Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima

An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.     

Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen.

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS-PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA-immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA, these CGA-processing products are released from the sheep spleen. The secretion of CGA-derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.     

Preparation of noradrenaline-storing organelles from bovine sympathetic ganglia: Biochemical and morphological evaluation of partly purified large dense cored vesicles

Large dense cored vesicles from bovine sympathetic ganglia were isolated and partly purified. Biochemical and morphological evaluation of the present vesicle-preparation revealed that it represents a convenient fraction for the characterization of perikaryal noradrenergic vesicles.

Homogenates of bovine stellate ganglia were subjected to differential centrifugation and D₂O-sucrose density gradient centrifugation. Biochemical evaluation of gradient fractions was performed by measuring marker enzyme activities reflecting subcellular contamination, while morphological evaluation was performed by electron microscopic analysis of the isolated fractions. Both techniques revealed that the vesicle-preparation was, at first, still considerably contaminated by mitochondria and lysosomes.

An improved purification could be achieved by subjecting this fraction to an additional centrifugation under iso-osmotic conditions, also applied for the preparation of highly purified splenic nerve vesicles. The resulting vesicle-fraction was almost complete free of contaminating enzyme activities and consisted merely of large dense cored vesicles as revealed by electron microscopic observations (50–70% purity). Neuropeptide Y and chromogranin A were enriched more than 50 times as compared to the total homogenate.

Although the purity of these vesicles was still not satisfactory for direct chemical analysis, this vesicle-preparation seemed very well suited for immunological characterization of perikaryal large dense cored vesicles.     

Serosurveillance for selected infectious disease agents in wild boars (Sus scrofa) and outdoor pigs in Switzerland

The large abundance of free-ranging wild boars (Sus scrofa) and a trend towards animal friendly outdoor management of domestic pigs lead to an increasing probability of disease transmission between those animal populations. In 2001, an active monitoring was started for classical swine fever (CSF), Aujeszky’s disease (AD) and porcine brucellosis (PB) in wild boars in Switzerland. The objective of this programme was to document the serological status of wild boars regarding the selected pathogens. To continue this serosurveillance, 1,060 wild boar samples were collected during two regular hunting seasons in 2004–2005. Furthermore, in a pilot study, 61 outdoor pigs from 14 farms located in areas with high wild boar densities were sampled in 2004 and serologically tested for AD and PB. All wild boar samples were negative for CSF. Seroprevalence for AD was 2.83% (95% CI 1.91–4.02%). Seroprevalence for PB was 13.5% (95% CI 10.7–16.7%) for the Rose Bengal test and 11.05% (95% CI 8.82–13.61%) for the indirect ELISA. There was no serological evidence for AD in domestic pigs. All tested animals from 13 piggeries were seronegative for PB, but three pigs from the same farm showed doubtful results. Further investigations on the farm did not indicate the presence of PB in the herd. These findings urge the need for better diagnostic tools to obtain reliable results concerning PB prevalence. Since contact and following transmission of infectious agents between infected wild boars and outdoor pigs might occur in the future, it is advisable to include outdoor pigs in areas at risk in routine surveillance programmes.     

Cross-linking Chemistry of Squid Beak

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (¹H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.     

Processing of gene expression data generated by quantitative real-time RT-PCR

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.     

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.     

RepGene: a spreadsheet template for the management of reporter gene assays.

One of the most powerful techniques in molecular biology is the controlled expression of specific proteins by transfection of eukaryotic cells. This method has become feasible and highly sensitive and, thus, suitable for high-throughput reporter gene assays in basic and applied research. Moreover, the limiting factors are neither the transfection efficiency nor the functional analysis, but rather the ability to manage complex experimental protocols when multiple genes are co-transfected and/or when the effects of several chemical compounds are investigated within the same experiment. Here, we describe an easy-to-use and highly flexible spreadsheet template intended to rationalize and expedite the organization and data management of multi-step reporter gene assays. The objectives of this spreadsheet template are the design of the transfection protocol, the coordination of the administration of test compounds, and the graphical presentation and statistical analysis of the results.     

Loss of NOTCH2 positively predicts survival in subgroups of human glial brain tumors

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas.     

Molecular Biocomputing Suite: a word processor add-in for the analysis and manipulation of nucleic acid and protein sequence data.

In all fields of molecular biology, researchers are increasingly challenged by experiments planned and evaluated on the basis of nucleic acid and protein sequence data generally retrieved from public databases. Despite the wide spectrum of available Web-based software tools for sequence analysis, the routine use of these tools has disadvantages, particularly because of the elaborate and heterogeneous ways of data input, output, and storage. Here we present a Visual Basic-encoded Microsoft Word Add-In, the Molecular BioComputing Suite (MBCS), available at the BioTechniques Software Library (www.BioTechniques.com). The MBCS software aims to manage and expedite a wide range of sequence analyses and manipulations using an integrated text editor environment including menu-guided commands. Its independence of sequence formats enables MBCS to be used as a pivotal application between other software tools for sequence analysis, manipulation, annotation, and editing.