Cell type-specificity of nonclassical estrogen signaling in the developing midbrain

Estrogens have widespread biological functions in the CNS involving the coordination of developmental processes, the regulation of cell physiology, and the control of neuroendocrine systems. In the midbrain, estrogens promote the survival, maturation, and function of neurons and, in particular, of dopamine cells. Aside from classical signaling through nuclear estrogen receptors, we have provided evidence that cellular transmission of estrogen effects in the midbrain comprises a complex intracellular signaling scenario. The major conclusion drawn from our studies is that estrogens interact with yet unidentified membrane receptor complexes which stimulate the phospholipase C and induce the formation of inosite-tri-phosphate (IP₃). This causes a rapid and transitory rise in intracellular free calcium. The modulation of calcium homeostasis is the primary nonclassical physiological response to estrogens in all cell types. Surprisingly, a different secondary downstream signaling cascade seems to be activated in each estrogen-responsive cell population, i.e. phosphatidylinositol-3 kinase (PI3-kinase) in GABAergic and cAMP/ protein kinase A (PKA) in dopaminergic neurons, mitogen-activated protein kinase (MAP-kinase) in astrocytes. The precise biological role of estrogens for the different cell types is still fragmentary. We assume that estrogens positively influence intracellular signaling mechanisms which are important for cell differentiation and survival. It remains to be elucidated what determines the cell type-specificity of these estrogen responses.     

Vaccination with tumor peptide in CpG adjuvant protects via IFN-gamma-dependent CD4 cell immunity

The low frequency of tumor Ag-specific T cells in vivo has made it challenging to directly measure their clonal sizes and cytokine signatures. We used a new generation ELISPOT approach to study the constitutive immunogenicity of the RMA tumor in syngeneic B6 mice and adjuvant-guided immunity against an MHC class II-restricted RMA peptide, H11.1. The RMA tumor was found to activate cells of the innate immune system and to induce a type 1 polarized, RMA-specific CD4 and CD8 T cell response. With clonal sizes approximately 10/10(6), the magnitude of this constitutively induced immune response did not suffice to control the tumor cell growth. In contrast, immunization with H11.1 peptide, using an immunostimulatory CpG oligonucleotide or CFA as adjuvant, engaged approximately 25- or approximately 10-fold higher clonal sizes of type 1 polarized CD4 cells, respectively. Therefore, the CpG oligonucleotide functioned as a stronger type 1 adjuvant and, unlike CFA, elicited protective immunity. The protection was IFN-gamma dependent, as it was not inducible in IFN-gamma knockout mice. Therefore, CpG adjuvant-guided induction of type 1 immunity against tumor Ags might be a promising subunit vaccination approach.     

Lack of strand bias in UV-induced mutagenesis in Escherichia coli

We have investigated whether UV-induced mutations are created with equal efficiency on the leading and lagging strands of DNA replication. We employed an assay system that permits measurement of mutagenesis in the lacZ gene in pairs of near-identical strains. Within each pair, the strains differ only in the orientation of the lacZ gene with respect to the origin of DNA replication. Depending on this orientation, any lacZ target sequence will be replicated in one orientation as a leading strand and as a lagging strand in the other orientation. In contrast to previous results obtained for mutations resulting from spontaneous replication errors or mutations resulting from the spontaneous SOS mutator effect, measurements of UV-induced mutagenesis in uvrA strains fail to show significant differences between the two target orientations. These data suggest that SOS-mediated mutagenic translesion synthesis on the Escherichia coli chromosome may occur with equal or similar probability on leading and lagging strands.     

Protofibrillar islet amyloid polypeptide permeabilizes synthetic vesicles by a pore-like mechanism that may be relevant to type II diabetes

Islet amyloid polypeptide (IAPP) and insulin are copackaged and cosecreted by pancreatic islet beta-cells. Non-insulin-dependent (type II) diabetes mellitus (NIDDM) is characterized by dysfunction and depletion of these beta-cells and also, in more than 90% of patients, amyloid plaques containing fibrillar IAPP. An aggregated but not necessarily fibrillar form of IAPP is toxic in cell culture, suggesting that prefibrillar oligomeric (protofibrillar) IAPP may be pathogenic. We report here that IAPP generates oligomeric species in vitro that are consumed as beta-sheet-rich fibrils grow. Protofibrillar IAPP, like protofibrillar alpha-synuclein, which is implicated in Parkinson's disease pathogenesis, permeabilizes synthetic vesicles by a pore-like mechanism. The formation of the IAPP amyloid pore is temporally correlated to the formation of early IAPP oligomers and its disappearance to the appearance of amyloid fibrils. Neither pores nor oligomers were formed by the nonfibrillogenic rat IAPP variant. The IAPP amyloid pore may be critical to the pathogenic mechanism of NIDDM, as other amyloid pores may be to Alzheimer's disease and Parkinson's disease.     

Two-dimensional electrophoresis patterns of total proteins,esterases and acid phosphatases from pollen,seeds and leaves of three cultivars of Helianthus annuus L

Total proteins, esterases and acid phosphatases from pollen, seeds and leaves of three sunflower cultivars were separated by 2-D electrophoresis. The characteristic peptides for each cultivar were identified. The seeds and pollen of the cultivar Wielkopolski contained 45 and 37 characteristic peptides, respectively, while the seeds and pollen of Coril contained 73 and 35 characteristic peptides. The cultivar Frankasol had the lowest total number of stained peptides in seeds and pollen, and the number of the characteristic peptides was only 61 and 25, respectively. Analyses of esterases and acid phosphatases from young leaves and pollen led to identification of isoenzymes characteristic of the three cultivars. Only for Frankasol no specific acid phosphatase was observed, both in leaves and in pollen.     

Site of the previous meiotic division defines cleavage orientation in the mouse embryo

The conservation of early cleavage patterns in organisms as diverse as echinoderms and mammals suggests that even in highly regulative embryos such as the mouse, division patterns might be important for development. Indeed, the first cleavage divides the fertilized mouse egg into two cells: one cell that contributes predominantly to the embryonic part of the blastocyst, and one that contributes to the abembryonic part. Here we show, by removing, transplanting or duplicating the animal or vegetal poles of the mouse egg, that a spatial cue at the animal pole orients the plane of this initial division. Embryos with duplicated animal, but not vegetal, poles show abnormalities in chromosome segregation that compromise their development. Our results show that localized factors in the mammalian egg orient the spindle and so define the initial cleavage plane. In increased dosage, however, these factors are detrimental to the correct execution of division.     

Difficulties in the deprotection of 1,2-ketals in nucleosides containing alkylidencarbazoyl groups

The synthesis of unprotected alkylidencarbazoyl nucleoside derivatives 8a-8d is shown. A direct deprotection route from readily available 2',3'-isopropylidene protected nucleosides 5a-5d. prepared from a chemoenzymatic procedure, did not give the selective cleavage of the ketal function without affecting the C=N bond. The next option tried was to look at the previous compound in the retrosynthetic route: 2',3'-protected carbazoyl nucleoside 4. However, in all cases we obtained unsatisfactory results. Stepping further back, the hydrolysis of compound 3a led us to unprotected carbonate nucleoside 9 in quantitative yield. With this compound available, the synthesis towards derivatives 8 was accomplished through a known procedure.     

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.     

Supramolecular ligands: Monomer structure and protein ligation capability

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of his azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to his azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.