The synthesis of <Emphasis Type="Italic">O</Emphasis>-substituted 3-oximes of 6α -methyl-16α,17α-cyclohexanopregn-4-ene-3,20-diones tritium-labeled in the 1,2-position

1,2-Tritium-labeled 3-(O-carboxypropyl)- and 3-(O-carbomethoxypropyl)-oximes of 6α-methyl-16α,17α-cyclohexanopregn-4-ene-3,20-diones were obtained by the homogeneous catalytic hydrogenation of 1,2-dehydroprecursors with gaseous tritium and the subsequent separation of the resulting mixtures by HPLC. The specific radioactivities of 50–55 Ci/mmol were prepared using tris-(triphenylphosphine)-rhodium chloride.     

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.     

Neurospecific cytoskeletal proteins

Data available in literature on neurospecific proteins of cytoskeletal structures--microtubules, microfilaments and intermediate filaments are generalized. Properties of tissue-specific cytoskeletal proteins which are typical of nerve cells are summarized. The structure, physicochemical properties, cell localization, metabolism and function of cytoskeletal proteins are characterized. The coexpression and interaction of different cytoskeletal structures are considered. An analysis of neurospecific cytoskeletal proteins is of great practical importance for neurobiology, neurooncology, neurosurgery. The proteins can be used as markers of different pathologies in the nervous system.     

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.     

New aspects of biosynthesis and metabolism of <Emphasis Type="Italic">N</Emphasis>-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.     

Postnatal development of corticosteroid function of the adrenals in C57BL/6J-Ay mice

We studied postnatal development of corticosteroid function of the adrenals in mice during the period of elevated activity of the hypothalamic-pituitary-adrenal system and the influence of mutant gene Ay on this process. Normally, a corticosterone peak in blood and increased basal and stimulated steroidogenesis in vitro are observed in 3-week old mice. In 3-week old Ay/a mice (hyperexpression of protein agouti) a corticosterone peak in blood is lowered and genotypic differences in steroidogenesis in vitro are absent, as compared to a/A mice (absence of agouti), while at the ages of 10 and 15 weeks, there were no genotypic differences in the blood level of corticosterone and steroidogenesis in vitro was elevated. Thus, a high level of corticosterone during the period of elevated activity of the hypothalamic-pituitary-adrenal system in 3-week old mice is determined by enhanced steroidogenic function of the adrenals. Mutant gene Ay in male mice affected the postnatal development of the adrenal function: the peak of corticosterone in blood was lowered during the period of elevated activity of the system.     

Stimulation,Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E₁-E₂ conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E₂, but their detailed mechanisms of action are different. PE accelerates the rate of E₁ → E₂P but does not affect E₂(2K)ATP → E₁3NaATP, whereas sphingomyelin inhibits the rate of E₂(2K)ATP → E₁3NaATP, with very little effect on E₁ → E₂P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.     

A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly

TP53 (tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.     

Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex

Results reported here indicate that adenovirus 5 exploits the cellular aggresome response to accelerate inactivation of MRE11-RAD50-NBS1 (MRN) complexes that otherwise inhibit viral DNA replication and packaging. Aggresomes are cytoplasmic inclusion bodies, observed in many degenerative diseases, that are formed from aggregated proteins by dynein-dependent retrograde transport on microtubules to the microtubule organizing center. Viral E1B-55K protein forms aggresomes that sequester p53 and MRN in transformed cells and in cells transfected with an E1B-55K expression vector. During adenovirus infection, the viral protein E4orf3 associates with MRN in promyelocytic leukemia protein nuclear bodies before MRN is bound by E1B-55K. Either E4orf3 or E4orf6 is required in addition to E1B-55K for E1B-55K aggresome formation and MRE11 export to aggresomes in adenovirus-infected cells. Aggresome formation contributes to the protection of viral DNA from MRN activity by sequestering MRN in the cytoplasm and greatly accelerating its degradation by proteosomes following its ubiquitination by the E1B-55K/E4orf6/elongin BC/Cullin5/Rbx1 ubiquitin ligase. Our results show that aggresomes significantly accelerate protein degradation by the ubiquitin-proteosome system. The observation that a normal cellular protein is inactivated when sequestered into an aggresome through association with an aggresome-inducing protein has implications for the potential cytotoxicity of aggresome-like inclusion bodies in degenerative diseases.