A new method testing the orthogonality of different protecting groups.

A new test was elaborated to identify a new set of orthogonal protecting groups. With the developed method eight different protecting groups were tested under various deprotection conditions and the complex reaction mixtures were analysed by HPLC. The developed method allows for quick identification of orthogonality using simple model structures.

Comparative solution and solid-phase glycosylations toward a disaccharide library.

A comparative study on solution-phase and solid-phase oligosaccharide synthesis was performed. A 16-member library containing all regioisomers of Glc-Glc, Glc-Gal, Gal-Glc, and Gal-Gal disaccharides was synthesized both in solution and on solid phase. The various reaction conditions for different approaches and corresponding yields are analyzed and discussed.

Two types of calcium channels in human ovarian endocrine cells: involvement in steroidogenesis.

Nature, regulation, and functional role of ion channels of human ovarian endocrine cells are not well known. In our present study, we show two types of voltage-activated Ca(2+) currents (I(Ca)) in cultured human luteinized granulosa cells (GCs), as assessed by whole-cell patch-clamp experiments. Electrophysiological properties, namely low threshold of activation, pronounced time-dependent inactivation, slow and voltage-dependent deactivation kinetics, insensitivity to SNX-482, and high sensitivity to Ni(2+), defined the predominant I(Ca) as a T-type Ca(2+) current (I(Ca.T)). In 4% of cells a Ni(2+)-insensitive I(Ca) was measured alone or together with I(Ca.T). This Ca(2+) current was high voltage activated and highly sensitive to dihydropyridine, indicative of an L-type Ca(2+) current. RT-PCR analysis demonstrated the presence of mRNA coding for alpha(1)-subunits of two different Ca(2+) channels (T-type Ca(v)3.2 and L-type Ca(v)1.2) in GCs. In addition, these two types were detected in the human corpus luteum by RT-PCR (Ca(v)3.2) and immunohistochemistry (Ca(v)1.2). Although stimulation of cultured GCs with human chorionic gonadotropin did not change the characteristics of recorded I(Ca.T), it markedly increased the percentage of cells displaying I(Ca) from 29 to 63% and significantly increased (2.2-fold) the density of I(Ca.T). Furthermore, the stimulatory effect of human chorionic gonadotropin on progesterone production was diminished by pharmacological blockage of I(Ca.T) by Ni(2+) or flunarizine. Thus, our study provides evidence that human GCs in vivo and in vitro express T- and L-type Ca(2+) channels and that the Ca(v)3.2 (also called alpha(1H)) isoform is involved in a fundamental endocrine function of these cells.

An Intrinsic gamma-aminobutyric acid (GABA)ergic system in the adrenal cortex: findings from human and rat adrenal glands and the NCI-H295R cell line.

gamma-Aminobutyric acid (GABA), a major neurotransmitter in the central nervous system, also acts as a paracrine or autocrine signaling molecule in endocrine tissues such as the pancreatic islets, adenohypophysis, and testis. In the present study, we describe local GABA production and functional GABA(B) receptors in the adrenal cortex, possibly forming an auto- or paracrine GABAergic system. Using immunohistochemistry and RT-PCR, we localized the GABA-synthesizing enzyme glutamate decarboxylase 67 and the vesicular GABA transporter in steroid-producing cells of the human and rat adrenal cortex. Immunocytochemistry, Western blots, and RT-PCR experiments demonstrated the presence of glutamate decarboxylase 67 in the human adrenocortical cell line NCI-H295R. Measurements of glutamate decarboxylase activity confirmed that, in these cells and in rat adrenals, glutamate is decarboxylated to form GABA. In addition, we found expression of the GABA(B(1a)), GABA(B(1e)), and GABA(B(2)) subunits of the heterodimeric GABA(B) receptor in NCI-H295R cells as shown by RT-PCR. GABA(B(1a)) and its truncated splice variant GABA(B(1e)) were also found in human and rat adrenal glands. Immunostaining for the GABA(B(2)) subunit revealed its presence in the human and rat adrenal cortex and in NCI-H295R cells. The GABA(B) receptors we identified were functional because the GABA(B) agonist baclofen inhibited T-type Ca(2+) currents in whole-cell patch clamp experiments on NCI-H295R cells. This effect was blocked by pertussis toxin. Furthermore, the alpha(2)-, alpha(3)-, beta(2)-, beta(3)- gamma(2)-, and epsilon-subunits of the GABA(A) receptor were detected in this cell line by RT-PCR. Hence, we conclude that GABA is synthesized and stored by steroid-producing cells of the adrenal cortex and may influence these cells in a paracrine or autocrine manner.