Acetylcholine responses of identified neurons in Helix pomatia--III. Ionic composition of the depolarizing currents induced by acetylcholine

The ionic composition of the currents underlying the acetylcholine (ACh) depolarizations in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia was analysed. The equilibrium potential of the ACh responses was -2.8 +/- 0.6 mV (N = 49) and -4.0 +/- 0.7 mV (N = 79; mean +/- SEM) in the neurons B1 and B3, respectively. Replacement of NaCl in the bath solution by sucrose shifted the ACh equilibrium potential into the negative direction. A similar but less pronounced shift occurred when Ca2+ was substituted for Na+. Substitution of Cl- in the bath solution by propionate or an increase of the intracellular Cl- concentration did not affect the ACh equilibrium potential. Changes of K+ concentration in the bath between 1 and 50 mmol/l left the ACh equilibrium potential nearly unaffected when the Na+ concentration was at the control level. With a simultaneous reduction of extracellular Na+ an increase of K+ concentration shifted the ACh equilibrium potential towards more positive potentials. The findings are compatible with calculated K+ permeabilities if a K+ redistribution across the cell membrane is considered. In the neurons B1 and B3, channels operated by ACh are permeable for K+, Na+ and Ca2+, with the relative permeabilities 1.6:1.0:0.1.     

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.     

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

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)     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.     

Characterization of a meta-Fluorotyrosine-Tolerant Cell Culture of Eschscholtzia californica Cham

A cell line of Eschscholtzia californica selected for meta-fluorotyrosine (MFT) tolerance was found to have 10-fold increased levels of phenylalanine and tyrosine compared to the parent line, while most other amino acids were only increased 2-fold. Tracer experiments with shikimic acid in the presence of MFT showed that the biosynthesis of the aromatic amino acids was not impaired in the tolerant line. Feeding experiments with phenylalanine, tyrosine, or shikimic acid also revealed a reduced turnover of the pools of the aromatic amino acids in the variant. Thus undisturbed de novo biosynthesis of the aromatic amino acids and dilution of toxic effects of MFT by the enlarged pool sizes seemed to be the main reason for the acquired tolerance. Despite the enlarged availability of the precursor tyrosine, formation of the benzophenanthridine alkaloids was enhanced neither in the growth nor in the production medium.     

The 5'' noncoding region of the human leukemia-associated oncogene BCR/ABL is a potent inhibitor of in vitro translation.

The mRNA encoding the chimeric BCR/ABL oncogene, which is transcribed from the Philadelphia chromosome in human chronic myelogenous leukemia, has a 5' noncoding sequence greater than 500 bases in length which is highly GC rich and contains a short open reading frame. This untranslated sequence has a dramatic inhibitory effect upon translational efficiency in vitro. However, when BCR/ABL message is expressed in certain cell types such as the NIH 3T3 cell line, the 5' noncoding region has little inhibitory effect on translational efficiency.     

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.