Parallel beta-sheets and polar zippers in amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p

We report the results of solid-state nuclear magnetic resonance (NMR) and atomic force microscopy measurements on amyloid fibrils formed by residues 10-39 of the yeast prion protein Ure2p (Ure2p(10)(-)(39)). Measurements of intermolecular (13)C-(13)C nuclear magnetic dipole-dipole couplings indicate that Ure2p(10)(-)(39) fibrils contain in-register parallel beta-sheets. Measurements of intermolecular (15)N-(13)C dipole-dipole couplings, using a new solid-state NMR technique called DSQ-REDOR, are consistent with hydrogen bonds between side chain amide groups of Gln18 residues. Such side chain hydrogen bonding interactions have been called "polar zippers" by M. F. Perutz and have been proposed to stabilize amyloid fibrils formed by peptides with glutamine- and asparagine-rich sequences, such as Ure2p(10)(-)(39). We propose that polar zipper interactions account for the in-register parallel beta-sheet structure in Ure2p(10)(-)(39) fibrils and that similar peptides will also exhibit parallel beta-sheet structures in amyloid fibrils. We present molecular models for Ure2p(10)(-)(39) fibrils that are consistent with available experimental data. Finally, we show that solid-state (13)C NMR chemical shifts for (13)C-labeled Ure2p(10)(-)(39) fibrils are insensitive to hydration level, indicating that the fibril structure is not affected by the presence or absence of bulk water.     

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.     

Feasibility of using the national hospital discharge survey to estimate the prevalence of selected birth defects

BACKGROUND: Nationally representative data on the prevalence of certain birth defects are largely unavailable. We evaluated the feasibility of using data from the National Hospital Discharge Survey (NHDS) to describe the prevalence of selected birth defects. METHODS: All live births recorded in the NHDS during 1999-2001 were included. The prevalence for selected birth defects was calculated using weighted ratio estimators. Prevalence ratios comparing the NHDS estimates to published national estimates from the National Birth Defects Prevention Network (NBDPN) were calculated. RESULTS: With the exception of common truncus, the NHDS prevalence for the selected defects was consistently lower than the NBDPN estimates. The prevalence ratios ranged from 0.38 for trisomy 18 and anopthalmia/micropthalmia to 1.16 for common truncus. The NHDS prevalence estimates for spina bifida without anencephaly (PR 0.89, 95% CI: 0.57-1.22) and gastroschisis/omphalocele (PR 0.94, 95% CF: 0.48-1.40) most closely approximated the NBDPN estimates. CONCLUSIONS: NHDS data underestimate the prevalence of most birth defects. Additional research is needed to determine whether NHDS estimates may be useful for evaluating trends in certain conditions. Surveillance systems employing active case-finding continue to provide more accurate estimates of birth defects prevalence.     

Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning

We report solid-state nuclear magnetic resonance (NMR) measurements on the peptide Vpu(1-40), comprising residues 1-40 of the 81-residue type 1 integral membrane protein Vpu encoded by the HIV-1 genome. On the basis of a combination of 13C and 15N NMR chemical shifts under magic-angle spinning (MAS), effects of local mobility on NMR signal intensities, site-specific MAS NMR line widths, and NMR-detected hydrogen-deuterium exchange, we develop a model for the structure and dynamics of the Vpu(1-40) monomer in phospholipid bilayer membranes. Our data are largely consistent with earlier structural studies of Vpu peptides by Opella and co-workers, in which solution NMR and solid-state NMR without MAS were used, but our data provide new information about local variations in the degree of mobility and structural order. In addition, our data indicate that the transmembrane alpha-helix of Vpu(1-40) extends beyond the hydrophobic core of the bilayer. We find no evidence for heterogeneity in the conformation and intermolecular contacts of the transmembrane alpha-helix, with the exception of two distinct chemical shifts observed for the C alpha and C beta atoms of A18 that may reflect distinct modes of helix-helix interaction. These results have possible implications for the supramolecular structure of Vpu oligomers that form cation-selective ion channels.     

Estrogen controls embryonic stem cell proliferation via store-operated calcium entry and the nuclear factor of activated T-cells (NFAT)

Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into all cell lineages. Calcium is a universal second messenger which regulates a number of cellular pathways. Previous studies showed that store-operated calcium channels (SOCCs) but not voltage-operated calcium channels are present in mouse ESCs (mESCs). In this study, store-operated calcium entry (SOCE) was found to exist in mESCs using confocal microscopy. SOCC blockers lanthanum, 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365 reduced mESC proliferation in a concentration-dependent manner, suggesting that SOCE is important for ESC proliferation. Pluripotent markers, Sox-2, Klf-4, and Nanog, were down-regulated by 2-APB, suggesting that self-renewal property of mESCs relies on SOCE. 17β-estradiol (E2) enhanced mESC proliferation. This enhanced proliferation was associated with an increment of SOCE. Both stimulated proliferation and increased SOCE could be reversed by SOCC blockers suggesting that E2 mediates its stimulatory effect on proliferation via enhancing SOCE. Also, cyclosporin A and INCA-6, inhibitors of calcineurin [phosphatase that de-phosphorylates and activates nuclear factor of activated T-cells (NFAT)], reversed the proliferative effect of E2, indicating that NFAT is involved in E2-stimulated proliferation. Interestingly, E2 caused the nuclear translocation of NFATc4, and this could be reversed by 2-APB. These results suggested that NFATc4 is the downstream target of E2-induced SOCE. The present investigation provides the first line of evidence that SOCE and NFAT are crucial for ESCs to maintain their unique characteristics. In addition, the present investigation also provides novel information on the mechanisms of how E2, an important female sex hormone, affects ESC proliferation.     

Photochemical nature of parietopsin

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV-visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an "evolutionary intermediate" between invertebrate and vertebrate visual opsins.     

The Stem Region of Premembrane Protein Plays an Important Role in the Virus Surface Protein Rearrangement during Dengue Maturation

Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111–131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.