Mass spectrometric analysis of head-to-tail connected cyclic peptides

Tandem mass spectrometry is an extremely useful tool for high sensitive sequence identification of peptides. In the case of cyclic peptides fragmentation can easily be performed for sequence analysis. However, analysis is usually tedious due to the lack of a defined beginning and end of the sequence. Since cyclic peptides are a highly interesting class of compounds especially for the pharmaceutical industry, ways have to be found to identify their strictures. In this work we demonstrate how software and dedicated analytical strategies can be used for detailed analysis of these substances.     

Semiquantitative Confocal Laser Scanning Microscopy Applied to Marine Invertebrate Ecotoxicology

Confocal laser scanning microscopy (CLSM) represents a powerful, but largely unexplored ecotoxicologic tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We describe here a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in the polycylic aromatic hydrocarbon chrysene. This method is based on fluorogenic labeling of embryo yolk and subsequent statistical analysis of areal pixel intensities over multiple Z-series using a general linear model (GLM)–nested analysis of variance. The fluorescent yolk-labeling method described here was able to detect statistically significant differences in yolk concentrations in marine copepod (Amphiascus tenuiremis) eggs or embryos from females exposed to ultraviolet light and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. In constrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV-exposed treatment with chrysene at 2500 ng/g sediment (65.7% higher) and the UV-exposed treatment with chrysene at 500 ng/g sediment (76.6% higher).     

Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced beta-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3' UTR of beta-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with beta-actin mRNA and translocates to axons and growth cones of motoneurons.     

Studies on the reaction mechanism of riboflavin synthase: X-ray crystal structure of a complex with 6-carboxyethyl-7-oxo-8-ribityllumazine

Riboflavin synthase catalyzes the disproportionation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. We have determined the structure of riboflavin synthase from Schizosaccharomyces pombe in complex with the substrate analog, 6-carboxyethyl-7-oxo-8-ribityllumazine at 2.1 A resolution. In contrast to the homotrimeric solution state of native riboflavin synthase, we found the enzyme to be monomeric in the crystal structure. Structural comparison of the riboflavin synthases of S. pombe and Escherichia coli suggests oligomer contact sites and delineates the catalytic site for dimerization of the substrate and subsequent fragmentation of the pentacyclic intermediate. The pentacyclic substrate dimer was modeled into the proposed active site, and its stereochemical features were determined. The model suggests that the substrate molecule at the C-terminal domain donates a four-carbon unit to the substrate molecule bound at the N-terminal domain of an adjacent subunit in the oligomer.     

Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition

CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA.     

The role of the plasmalemmal dopamine and vesicular monoamine transporters in methamphetamine-induced dopaminergic deficits

Amphetamine (AMPH) and methamphetamine (METH) are members of a collection of phenethylamine psychostimulants that are commonly referred to collectively as "amphetamines." Amphetamines exert their effects, in part, by affecting neuronal dopamine transport. This review thus focuses on the effects of AMPH and METH on the plasmalemmal dopamine transporter and the vesicular monoamine transporter-2 in animal models with a particular emphasis on how these effects, which may vary for the different stereoisomers, contribute to persistent dopaminergic deficits.