Prenatal exposure to medicines and the risk of childhood brain tumor

Background: Childhood brain tumors are associated with high mortality and morbidity but little is known about its causes. About half of women use medicines when pregnant and some of the medicines commonly used might be carcinogenic. Objective: The aim with this population-based case–control study was to analyze associations between specific groups of medicines taken during pregnancy and the risk of brain tumor in the offspring. Methods: All children, up to 15 years of age, born in Sweden between 1975 and 1984 were eligible for the study. Cases (N = 512) were children diagnosed with brain tumor and controls (N = 525) were randomly selected from the Medical Birth Register. Exposure data on medicines was extracted blindly from antenatal medical records and grouped according to Anatomical Therapeutic Chemical (ATC) code. Information on maternal reproductive history was received from the Medical Birth Register. We used logistic regression to estimate associations between fetal exposure to medicines and childhood brain tumor. Results: No significant changes in risk were noted after exposure to iron supplementation, antiemetics, analgesics, antibiotics or any other main ATC group. A tendency of protective effect was seen for prenatal exposure to folic acid (adjusted OR 0.6, 95% CI 0.3–1.1). Ten children with a diagnosis of brain tumor had been exposed to β-blocking agents in fetal life as compared to two children without brain tumor (adjusted OR 5.3, 95% CI 1.2–24.8). Conclusions: In this case–control study, an increased risk of brain tumor was seen in children exposed to β-blocking agents during fetal life. However, due to the low number of exposed the interpretation of this finding should be made with caution.     

New method for toxicity assessment in marine and brackish environments using the macroalga Gracilaria tenuistipitata (Gracilariales,Rhodophyta)

A growth inhibition test method was developed using the macroalga Gracilaria tenuistipitata as the test organism. This alga was chosen because of its high laboratory growth rates, commonly 30–40% d^–1, which are reached in salinities between 5 and 40, and its epiphyte resistance. The toxicity of a number of substances, including heavy metals, herbicides and complex wastewaters towards the alga was assayed. Anti-fouling paints were tested with a modification of the method. EC50 values for heavy metals varied between 0.05 and 17 mg l^–1 and for herbicides between 0.002 and 0.02 mg l^–l. The sensitivity to the toxicant was generally higher at low salinity. Omitting nitrogen and phosphorus additions to the test medium increased the sensitivity and a semi-static performance was possible with maintained or increased sensitivity. Preliminary tests done with a computerised photosynthesis inhibition method produced promising results.In conclusion, this is a simple, sensitive and reproducible test method for assessing the toxicity of substances, wastewaters and anti-fouling paints in brackish and marine environments.     

Comparative proteomics of industrial lager yeast reveals differential expression of the cerevisiae and non-cerevisiae parts of their genomes

The proteomes of three industrial lager beer strains, CMBS33, OG2252 and A15, were analysed under standardised laboratory growth conditions. Protein spots in the 2-DE pattern of the lager strains were subjected to MS/MS to identify protein variants. We found the protein composition of the three lager strains to be qualitatively rather similar, while being substantially different from the Saccharomyces cerevisiae strain BY4742. Database searches using several fully sequenced genomes from the Saccharomyces genera indicated that the non-cerevisiae proteins in the 2-D pattern of lager strains were most closely related to S. bayanus. For many proteins the regulation of the bayanus-like protein and its cerevisiae counterpart varied in a strain-dependent manner, e.g. the bayanus-like form of Tdh3p was roughly eight-fold more abundant than the cerevisiae form in the OG2252 strain. We also found differential regulation of cerevisiae- and bayanus-like proteins during various stress conditions like low temperature growth, and adaptation to high temperatures or high salinity, e.g. for Arg1p, Sti1p and Pdc1p. Our data on the differential regulation of the two genomes in these hybrid strains may have important industrial implications for strain improvement and strain protection.     

