Assessment of dietary intake: NuGO symposium report

Advances in genomics science and associated bioinformatics and technology mean that excellent tools are available for characterising human genotypes. At the same time, approaches for characterising individual phenotypes are developing rapidly. In contrast, there has been much less investment in novel methodology for measuring dietary exposures so that there is now a significant gap in the toolkit for those investigating how diet interacts with genotype to determine phenotype. This symposium reviewed the strengths and limitations of current tools used in assessment of dietary intake and the potential to improve these tools through, for example, the use of statistical techniques that combine information from different sources (such as modelling and calibration methods) to ameliorate measurement error and to provide validity checks. Speakers examined the use of approaches based on technologies such as mobile 'phones, digital cameras and Web-based systems which offer the potential for more acceptable (for study participants) and less laborious (for researchers and participants) routes to more robust data collection. In addition, the application of omics, especially metabolomics, tools to biofluids to identify new biomarkers of intake offers great potential to provide objective measures of food consumption with the advantage that data may be collected in forms that can be integrated readily with other high throughput (nutrigenomic) technologies.     

Application of Petri net based analysis techniques to signal transduction pathways

Background  

Signal transduction pathways are usually modelled using classical quantitative methods, which are based on ordinary differential equations (ODEs). However, some difficulties are inherent in this approach. On the one hand, the kinetic parameters involved are often unknown and have to be estimated. With increasing size and complexity of signal transduction pathways, the estimation of missing kinetic data is not possible. On the other hand, ODEs based models do not support any explicit insights into possible (signal-) flows within the network. Moreover, a huge amount of qualitative data is available due to high-throughput techniques. In order to get information on the systems behaviour, qualitative analysis techniques have been developed. Applications of the known qualitative analysis methods concern mainly metabolic networks. Petri net theory provides a variety of established analysis techniques, which are also applicable to signal transduction models. In this context special properties have to be considered and new dedicated techniques have to be designed.     

Gdnf Haploinsufficiency Causes Hirschsprung-Like Intestinal Obstruction and Early-Onset Lethality in Mice

Hirschsprung disease (HSCR) is a common congenital disorder that results in intestinal obstruction and lethality, as a result of defective innervation of the gastrointestinal (GI) tract. Despite its congenital origin, the molecular etiology of HSCR remains elusive for >70% of patients. Although mutations in the c-RET receptor gene are frequently detected in patients with HSCR, mutations in the gene encoding its ligand (glial cell line-derived neurotrophic factor [GDNF]), are rarely found. In an effort to establish a possible link between human HSCR and mutations affecting the Gdnf locus, we studied a large population of mice heterozygous for a Gdnf null mutation. This Gdnf(+/-) mutant cohort recapitulates complex features characteristic of HSCR, including dominant inheritance, incomplete penetrance, and variable severity of symptoms. The lack of one functioning Gdnf allele causes a spectrum of defects in gastrointestinal motility and predisposes the mutant mice to HSCR-like phenotypes. As many as one in five Gdnf(+/-) mutant mice die shortly after birth. Using a transgenic marking strategy, we identified hypoganglionosis of the gastrointestinal tract as a developmental defect that renders the mutant mice susceptible to clinical symptoms of HSCR. Our findings offer a plausible way to link an array of seemingly disparate features characteristic of a complex disease to a much more narrowly defined genetic cause. These findings may have general implications for the genetic analysis of cause and effect in complex human diseases.     

On the evidence of a diffusion barrier in the outer cortex apoplast of cress-roots (Lepidium sativum), demonstrated by analytical electron microscopy

To mark the apoplastic pathway of ions in the root of the dicotyledonous plant Lepidium sativum we used the heavy element lanthanum, which can be identified by analytical electron microscopy (EELS and ESI). In the front root tip, the primary walls of all meristematic cells contained lanthanum. 10-15 mm behind the root apex, lanthanum was found in the cortex cell walls up to the endodermis, but not in the stele. 20-25 mm from the tip, lanthanum was accumulated in the radial cell walls of the hypodermis, which, however, is not a complete diffusion barrier for ions, so that traces of lanthanum also were found in the cortex cell walls up to the endodermis. This study provides evidence for the presence of two apolastic diffusion barriers in the region of highest water uptake in cress roots.     

Proteolysis of SNAP-25 Isoforms by Botulinum Neurotoxin Types A, C, and E

Abstract : Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg¹⁹⁸ and Ala¹⁹⁹, depends on the presence of regions Asn⁹³ to Glu¹⁴⁵ and Ile¹⁵⁶ to Met²⁰², and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex ( K _D = 1.9 × 10^-7 M ) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met¹⁴⁶-Gln¹⁹⁷, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile¹⁵⁶-Asp¹⁸⁶. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys¹⁸⁵Asp or Pro¹⁸²Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

A Conjugate of Cellulase with Fluorescein Isothiocyanate: A Specific Stain for Cellulose

A fluorescent technique has been developed for in situ staining of cellulose. The staining agent is a conjugate of cellulase and fluorescein isothiocyanatc (FITC). Application of this agent does not disturb intercellular or intracellular substances. The technique depends on the specific binding of the fluorescent labeled enzyme to its substrate. The stain has been tested on cell-free noncellulose polysaccharides similar to cellulose and does not stain them. The technique has been used to localize cellulose during the life cycle of Dictyostelium discoideum with results that correspond to previous work using other methods.