The role of nutrition on epigenetic modifications and their implications on health

Nutrition plays a key role in many aspects of health and dietary imbalances are major determinants of chronic diseases including cardiovascular disease, obesity, diabetes and cancer. Adequate nutrition is particularly essential during critical periods in early life (both pre- and postnatal). In this regard, there is extensive epidemiologic and experimental data showing that early sub-optimal nutrition can have health consequences several decades later.     

Long-term effects of dietary isoflavones on uterine gene expression profiles

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects.Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 μg/g diet) and an ISO-high diet (232 μg daidzein and 240 μg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring.We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018 ± 350 mg/kg BW) than with ISO-high diet (497 ± 133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects.In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.     

Assessment of Optimal Selected Prognostic Factors

The identification and assessment of prognostic factors is one of the major tasks in clinical research. The assessment of one single prognostic factor can be done by recently established methods for using optimal cutpoints. Here, we suggest a method to consider an optimal selected prognostic factor from a set of prognostic factors of interest. This can be viewed as a variable selection method and is the underlying decision problem at each node of various tree building algorithms. We propose to use maximally selected statistics where the selection is defined over the set of prognostic factors and over all cutpoints in each prognostic factor. We demonstrate that it is feasible to compute the approximate null distribution. We illustrate the new variable selection test with data of the German Breast Cancer Study Group and of a small study on patients with diffuse large B‐cell lymphoma. Using the null distribution for a p‐value adjusted regression trees algorithm, we adjust for the number of variables analysed at each node as well. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Iodine binding and peroxidase activity in the endostyle of Salpa fusiformis,Thalia democratica,Dolioletta gegenbauri and Doliolum nationalis (Tunicata,Thaliacea)

Summary The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electronmicroscopic levels. To reveal the primary binding site for iodine, autoradiography was carried out on endostylar tissue from animals that had been incubated with high activity ¹²⁵I^- over a short period of time. The specific iodine binding enzyme, a peroxidase, was traced by its reaction with DAB. In accordance with previous findings, the iodinebinding cells proved to be the same as those containing the peroxidase. There were also strong indications of a secondary uptake of iodinated compounds and subsequent release into the body fluid. Together with the ultrastructural features, the data provided strong evidence indicating that these cells constitute a protothyroid region, which partly functions as an endocrine organ, possibly homologous with the vertebrate thyroid gland. Since the number of zones varied between the species, the numeration of the protothyroid region also varied. However, in all the examined endostyles, the protothyroid region was seen to be situated dorsolaterally to the glandular regions of the endostyle concerned with food capture.     

Assessment of membrane potential changes using the carbocyanine dye,diS-C3-(5): synchronous excitation spectroscopy studies

The fluorescence of the voltage sensitive dye, diS-C₃-(5), has been analyzed by means of synchronous excitation spectroscopy. Using this rather rare fluorescence technique we have been able to distinguish between the slightly shifted spectra of diS-C₃-(5) fluorescence from cells and from the supernatant. It has been found that diS-C₃-(5) fluorescence in the supernatant can be selectively monitored at _exc = 630 nm and _em= 650 nm, while the cell associated fluorescence can be observed at _exc= 690 nm and _em = 710 nm. A modified theory for the diSC₃-(5) fluorescence response to the membrane potential is presented, according to which a linear relationship exists between the logarithmic increment of the dye fluorescence intensity in the supernatant, In I/I°, and the underlying change in the plasma membrane potential, _p=_p-°_p. The theory has been tested on human myeloid leukemia cells (line ML-1) in which membrane potential changes were induced by valinomycin clamping in various K⁺ gradients. It has been demonstrated that the membrane potential change, _p,can be measured on an absolute scale. Offprint requests to: J. Plasek     

Nasal Tumor in a Fallow Deer (Dama Dama L)

A nasal, so called ethmoidal, tumor from a fallow deer is described. It appears to be the first reported case of that species. The etiology is discussed.     

Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.     

Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.     

Three fluorescent probes for the flow-cytometric assessment of membrane potential inSaccharomyces cerevisiœ

Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3′-dipropylthiacarbocyanine iodide (diS-C₃(3)) and 3,3′-dipropyloxacarbocyanine iodide (diO-C₃(3)), were tested for their suitability as fluorescent indicators of membrane potential inSaccharomyces cerevisiœ in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60–3000 nmol/L. The optimum staining period was 15–20 min for diS-C₃(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.