Activin-like kinase receptor 1 (ALK1) in atherosclerotic lesions and vascular mesenchymal cells

OBJECTIVE: Activin-like kinase receptor 1 (ALK1) is a transforming growth factor (TGF)-beta type I receptor expressed in vascular mesenchyme, yet its function in vascular mesenchymal cells (VMC) is unclear. We examined ALK1 expression in human coronary atherosclerotic lesions and bovine and human VMC undergoing cellular condensation in vitro. We also examined the effect of activated ALK1 on cell proliferation and smooth muscle cell (SMC) differentiation. METHODS AND RESULTS: Our results showed that ALK1 was expressed in human coronary atherosclerotic lesions as determined by immunohistochemistry. ALK1 was also expressed in cellular condensations of bovine and human VMC as determined by real-time PCR and immunocytochemistry. Bone morphogenetic protein (BMP)-2, which is known to increase condensation size, increased ALK1 expression when induced from a BMP-2 adenoviral vector. In turn, activated ALK1 induced expression of matrix GLA protein (MGP), a BMP-2 inhibitor known to limit condensation size. Activated ALK1 enhanced proliferation of VMC as determined by 3H-thymidine incorporation, whereas MGP decreased proliferation. Activated ALK1 also enhanced expression of SMC lineage markers and ALK5, another TGF-beta type I receptor, as determined by immunoblotting, real-time PCR and immunocytochemistry. Anti-TGF-beta antibodies abolished expression of SMC markers in the presence of constitutively active ALK1, suggesting that ALK1 activation alone is not sufficient to promote SMC differentiation. CONCLUSIONS: We conclude that there is a balance between the actions of BMP-2 and MGP in the initiation of vascular mesenchymal cell condensation and SMC differentiation, and that targeting ALK1, BMP2 and/or MGP may lead to novel concepts of atherosclerosis treatment.     

The Y152X MC1R gene mutation: occurrence in ethnically diverse Jewish malignant melanoma patients

MC1R sequence variants are associated with malignant melanoma risk, and most commonly are missense mutations. Few (n=9) truncating mutations have been described in this gene as predisposing to malignant melanoma. In this study, three Jewish individuals were found to harbor an identical truncating MC1R mutation--Y152X: an Ashkenazi patient with two malignant melanomas, a non-Ashkenazi malignant melanoma patient with familial malignant melanoma and her asymptomatic mother. Both malignant melanoma patients carried additional, seemingly pathogenic MC1R variants. Haplotype analysis revealed that all three mutation carriers shared the same haplotype. This sequence variant was previously described in ethnically diverse, non-Jewish individuals and in all likelihood represents an error-prone domain that, in conjunction with other genetic and environmental factors, increases malignant melanoma risk.     

Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.     

Choleretic effects of the Mongolian medicinal plant Saussurea amara in the isolated perfused rat liver

Saussurea amara is used in traditional Mongolian medicine for the treatment of hepato-biliary disorders. To determine the plant's effect on the bile-salt independent bile flow (hydrocholeresis) as a measure of liver exocrine functions, different extracts were investigated in the isolated rat liver perfusion system. The methanolic extract (3) exerted a dose-dependent increase in bile flow (16%, 37%, 53%, 61%) in concentrations of 50 mg/L, 100 mg/L, 250 mg/L and 500 mg/L. The aqueous crude extract (1) and the ethyl acetate extract (2) also showed a dose-dependent increase, whereas at the highest concentrations (1000 mg/L and 100 mg/L, respectively) a continuous decrease in bile flow could be observed. Cynaropicrin also provoked a dose-dependent increase in bile flow, but caused liver damage at the highest dose tested (20 mg/L). Apigenin 7- O-glucoside, present in extracts 2 and 3, induced a dose-dependent increase of 20%, 30% and 40% (5 mg/L, 10 mg/L, 20 mg/L) and showed a significantly higher effect than the reference substance cynarin. The total flavonoid content was determined by spectrophotometry. To quantify the absolute amount of cynaropicrin in the crude drug and in the tested extracts, an HLPC system was established with santonin as internal standard.     

