The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay

The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.     

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.     

The genetics of the opioid system and specific drug addictions

Addiction to drugs is a chronic, relapsing brain disease that has major medical, social, and economic complications. It has been established that genetic factors contribute to the vulnerability to develop drug addiction and to the effectiveness of its treatment. Identification of these factors may increase our understanding of the disorders, help in the development of new treatments and advance personalized medicine. In this review, we will describe the genetics of the major genes of the opioid system (opioid receptors and their endogenous ligands) in connection to addiction to opioids, cocaine, alcohol and methamphetamines. Particular emphasis is given to association and functional studies of specific variants. We will provide information on the sample populations and the size of each study, as well as a list of the variants implicated in association with addiction-related phenotypes, and with the effectiveness of pharmacotherapy for addiction.     

Subcellular localization of RNA and proteins in prokaryotes

The field of bacterial cell biology has been revolutionized in the last decade by improvements in imaging capabilities which have revealed that bacterial cells, previously thought to be non-compartmentalized, possess an intricate higher-order organization. Many bacterial proteins localize to specific subcellular domains and regulate the spatial deployment of other proteins, DNA and lipids. Recently, the surprising discovery was made that bacterial RNA molecules are also specifically localized. However, the mechanisms that underlie bacterial cell architecture are just starting to be unraveled. The limited number of distribution patterns observed thus far for bacterial proteins and RNAs, and the similarity between the patterns exhibited by these macromolecules, suggest that the processes that underlie their localization are inextricably linked. We discuss these spatial arrangements and the insights that they provide on processes, such as localized translation, protein complex formation, and crosstalk between bacterial machineries.     

The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.     

Dihydrotestosterone and estradiol-17β mutually neutralize their inhibitory effects on human vascular smooth muscle cell growth in vitro

We reported previously that high concentrations of either estradiol-17β (E₂) or dihydrotestosterone (DHT) inhibit growth of human cultured vascular smooth muscle cells (VSMC), mediated by cell membrane receptors and MAP-kinase–kinase activity (MEK). We now tested whether the presence of the opposite gender's dominant sex hormone modifies these effects. We incubated VSMC with various concentrations of E₂ and DHT or protein bound hormones (E₂–BSA or T–BSA), alone or in various combinations. High concentration of E₂ or E₂–BSA inhibited VSMC growth and stimulated MEK. In the presence of 3 nM DHT, high concentration of E₂ no longer inhibited ³[H] thymidine incorporation or increased MEK. Moreover, when high DHT concentration (300 nM) was added to VSMC exposed to high E₂, VSMC growth actually increased without change in MEK. DHT at 300 nM suppressed VSMC growth and increased MEK while 0.3 nM E₂ had only marginal effect on this interaction, and 30 nM E₂ reversed the inhibitory effect of DHT on cell growth. The inhibitory effects of both E₂ and DHT on VSMC cell growth and the stimulation of MEK was apparently mediated by cell membrane receptors, as it persisted when bovine serum albumin (BSA)-bound hormones were used. Further, inhibition of VSMC growth induced by E₂–BSA was reversed in the presence of T–BSA and vice versa. These results suggest that while female and male sex hormones affect VSMC growth similarly, they interfere in a dose-, hormone- and MEK-dependent manner with each other's effect.