Oolemmal proteomics – identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane

BACKGROUND: The mature mouse egg contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins remain to be characterized, therefore in this study we have identified highly abundant egg proteins using a proteomic approach and found that several of these proteins also appear to localize to the egg surface. Characterization of such molecules will provide important insight into the cellular events of fertilization and early development. METHODS: In order to identify some of the more abundant egg proteins, whole egg extracts were resolved on coomassie-stained two-dimensional (2D) PAGE gels. Several highly abundant protein spots were cored and microsequenced by tandem mass spectrometry (TMS), and determined to be molecular chaperone proteins. Concurrent experiments were performed to identify oolemmal proteins using 2D avidin blotting. Proteins spots that appeared to be surface labeled by biotinylation were correlated with the initial coomassie-stained reference gel. Surprisingly, some of the surface labelled proteins corresponded to those abundant chaperone proteins previously identified. To confirm whether these molecules are accumulating at the oolemmal surface in eggs, we performed immunofluoresence on live, zona-free eggs using antibodies to HSP70, HSP90, GRP94, GRP78, calreticulin and calnexin. RESULTS: The putative surface-labeled proteins identified by biotinylation included the molecular chaperones HSP70 (MW 70 KDa, pI 5.5), HSP90a (MW 85 KDa, pI 4.9), GRP94 (MW 92 KDa, pI 4.7), GRP78 (MW 72 KDa, pI 5.0), Oxygen regulated protein 150 (ORP150; MW 111 KDa, pI 5.1), Calreticulin (MW 48 KDa, pI 4.3), Calnexin (MW 65 KDa, pI 4.5), and Protein disulfide isomerase (PDI; MW 57 KDa, pI 4.8). Immunofluoresence results showed that antibodies to HSP90, GRP94, GRP78 and calreticulin were reactive with oolemmal proteins. We were unable to confirm surface localization of HSP70 or calnexin by this method. CONCLUSIONS: We report here the identification of nine highly abundant molecular chaperones in the mouse egg proteome. In addition, we present preliminary data suggesting that these molecules localize to the oolemma of the mature mouse egg

Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation

The mechanisms involved in the regulation of mammalian sperm motility are not well understood. Calcium ions (Ca(2+)) have been suggested to play a key role in the maintenance of motility; nevertheless, how Ca(2+) modulates this process has not yet been completely characterized. Ca(2+) can bind to calmodulin and this complex regulates the activity of multiple enzymes, including Ca(2+)/calmodulin-dependent protein kinases (CaM kinases). Results from this study confirmed that the presence of Ca(2+) in the incubation medium is essential for maintaining human sperm motility. The involvement of CaM kinases in Ca(2+) regulation of human sperm motility was evaluated using specific inhibitors (KN62 and KN93) or their inactive analogues (KN04 and KN92 respectively). Sperm incubation in the presence of KN62 or KN93 led to a progressive decrease in the percentage of motile cells; in particular, incubation with KN62 also reduced sperm motility parameters. These inhibitors did not alter sperm viability, protein tyrosine phosphorylation or the follicular fluid-induced acrosome reaction; however, KN62 decreased the total amount of ATP in human sperm. Immunological studies showed that Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) is present and localizes to the human sperm flagellum. Moreover, CaMKIV activity increases during capacitation and is inhibited in the presence of KN62. This report is the first to demonstrate the presence of CaMKIV in mammalian sperm and suggests the involvement of this kinase in the regulation of human sperm motility.Fil: Marin Briggiler, Clara Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Jha, Kula N.. University of Virginia; Estados UnidosFil: Chertihin, Olga. University of Virginia; Estados UnidosFil: Buffone, Mariano Gabriel. Laboratorio de Estudios en Reproducción; ArgentinaFil: Herr, John C,. University of Virginia; Estados UnidosFil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Visconti, Pablo E.. University of Massachussets; Estados Unido

DEVELOPMENT OF STANDARDS FOR PLACEMENT OF STEEL GUARDRAIL POSTS IN ROCK

A steel post W-beam guardrail system was developed for installation in rock-soil foundations. The guardrail system was constructed with a 2.66-mm (12-gauge) thick W-beam rail, 53.34 m in length. The W-beam guardrail was supported by twenty-seven W152x13.4 by 1,346-mm long steel posts, spaced at 1,905 mm on center. The posts were installed in drilled holes in concrete, constructed by drilling three 203-mm diameter holes on 165- mm centers to a depth of 610 mm. The drilled holes were backfilled with compacted ASTM C33 coarse aggregate, size no. 57. One full-scale vehicle crash test, using a 3⁄4-ton pickup truck, was performed on the W-beam guardrail system. The test was conducted and reported in accordance with the requirements specified in the National Cooperative Highway Research Program (NCHRP) Report No. 350, Recommended Procedures for the Safety Performance Evaluation of Highway Features. The safety performance of the W-beam guardrail system with post placed in rock was determined to be acceptable according to the Test Level 3 (TL-3) evaluation criteria specified in NCHRP Report No. 350. Further, guardrail post placement recommendations were also developed for situations where rock is located below the surface. These recommendations were developed through an analysis of bogie testing of posts

