The proteasomal system and HNE-modified proteins

Metabolic processes and environmental conditions cause the constant formation of oxidizing species over the lifetime of cells and organisms. This leads to a continuous oxidation of intracellular components, including lipids, DNA and proteins. During the extensively studied process of lipid peroxidation, several reactive low-molecular weight products are formed, including reactive aldehydes as 4-hydroxynonenal (HNE). These aldehydic lipid peroxidation products in turn are able to modify proteins. The degradation of oxidized and oxidatively modified proteins is an essential part of the oxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasomal system. The proteasomal system by itself is a multicomponent system responsible for the degradation of the majority of intracellular proteins. It has been shown that some, mildly cross-linked, HNE-modified proteins are preferentially degraded by the proteasome, but extensive modification with this cross-linking aldehyde leads to the formation of protein aggregates, that can actually inhibit the proteasome. This review summarizes our knowledge of the interactions between lipid peroxidation products, proteins, and the proteasomal system.     

Total biosynthesis of hydrocortisone from a simple carbon source in yeast

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.     

The multiple forms of brain acetylcholinesterase

Summary The pattern of the multiple forms of the acetylocholinesterase (AChE, E.C. 3.1.1.7) of the rat brain is investigated using polyacrylamide gradient micro-gel electrophoresis with regard to a possible functional importance of this individual forms. The patterns of the AChE-forms of selected regions of the CNS are compared and certain differences could be shown. After increased cholinergic input (into the hippocampus by electrical stimulation of the nc. septi medialis) an aggregation of AChE subunits is detectable. Subletal intoxication with an irreversible inhibitor of AChE is followed by a faster recovery of the smaller forms. A suggestion of a possible functional role of the multiple forms of AChE is discussed.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

Resting site selection by large herbivores – The case of European bison (Bison bonasus) in Białowieża Primeval Forest

Resting site selection by European bison (Bison bonasus, L., 1758), the largest terrestrial mammal of Europe, was studied in the free-ranging population in Bia?owie?a Primeval Forest (Poland) in 2009–2010. In total, 104 sites of 21 bison (both collared and uncollared) were analysed to determine the most important microhabitat characteristics selected by resting bison during summer and winter and to study the influence of supplementary feeding on resting behaviour of this herbivore. Resting sites were identified on the basis of GPS locations and activity records collected by GPS collars, as well as direct observations of bison, and were compared with control sites. Microhabitat selection by bison did not differ significantly between the sexes. During summer and winter, bison resting sites displayed a high tree density, low visibility and high complexity (structures providing cover). Summer resting sites were also characterised by a significantly lower abundance of blood-sucking insects and denser canopy than control sites. Winter resting sites showed a lower complexity and higher visibility than summer sites, and were less often located in mixed forest habitats. During winter, bison rested more frequently in forest below 50 years of age than in older forest. Resting sites of non-fed bison were more often located in young coniferous forests, were lower in visibility and situated closer to open areas than sites of bison using supplementary winter feeding, suggesting a trade-off between food and cover. The results indicate that European bison select their resting sites in areas of mosaic habitat structure providing cover from disturbances with access to profitable natural forage grounds.     

Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II

Endoglin is an auxiliary component of the transforming growth factor-beta (TGF-beta) receptor system, able to associate with the signaling receptor types I (TbetaRI) and II (TbetaRII) in the presence of ligand and to modulate the cellular responses to TGF-beta1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TbetaRI or TbetaRII. This study shows that full-length endoglin interacts with both TbetaRI and TbetaRII, independently of their kinase activation state or the presence of exogenous TGF-beta1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TbetaRI interaction was different from that of endoglin/TbetaRII. TbetaRII interacts with the amino acid region 437-558 of the extracellular domain of endoglin, whereas TbetaRI interacts not only with the region 437-558 but also with the protein region located between amino acid 437 and the N terminus. Both TbetaRI and TbetaRII interact with the cytoplasmic domain of endoglin, but TbetaRI only interacts when the kinase domain is inactive, whereas TbetaRII remains associated in its active and inactive forms. Upon association, TbetaRI and TbetaRII phosphorylate the endoglin cytoplasmic domain, and then TbetaRI, but not TbetaRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TbetaRII, TbetaRI, and downstream Smad proteins as well as a modulation of TGF-beta signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-beta responses.     

Artificial insemination in cattle with DNA‐treated sperm

Abstract

We have investigated the use of sperm cells as vectors for transferring exogenous DNA into the genome of cattle by artificial insemination with DNA‐treated sperm. First we demonstrated the DNA‐binding ability of cattle sperm with radioactively labeled DNA. For artificial insemination ejaculated semen was washed and incubated with 1 μg DNA/10⁶ sperm for one hour at 37°C. Three hundred synchronized heifers were inseminated once with a dose of 40×10⁶ sperm. Forty‐five calves and 41 fetuses were obtained. Southern analysis revealed in one calf a signal after probing with the 1 kb Pst I fragment of pSV2‐cat.