N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world''s three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.Glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans, that cover all human cells. Around 1% of the human genome participates in the biosynthesis of glycans(1). This biosynthesis is the most complex post-translational modification of proteins, and the great variability in glycan structures contains a tremendous ability to fine-tune the chemical and biological properties of glycoproteins. The glycosylation process occurs most abundantly in the Golgi apparatus and the endoplasmic reticulum, but also occurs in the cytoplasm and nucleus (2). Most glycoconjugates are localized to cell surfaces, where glycans participate in events essential for cell viability and function, such as cell adhesion, motility, and intracellular signaling (2). Changes in these functions are key steps seen when normal cells transform to malignant ones, and these are also reflected in changes of a cell''s glycan profile, observed in many cancers (3, 4). Specific structural changes in glycans may serve as cancer biomarkers (5, 6), and changes in glycosylation profiles are related to aggressive behavior in tumor cells (7–9).Cancer-associated asparagine-linked glycan (N-glycan) structures may play specific roles in supporting tumor progression; growth (10, 11), invasion (12, 13), and angiogenesis (14). Changes in the N-glycan profile emerge in numerous cancers, including lung (15, 16), breast (17), and colorectal cancer (CRC)¹ (16, 18). Balog et al. (18) comparing the N-glycomic profile of CRC tissue to adjacent normal mucosa, reported differences in specific glycan structures. Moreover, serum N-glycosylation profile from patients with CRC differ from those of healthy controls (19).Colorectal cancer is the third most common cause of cancer-related death worldwide and its incidence is rising; 40% of CRCs are of rectal origin. Roughly 40% of patients have localized disease (stage I–II; Dukes A–B), another 40% loco regional disease (stage III; Dukes C), and 20% metastasized disease (stage IV; Dukes D) (20). Although stage at diagnosis is the most important factor determining prognosis, clinical outcome, and response to adjuvant treatment can markedly vary within each stage. Adjuvant therapy routinely goes to stage III patients, but the benefit of adjuvant treatment for stage II patients is unclear. Of stage II patients, 80% are cured by radical surgery alone. To identify patients who will benefit from postoperative treatment, we need novel biomarkers. The glycan profile of the tumor tissue could provide new biomarkers for diagnosis and prognosis of cancer.In this study, we characterized the N-glycomic profiles of rectal adenomas and carcinomas by MALDI-TOF mass spectrometric (MS) profiling of asparagine-linked glycans. Our aim was to identify differences between adenomas and carcinomas, and also between cancers of different stages. Based on glycan profiling, we also chose, for immunohistochemical expression studies of a series of 220 CRC patients, two glycan markers: sialyl Lewis a and pauci-mannose.     

Serotonin 5-HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confocal fluorescence resonance energy transfer

Dimerization is a common property of G-protein-coupled receptors (GPCR). While the formation of GPCR dimers/oligomers has been reported to play important roles in regulating receptor expression, ligand binding, and second messenger activation, less is known about how and where GPCR dimerization occurs. The present study was performed to identify the precise cellular compartment in which class A GPCR dimer/oligomer biogenesis occurs. We addressed this issue using confocal microscopy and fluorescence resonance energy transfer (FRET) to monitor GPCR proximity within discrete intracellular compartments of intact living cells. Time-lapse confocal imaging was used to follow CFP- and YFP-tagged serotonin 5-HT2C receptors during biosynthesis in the endoplasmic reticulum (ER), trafficking through the Golgi apparatus and subsequent expression on the plasma membrane. Real-time monitoring of FRET between CFP- and YFP-tagged 5-HT2C receptors was performed by acceptor photobleaching within discrete regions of the ER, Golgi, and plasma membrane. The FRET signal was dependent on the ratio of CFP- to YFP-tagged 5-HT2C receptors expressed in each region and was independent of receptor expression level, as predicted for proteins in a non-random, clustered distribution. FRET efficiencies measured in the ER, Golgi, and plasma membrane were similar. These experiments provide direct evidence for homodimerization/oligomerization of class A GPCR in the ER and Golgi of intact living cells, and suggest that dimer/oligomer formation is a naturally occurring step in 5-HT2C receptor maturation and processing.     

Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes

We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin-remazol-brilliant violet) and a small, presumably amorphous, subfraction of alpha-chitin and beta-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472-484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (- 2 to + 2), as opposed to six (- 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study.     

Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.