities. All 5 developed a first severe ketoacidotic crisis with blood gas pH < 7.1, and experienced multiple ketoacidotic decompensations (two of them had seven such episodes). In general, the outcome was good even following multiple ketoacidotic events. Permanent ketosis or ketonuria is considered a pathognomonic feature of SCOT deficiency. However, this condition depends not only on residual activity but also on environmental factors.     

Patients' direct experiences as central elements of placebo analgesia

Placebo analgesic effects appear to be related to patients' perception of the therapeutic intervention. In this paper, we review quantitative findings of how the relationship with the physician and the verbal suggestions given for relief may influence patients' perception of a treatment and how patients' expectations and emotional feelings may affect treatment outcome. We also present qualitative data from interviews with patients who have experienced pain relief following a placebo or an active treatment. A special focus is given to the temporal development of placebo analgesia at psychological and neurophysiological levels. Finally, we discuss the extent to which the quantitative and qualitative findings supplement or contrast with each other, and we touch upon possible implications of patients' direct experience as central for placebo analgesia.     

The structural basis for the function of two anti-VEGF receptor 2 antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.     

Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrP(Sc) in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrP(Sc) in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrP(Sc) in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrP(Sc) was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrP(Sc) to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.     

Diazoxide preserves hypercapnia-induced arteriolar vasodilation after global cerebral ischemia in piglets

Diazoxide (Diaz), an activator of mitochondrial ATP-sensitive K+ (mitoKATP) channels, is neuroprotective, but the mechanism of action is unclear. We tested whether Diaz preserves endothelium-dependent (hypercapnia) or -independent [iloprost (Ilo)] cerebrovascular dilator responses after ischemia-reperfusion (I/R) in newborn pigs and whether the effect of Diaz is sensitive to 5-hydroxydecanoate (5-HD), an inhibitor of mitoKATP channels. Anesthetized, ventilated piglets (n = 48) were equipped with closed cranial windows. Changes in diameter of pial arterioles were determined with intravital microscopy in response to graded hypercapnia (5-10% CO2 - 21% O2-balance N2, n = 25) or Ilo (0.1-1 microg/ml, n = 18) before and 1 h after 10 min of global I/R. Experimental groups were pretreated with vehicle, NS-398 (a selective cyclooxygenase-2 inhibitor, 1 mg/kg), Diaz (3 mg/kg), or 5-HD (20 mg/kg) + Diaz. Potential direct effects of Diaz and 5-HD on hypercapnic vasodilation were also tested in the absence of I/R (n = 5). To confirm the direct effect of Diaz on mitochondria, mitochondrial membrane potential in cultured piglet cerebrovascular endothelial cells was monitored using Mito Tracker Red. Hypercapnia resulted in dose-dependent pial arteriolar vasodilation, which was attenuated by approximately 70% after I/R in vehicle- and NS-398-treated animals. Diaz and 5-HD did not affect the CO2 response. Diaz significantly preserved the postischemic vasodilation response to hypercapnia, but not to Ilo. Diaz depolarized mitochondria in cultured piglet cerebrovascular endothelial cells, and 5-HD completely abolished the protective effect of Diaz, both findings indicate a role for mitoKATP channels. In summary, preservation of arteriolar dilator responsiveness by Diaz may contribute to neuroprotection.