Adrenal expression of orexin receptor subtypes is differentially regulated in experimental streptozotocin induced type-1 diabetes

Orexins (hypocretins) are involved in the regulation of energy homeostasis and sleeping behavior. Orexins were also implicated in the regulation of neuroendocrine and autonomic functions. Recent data show the expression of orexin receptors within the hypothalamic-pituitary-adrenal (HPA) axis and suggest specific actions of orexins at the pituitary and adrenal glands. To further evaluate the role of orexin in the HPA axis, we investigated the mRNA expression of prepro-orexin (PPO) and orexin receptors within the HPA axis of streptozotocin-injected (STZ) rats showing type-1 like diabetes. PPO, as well as OX(1) and OX(2) receptor levels were analyzed by quantitative real-time PCR (qPCR). STZ rats were characterized by decreased body weight, plasma insulin, and leptin levels and by increased plasma glucose. Hypothalamic PPO mRNA levels were significantly reduced in STZ compared to non-diabetic control rats. No differences were found in the mRNA levels of hypothalamic or pituitary OX(1) and OX(2) receptors between control and STZ rats. In adrenals, OX(1) receptor mRNA levels were significantly elevated in STZ rats while OX(2) receptors were significantly reduced. Our results imply distinct functions of adrenal orexin receptor subtypes during type-1 like diabetes.     

Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria.

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.     

Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Synthesis of spirocyclic σ1 receptor ligands as potential PET radiotracers,structure–affinity relationships and in vitro metabolic stability

Several 3H-spiro[[2]benzofuran-1,4′-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF₃·OEt₂ catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure–affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar σ₁ affinity (K_i = 0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the σ₂ subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [¹⁸F]2a and [¹⁸F]2e two different radiosynthetic approaches were followed.     

Structures of sortase B from Staphylococcus aureus and Bacillus anthracis reveal catalytic amino acid triad in the active site

Surface proteins attached by sortases to the cell wall envelope of bacterial pathogens play important roles during infection. Sorting and attachment of these proteins is directed by C-terminal signals. Sortase B of S. aureus recognizes a motif NPQTN, cleaves the polypeptide after the Thr residue, and attaches the protein to pentaglycine cross-bridges. Sortase B of B. anthracis is thought to recognize the NPKTG motif, and attaches surface proteins to m-diaminopimelic acid cross-bridges. We have determined crystal structure of sortase B from B. anthracis and S. aureus at 1.6 and 2.0 A resolutions, respectively. These structures show a beta-barrel fold with alpha-helical elements on its outside, a structure thus far exclusive to the sortase family. A putative active site located on the edge of the beta-barrel is comprised of a Cys-His-Asp catalytic triad and presumably faces the bacterial cell surface. A putative binding site for the sorting signal is located nearby.     

Angiotensin II stimulates the reactivity of the pituitary-adrenal axis in leptin-resistant Zucker rats, thereby influencing the glucose utilization

The HPA axis is hyperactive under conditions of leptin and insulin resistance as well as after ANG II administration. We hypothesized that a hyperreactivity of the HPA axis to ANG contributes to an impaired glucose utilization in obesity, since leptin resistance and an overactive renin-angiotensin-aldosterone system are features of obesity. Zucker rats were treated with ANG via subcutaneous minipumps (0, 0.9, and 9.0 mug/h; 4 wk). PA axis reactivity and glucose homeostasis were characterized after CRH treatment and during an oral glucose tolerance test (OGTT). The elevated plasma profile of corticosterone after CRH stimulation in saline-treated OZR compared with LZR confirmed that the sensitization of the PA axis depended on leptin resistance. Irrespective of the rat strain, circulating ANG levels and blood pressure were selectively increased after administration of 9 mug/h ANG (high ANG). Only high ANG induced an elevation of the corticosterone and glucose response after CRH stimulation in OZR but did not affect the ACTH secretion. During OGTT, corticosterone and consequently glucose increased in OZR after high ANG, whereas the insulin secretion was decreased. In the adrenal glands of OZR, AT(1A) receptor mRNA levels increased after high ANG. We conclude that the impairment of glucose utilization after ANG stimulation is potentiated in leptin-resistant rats as a result of a hyperreactive PA axis, thereby confirming the functional importance of a dysregulation within the HPA axis in metabolic syndrome or obesity. The ACTH-independent stimulation of corticosterone release and the selective increase of AT(1A) receptor mRNA in the adrenals of OZR indicated a sensitization of adrenals toward ANG, causing a stimulation of the PA axis.     

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.     

Novel versus unsupported clades: assessing the qualitative support for clades in MRP supertrees

Matrix representation with parsimony (MRP) supertree construction has been criticized because the supertree may specify clades that are contradicted by every source tree contributing to it. Such unsupported clades may also occur using other supertree methods; however, their incidence is largely unknown. In this study, I investigated the frequency of unsupported clades in both simulated and empirical MRP supertrees. Here, I propose a new index, QS, to quantify the qualitative support for a supertree and its clades among the set of source trees. Results show that unsupported clades are very rare in MRP supertrees, occurring most often when there are few source trees that all possess the same set of taxa. However, even under these conditions the frequency of unsupported clades was <0.2%. Unsupported clades were absent from both the Carnivora and Lagomorpha supertrees, reflecting the use of large numbers of source trees for both. The proposed QS indices are correlated broadly with another measure of quantitative clade support (bootstrap frequencies, as derived from resampling of the MRP matrix) but appear to be more sensitive. More importantly, they sample at the level of the source trees and thus, unlike the bootstrap, are suitable for summarizing the support of MRP supertree clades.     

‘Further Development’ of Mendel’s legacy? Erich von Tschermak-Seysenegg in the context of Mendelian–biometry controversy, 1901–1906

The contribution of Erich von Tschermak-Seysenegg (1871?C1962) to the beginning of classical genetics is a matter of dispute. The aim of this study is to analyse, based on newly accessible archive materials, the relevance of his positions and theoretical views in a debate between advocates of early Mendelian explanation of heredity and proponents of biometry, which took place in England around 1901?C1906. We challenge not only his role of an ??external consultant??, which at the time de facto confirmed his status of ??rediscoverer?? of Mendel??s work but also analyse his ambivalent positions which are to be seen as a part of ??further development?? (Weiterführung), a development of Mendel??s legacy as he understood it. Second, there is an interesting aspect of establishing connections within an ??experimental culture?? along the Mendel??s lines of thought that was parallel to the first step of institutionalizing the new discipline of Genetics after 1905/06. Part of the study is also the analysis of contribution of his older brother Armin von Tschermak-Seysenegg (1870?C1952) who??much like in the case of ??rediscovery?? of 1900?C1901??was for his younger brother an important source of theoretical knowledge. In this particular case, it regarded Bateson??s ??Defence?? of Mendel from 1902.