Identification of Unknown Protein Function Using Metabolite Cocktail Screening

SummaryProteins of unknown function comprise a significant fraction of sequenced genomes. Defining the roles of these proteins is vital to understanding cellular processes. Here, we describe a method to determine a protein function based on the identification of its natural ligand(s) by the crystallographic screening of the binding of a metabolite library, followed by a focused search in the metabolic space. The method was applied to two protein families with unknown function, PF01256 and YjeF_N. The PF01256 proteins, represented by YxkO from Bacillus subtilis and the C-terminal domain of Tm0922 from Thermotoga maritima, were shown to catalyze ADP/ATP-dependent NAD(P)H-hydrate dehydratation, a previously described orphan activity. The YjeF_N proteins, represented by mouse apolipoprotein A-I binding protein and the N-terminal domain of Tm0922, were found to interact with an adenosine diphosphoribose-related substrate and likely serve as ADP-ribosyltransferases. Crystallographic screening of metabolites serves as an efficient tool in functional analyses of uncharacterized proteins

Report of the 1995 bunch train study group

In order to raise the luminosity LEP was operated in 1995 with four equidistant trains of bunches in each beam, instead of the usual four or eight single bunches. Each train consisted of up to four bunches. The bunch spacing was about 74 m. A comparison is made between the plans and expectations in the 1994 Bunch Train Report, and the actual implementation and observations in 1995. The observations made during machine development sessions and during routine operation for physics are discussed. The effects of the scheme on the background in the LEP experiments are anlysed. The performance of LEP equipment, in particular of beam instrumentation electrostatic separators, and the RF systems is presented. The plans for running LEP with bunch trains in 1996 are briefly outlined

An Integrated-Photonics Optical-Frequency Synthesizer

Integrated-photonics microchips now enable a range of advanced functionalities for high-coherence applications such as data transmission, highly optimized physical sensors, and harnessing quantum states, but with cost, efficiency, and portability much beyond tabletop experiments. Through high-volume semiconductor processing built around advanced materials there exists an opportunity for integrated devices to impact applications cutting across disciplines of basic science and technology. Here we show how to synthesize the absolute frequency of a lightwave signal, using integrated photonics to implement lasers, system interconnects, and nonlinear frequency comb generation. The laser frequency output of our synthesizer is programmed by a microwave clock across 4 THz near 1550 nm with 1 Hz resolution and traceability to the SI second. This is accomplished with a heterogeneously integrated III/V-Si tunable laser, which is guided by dual dissipative-Kerr-soliton frequency combs fabricated on silicon chips. Through out-of-loop measurements of the phase-coherent, microwave-to-optical link, we verify that the fractional-frequency instability of the integrated photonics synthesizer matches the $7.0*10^{-13}$ reference-clock instability for a 1 second acquisition, and constrain any synthesis error to $7.7*10^{-15}$ while stepping the synthesizer across the telecommunication C band. Any application of an optical frequency source would be enabled by the precision optical synthesis presented here. Building on the ubiquitous capability in the microwave domain, our results demonstrate a first path to synthesis with integrated photonics, leveraging low-cost, low-power, and compact features that will be critical for its widespread use.Comment: 10 pages, 6 figure

Search for composite and exotic fermions at LEP 2

A search for unstable heavy fermions with the DELPHI detector at LEP is reported. Sequential and non-canonical leptons, as well as excited leptons and quarks, are considered. The data analysed correspond to an integrated luminosity of about 48 pb^{-1} at an e^+e^- centre-of-mass energy of 183 GeV and about 20 pb^{-1} equally shared between the centre-of-mass energies of 172 GeV and 161 GeV. The search for pair-produced new leptons establishes 95% confidence level mass limits in the region between 70 GeV/c^2 and 90 GeV/c^2, depending on the channel. The search for singly produced excited leptons and quarks establishes upper limits on the ratio of the coupling of the excited fermio

Study of Tau-pair Production in Photon-Photon Collisions at LEP and Limits on the Anomalous Electromagnetic Moments of the Tau Lepton

Tau-pair production in the process e+e- -> e+e-tau+tau- was studied using data collected by the DELPHI experiment at LEP2 during the years 1997 - 2000. The corresponding integrated luminosity is 650 pb^{-1}. The values of the cross-section obtained are found to be in agreement with QED predictions. Limits on the anomalous magnetic and electric dipole moments of the tau lepton are deduced.Comment: 20 pages, 9 figures, Accepted by Eur. Phys. J.

CP asymmetry in B→ϕKSB \to \phi K_S in a general two-Higgs-doublet model with fourth-generation quarks

We discuss the time-dependent CP asymmetry of decay $B \to \phi K_S$ in an extension of the Standard Model with both two Higgs doublets and additional fourth-generation quarks. We show that although the Standard Model with two-Higgs-doublet and the Standard model with fourth generation quarks alone are not likely to largely change the effective $\sin 2 \beta$ from the decay of $B \to \phi K_S$, the model with both additional Higgs doublet and fourth-generation quarks can easily account for the possible large negative value of $\sin 2 \beta$ without conflicting with other experimental constraints. In this model, additional large CP violating effects may arise from the flavor changing Yukawa interactions between neutral Higgs bosons and the heavy fourth generation down type quark, which can modify the QCD penguin contributions. With the constraints obtained from $b \to s \bar{s} s$ processes such as $B \to X_s \gamma$ and $\Delta m_{B_s^0}$, this model can lead to the effective $\sin 2 \beta$ to be as large as $- 0.4$ in the CP asymmetry of $B \to \phi K_S$.Comment: 13 pages, 5 figures, references added, to appear in Eur.Phys.J.