In vivo processed fragments of IGF binding protein-2 copurified with bioactive IGF-II

Proteolysis of insulin-like growth factor binding proteins (IGFBPs), the major carrier of insulin-like growth factors (IGFs) in the circulation, is an essential mechanism to regulate the bioavailability and half-live of IGFs. Screening for peptides in human hemofiltrate, stimulating the survival of PC-12 cells, resulted in the isolation of C-terminal IGFBP-2 fragments and intact IGF-II co-eluting during the chromatographic purification procedure. The IGFBP-2 fragments exhibited molecular masses of 12.7 and 12.9kDa and started with Gly169 and Gly167, respectively. The fragments were able to bind both IGFs. The stimulatory effect of the purified fraction on the survival of the PC-12 cells could be assigned exclusively to IGF-II, since it was abolished by the addition of neutralizing IGF-II antibodies. We suggest that in the circulation IGF-II is not only complexed with intact IGFBP but also with processed IGFBP-2 fragments not impairing the biological activity of IGF-II.     

Modulation of MUC1 and blood group antigen expression in gastric adenocarcinoma cells by cytokines

Immunohistological studies demonstrated that MUC1 expression in gastric cancer is associated with a poor prognosis. As a mediator of cell-cell interactions, MUC1 may also be involved in metastasis. However, these aspects are of relevance since cytokine levels are locally increased as a consequence of peritumorous inflammatory response and coexisting chronic gastritis. Therefore we analyzed the potential influence of several cytokines on the expression of tumor-associated MUC1 and Lewis blood group antigens in gastric carcinoma cells. Gastric cancer cell lines AGS and KATOIII were incubated with the cytokines interleukin-1beta, interferon-gamma, tumor necrosis factor-alpha (TNF-alpha), and hepatocyte growth factor over a period of 72 h. Expressions of mucin antigens and cytokine secretion were measured by immunocytochemistry and/or enzyme-linked immunosorbent assay (ELISA). Analysis by fluorescence-activated cell sorter (FACS) demonstrated that MUC1 and sialyl Lewis A reactivities of AGS cells were increased significantly following TNF-alpha stimulation but not by other cytokines. Expression of mucin-associated antigens by cell line KATOIII was not affected by any of the employed cytokines. These data provide evidence that TNF-alpha can raise the expression of important mucin peptide as well as mucin-associated carbohydrate antigens and thereby potentially influence the progression of gastric carcinomas.     

Bioadhesion of supramolecular structures at supported planar bilayers as studied by the quartz crystal microbalance

A quartz crystal microbalance (QCM) was used to study the adhesion behavior of supramolecular aggregates at supported planar bilayers (SPBs). The QCM technique is a suitable method to detect the adsorption of biomolecules at the quartz surface owing to its sensitivity for changes in mass and viscoelastic properties. To simulate biomembranes, the quartz plates were coated with highly ordered lipid films. Therefore, a combination of self-assembled monolayers and Langmuir-Blodgett films was used. Firstly, the adsorption of liposomes coupled with the lectin concanavalin A was investigated at glycolipid-containing model membranes. Using different carbohydrates, it was possible to determine specific and nonspecific parts of the interactions. The adhesion occurred owing to specific lectin-carbohydrate interactions (about 20%) and to nonspecific interactions (about 80%). The composition of the liposomes was changed to simulate the structure of a native biomembrane consisting of the glycocalix, the lipid-protein bilayer, and the cytoskeleton. An artificial glycocalix was created by incorporating poly(ethylene glycol) into the liposomes. Liposomes which were intravesicular polymerized with polyacrylamide or polyacrylcholate simulated the cytoskeleton. It was determined that the modified liposomes had significant lower interactions with SPBs. The adsorption was reduced by approximately 80% compared to unmodified liposomes. Secondly, a model was developed for the detection of interactions between simple or mixed bile salt micelles and model membranes. It was found that simple bile salts did not adsorb at model membranes. Binary systems consisting of bile salt and phospholipid induced only small interactions. On the other hand, ternary systems consisting of bile salt, phospholipid, and fatty acid showed strong interactions. A dependence on the chain length of the fatty acid was observed. Thirdly, the interaction between ganglioside-containing model membranes and cholera toxin (beta-subunit) was investigated. Different ganglioside fractions showed varying adsorption in the following sequence: GM1 > GD1a > GD1b > GT1b.     

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.     

Atherosclerotic Plaque Destabilization in Mice: A Comparative Study

Atherosclerosis-associated diseases are the main cause of mortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions that may become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animal models mimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models.     

Enhancing single-molecule fluorescence with nanophotonics

Single-molecule fluorescence spectroscopy has become an important research tool in the life sciences but a number of limitations hinder the widespread use as a standard technique. The limited dynamic concentration range is one of the major hurdles. Recent developments in the nanophotonic field promise to alleviate these restrictions to an extent that even low affinity biomolecular interactions can be studied. After motivating the need for nanophotonics we introduce the basic concepts of nanophotonic devices such as zero mode waveguides and nanoantennas. We highlight current applications and the future potential of nanophotonic approaches when combined with biological systems and single-molecule spectroscopy.     

The head-fixed behaving rat--procedures and pitfalls

This paper describes experimental techniques with head-fixed, operantly conditioned rodents that allow the control of stimulus presentation and tracking of motor output at hitherto unprecedented levels of spatio-temporal precision. Experimental procedures for the surgery and behavioral training are presented. We place particular emphasis on potential pitfalls using these procedures in order to assist investigators who intend to engage in this type of experiment. We argue that head-fixed rodent models, by allowing the combination of methodologies from molecular manipulations, intracellular electrophysiology, and imaging to behavioral measurements, will be instrumental in combining insights into the functional neuronal organization at different levels of observation. Provided viable behavioral methods are implemented, model systems based on rodents will be complementary to current primate models--the latter providing highest comparability with the human brain, while the former offer hugely advanced methodologies on the lower levels of organization, for example, genetic alterations, intracellular electrophysiology, and imaging.     

A suggested role for mitochondria in Noonan syndrome

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders.     

Autonomous plasmid‐like replication of Bacillus ICEBs1: a general feature of integrative conjugative elements?

Integrative conjugative elements (ICEs) occur frequently in Gram‐positive and Gram‐negative bacteria. In contrast to plasmids, they are stably integrated in the bacterial genome, often inserted in a tRNA gene. They are excised from the host chromosome upon induction in order to be transferred to a recipient cell. When conjugative transfer is completed, they stably reintegrate in the chromosome. It is generally thought that ICEs are incapable of autonomous replication, instead relying on replication and segregation along with the host chromosome. In this issue of Molecular Microbiology Lee and co‐workers demonstrate that ICEBs1 from Bacillus subtilis is capable of autonomous plasmid‐like replication in its circular form after excision. The authors show that ICEBs1 replication is unidirectional; it initiates at oriT_ICEBs1 and requires the ICEBs1‐encoded conjugative relaxase NicK. Replication also requires the catalytic subunit of the host DNA polymerase PolC, the host processivity clamp DnaN and the host‐encoded alternative helicase PcrA. Autonomous replication of ICEBs1 appears to be important for its stable maintenance, but not for horizontal transfer of the element. Lee and co‐workers argue that plasmid‐like replication is likely a common property of ICEs, probably contributing to stability and maintenance of ICEs in bacterial populations. I discuss these findings in context with data on other ICEs from Gram‐positive and Gram‐negative bacteria and with respect to possible consequences of the findings for basic research on mobile genetic elements from Gram‐positive bacteria and their applications in biotechnology.