Automatic and controlled processes and the development of addictive behaviors in adolescents: a review and a model

This paper presents a review and a model of the development of addictive behaviors in (human) adolescents, with a focus on alcohol. The model proposes that addictive behaviors develop as the result of an imbalance between two systems: an appetitive, approach-oriented system that becomes sensitized with repeated alcohol use and a regulatory executive system that is not fully developed and that is compromised by exposure to alcohol. Self-regulation critically depends on two factors: ability and motivation to regulate the appetitive response tendency. The motivational aspect is often still weak in heavy drinking adolescents, who typically do not recognize their drinking as problematic. Motivation to regulate use often develops only years later, after the individual has encountered serious alcohol-related problems. Unfortunately, at that point behavioral change becomes harder due to several neurocognitive adaptations that result from heavy drinking. As we document, there is preliminary support for the central elements of the model (appetitive motivation vs. self-regulation), but there is a paucity of research directly addressing these mechanisms in human adolescents. Further, we emphasize that adolescent alcohol use primarily takes place in a social context, and that therefore studies should not solely focus on intra-individual factors predicting substance use and misuse but also on interpersonal social factors. Finally, we discuss implications of the model for interventions.     

Behavioral and biochemical evaluation of sub-lethal inhalation exposure to VX in rats

We evaluated the effects of low-level inhalation exposures (whole body, 60min duration) to the chemical warfare nerve agent VX (0.016, 0.15, 0.30 or 0.45mg/m(3)) in rats. The range of concentrations was approximately equivalent to 0.02-0.62 times 1.0 LC50. Biochemical effects were assessed by evaluating blood acetylcholinesterase (AChE) activity and by a regeneration assay that quantified the amount of VX (as the G analog) present in blood. Behavioral effects were assessed using a variable-interval 56-s schedule of reinforcement (VI56), in which rats were trained to press a lever to receive a food reward. VI56 training was established before exposure and evaluations continued after exposure. Additionally, after exposure, acquisition and maintenance of an eight-arm radial maze (RAM) task was evaluated in which rats learned to locate the four arms of the maze that presented a single food pellet reward. Behavioral assessments were conducted over approximately 3 months following exposure. Transient miosis was observed following exposure to all concentrations of VX and exposures to the 0.45mg/m(3) concentration also produced mild and temporary signs of toxicity (i.e., slight tremor and ataxia) in some subjects. All concentrations of VX also inhibited circulating AChE and the highest concentration inhibited AChE activity to less than 10% of pre-exposure values. Regenerated VX-G was found in red blood cell (RBC) and plasma blood fractions. In this respect, more VX-G was seen in plasma than RBC. Only small disruptions were observed on the VI56 or RAM following some VX exposures. In general, however, behavioral effects were minor and not clearly systematic. Taken together these results demonstrate that largely asymptomatic exposures to VX vapors in rats can produce substantial biochemical effects while having only minor performance effects on a previously learned behavioral task and on the acquisition of a new behavioral task.     

Influence of a ward-based pharmacist on the medication quality of geriatric inpatients: a before–after study

Background Despite several international studies demonstrating that ward-based pharmacists improve medication quality, ward pharmacists are not generally established in German hospitals. Aim We assessed the effect of a ward-based clinical pharmacist on the medication quality of geriatric inpatients in a German university hospital. Method The before-after study with a historic control group was conducted on the geriatric ward. During the control phase, patients received standard care without the involvement of a pharmacist. The intervention consisted of a clinical pharmacist providing pharmaceutical care from admission to discharge. Medication quality was measured on admission and discharge using the Medication Appropriateness Index (MAI). A linear regression analysis was conducted to calculate the influence of the intervention on the MAI. Results Patients in the intervention group (n?=?152, mean 83 years) were older and took more drugs at admission compared to the control group (n?=?159, 81 years). For both groups, the MAI per patient improved significantly from admission to discharge. Although the intervention did not influence the summated MAI score per patient, the intervention significantly reduced the MAI criteria Dosage (p?=?0.006), Correct Directions (p?=?0.016) and Practical Directions (p?=?0.004) as well as the proportion of overall inappropriate MAI ratings (at least 1 of 9 criteria inappropriate) (p?=?0.015). Conclusion Although medication quality was already high in the control group, a ward-based clinical pharmacist could contribute meaningfully to the medication quality on an acute geriatric ward.