Benzoic acid and specific 2-oxo acids activate hepatic efflux of glutamate at OAT2

The liver is the principal source of glutamate in blood plasma. Recently we have discovered that efflux of glutamate from hepatocytes is catalyzed by the transporter OAT2 (human gene symbol SLC22A7). Organic anion transporter 2 (OAT2) is an integral membrane protein of the sinusoidal membrane domain; it is primarily expressed in liver and much less in kidney, both in rats and humans. Many years ago, Häussinger and coworkers have demonstrated in isolated perfused rat liver that benzoic acid or specific 2-oxo acid analogs of amino acids like e.g. 2-oxo-4-methyl-pentanoate (‘2-oxo-leucine’) strongly stimulate release of glutamate (up to 7-fold); ‘2-oxo-valine’ and the corresponding amino acids were without effect. The molecular mechanism of efflux stimulation has remained unclear. In the present study, OAT2 from human and rat were heterologously expressed in 293 cells. Addition of 1 mmol/l benzoic acid to the external medium increased OAT2-specific efflux of glutamate up to 20-fold; ‘2-oxo-leucine’ was also effective, but not ‘2-oxo-valine’. Similar effects were seen for efflux of radiolabeled orotic acid. Expression of OAT2 did not increase uptake of benzoic acid; thus, benzoic acid is no substrate, and trans-stimulation can be excluded. Instead, further experiments suggest that increased efflux of glutamate is caused by direct interaction of benzoic acid and specific 2-oxo acids with OAT2. We propose that stimulators bind to a distinct extracellular site and thereby accelerate relocation of the empty substrate binding site to the intracellular face. Increased glutamate efflux at OAT2 could be the main benefit of benzoate treatment in patients with urea cycle defects.     

Loss of TRAIL-Receptors Is a Recurrent Feature in Pancreatic Cancer and Determines the Prognosis of Patients with No Nodal Metastasis after Surgery

Introduction

Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.

Aims and Methods

Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.

Results

The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.

Conclusion

This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.     

Case-control study of birth characteristics and the risk of hepatoblastoma

Background: Hepatoblastoma is a malignant embryonal tumor typically diagnosed in children younger than five years of age. Little is known on hepatoblastoma etiology. Methods: We matched California Cancer Registry records of hepatoblastomas diagnosed in children younger than age 6 from 1988 to 2007 to birth records using a probabilistic record linkage program, yielding 261 cases. Controls (n = 218,277), frequency matched by birth year to all cancer cases in California for the same time period, were randomly selected from California birth records. We examined demographic and socioeconomic information, birth characteristics, pregnancy history, complications in pregnancy, labor and delivery, and abnormal conditions and clinical procedures relating to the newborn, with study data taken from birth certificates. Results: We observed increased risks for hepatoblastoma among children with low [1500–2499 g, Odds Ratio (OR) = 2.02, 95% confidence interval (CI) 1.29–3.15] and very low birthweight (<1500 g, OR = 15.4, 95% CI 10.7–22.3), preterm birth <33 weeks (OR = 7.27, 95% CI 5.00, 10.6), small size for gestational age (OR = 1.75, 95% CI 1.25–2.45), and with multiple birth pregnancies (OR = 2.52, 95% CI 1.54–4.14). We observed a number of pregnancy and labor complications to be related to hepatoblastoma, including preeclampsia, premature labor, fetal distress, and congenital anomalies. Conclusion: These findings confirm previously reported associations with low birthweight and preeclampsia. The relation with multiple birth pregnancies has been previously reported and may indicate a relation to infertility treatments.     

Sporadic Retinoblastoma and Parental Smoking and Alcohol Consumption before and after Conception: A Report from the Children’s Oncology Group

Background

Retinoblastoma is the most frequent tumor of the eye in children and very little is known about the etiology of non-familial (sporadic) retinoblastoma. In this study we examined whether parental tobacco smoking or alcohol consumption (pre- or post-conception) contribute to the two phenotypes (bilateral or unilateral) of sporadic retinoblastoma.

Methods

Two large multicenter case-control studies identified 488 cases through eye referral centers in the United States and Canada or through the Children’s Oncology Group. Controls (n = 424) were selected from among friends and relatives of cases and matched by age. Risk factor information was obtained via telephone interview. We employed multivariable logistic regression to estimate the effects of parental tobacco smoking and alcohol consumption on retinoblastoma.

Findings

Maternal smoking before and during pregnancy contributed to unilateral retinoblastoma risk in the child: year before pregnancy conditional Odds Ratio (OR), 8.9; 95% confidence interval (CI) 1.5–51, and unconditional OR, 2.4; 95% CI, 1.3–4.7; month before or during pregnancy, conditional OR, 3.3; 95% CI, 0.5–20.8, and unconditional OR, 2.8; 95% CI, 1.1–7.0. No association was found for maternal or paternal alcohol consumption.

Conclusion

The results of this study indicate that maternal active smoking during pregnancy may be a risk factor for sporadic retinoblastoma. Our study supports a role for tobacco exposures in embryonal tumors.     

Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.     

Simple machine-assisted protocol for solid-phase synthesis of depsipeptides

A straightforward machine-assisted protocol for the synthesis of linear depsipeptides is reported. The synthesis was performed on a 433A Peptide Synthesizer (Applied Biosystems) using preprogrammed and optimized modules for Boc chemistry and without any need for hardware modification. The robustness of the protocol was demonstrated with 12 examples of 26-membered depsipeptides with single and multiple (up to 6) ester backbone substitutions.     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.