Single vibronic level emission spectroscopic studies of the ground state energy levels and molecular structures of jet-cooled HGeBr, DGeBr, HGeI, and DGeI

Single vibronic level dispersed fluorescence spectra of jet-cooled HGeBr, DGeBr, HGeI, and DGeI have been obtained by laser excitation of selected bands of the à A″1-X̃ A′1 electronic transition. The measured ground state vibrational intervals were assigned and fitted to anharmonicity expressions, which allowed the harmonic frequencies to be determined for both isotopomers. In some cases, lack of a suitable range of emission data necessitated that some of the anharmonicity constants and vibrational frequencies be estimated from those of HGeCl∕DGeCl and the corresponding silylenes (HSiX). Harmonic force fields were obtained for both molecules, although only four of the six force constants could be determined. The ground state effective rotational constants and force field data were combined to calculate average (rz) and approximate equilibrium (rze) structures. For HGeBr rze(GeH)=1.593(9)Å, rze(GeBr)=2.325(21)Å, and the bond angle was fixed at our CCSD(T)/aug-cc-pVTZ ab initio value of 93.6°. For HGeI we obtained rze(GeH)=1.589(1)Å, rze(GeI)=2.525(5)Å, and bond angle=93.2°. Franck-Condon simulations of the emission spectra using ab initio Cartesian displacement coordinates reproduce the observed intensity distributions satisfactorily. The trends in structural parameters in the halogermylenes and halosilylenes can be readily understood based on the electronegativity of the halogen substituent. ACKNOWLEDGMENT

Proteomic measures of gamma oscillations

Background: Gamma oscillations serve complex processes, and the first stage of their generation is the reticular activating system (RAS), which mediates the gamma-activity states of waking and paradoxical sleep. We studied whether the pedunculopontine nucleus (PPN), part of the RAS in which every cell manifests intrinsic gamma oscillations, undergoes changes resulting in distinctive protein expression. New method: We previously found that a histone deacetylation inhibitor, trichostatin A (TSA), acutely (30 min) blocked these oscillations. We developed a proteomic method for sampling stimulated and unstimulated PPN and determining protein expression in 1 mm punches of tissue from brain slices subjected to various treatments. Results: We compared brain slices exposed for 30 min to TSA (unstimulated), to the cholinergic agonist carbachol (CAR), known to induce PPN gamma oscillations, or exposed to both TSA + CAR. Comparison with existing methods: Label-free proteomics provides an unbiased and sensitive method to detect protein changes in the PPN. Our approach is superior to antibody-based methods that can lack specificity and can only be done for known targets. Proteomics methods like these have been leveraged to study molecular pathways in numerous systems and disease states. Conclusions: Significant protein changes were seen in two functions essential to the physiology of the PPN: cytoskeletal and intracellular [Ca2+] regulation proteins. TSA decreased, while CAR increased, and TSA + CAR had intermediate effects, on expression of these proteins. These results support the feasibility of the methods developed for determining proteomic changes in small samples of tissue participating in the most complex of brain processes.Fil: Byrum, Stephanie D.. Arkansas Children's Research Institute; Estados UnidosFil: Washam, Charity L.. Arkansas Children's Research Institute; Estados UnidosFil: Tackett, Alan J.. Arkansas Children's Research Institute; Estados UnidosFil: Garcia Rill, Edgar. University of Arkansas for Medical Sciences; Estados UnidosFil: Bisagno, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Urbano Suarez, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome

Time-resolved ChIP-chip can be utilized to monitor the genome-wide dynamics of the GINS complex, yielding quantitative information on replication fork movement.Replication forks progress at remarkably uniform rates across the genome, regardless of location.GINS progression appears to be arrested, albeit with very low frequency, at sites of highly transcribed genes.Comparison of simulation with data leads to novel biological insights regarding the dynamics of replication fork progressio

A proteomic study of human Merkel Cell Carcinoma

Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies

A PWWP Domain-Containing Protein Targets the NuA3 Acetyltransferase Complex via Histone H3 Lysine 36 trimethylation to Coordinate Transcriptional Elongation at Coding Regions

Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5′-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with identifier PXD001156