Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Azimuthal asymmetry in the risetime of the surface detector signals of the Pierre Auger Observatory.

The azimuthal asymmetry in the risetime of signals in Auger surface detector stations is a source of information on shower development. The azimuthal asymmetry is due to a combination of the longitudinal evolution of the shower and geometrical effects related to the angles of incidence of the particles into the detectors. The magnitude of the effect depends upon the zenith angle and state of development of the shower and thus provides a novel observable, (sec theta)(max), sensitive to the mass composition of cosmic rays above 3 x 10(18) eV. By comparing measurements with predictions from shower simulations, we find for both of our adopted models of hadronic physics (QGSJETII-04 and EPOS-LHC) an indication that the mean cosmic-ray mass increases slowly with energy, as has been inferred from other studies. However, the mass estimates are dependent on the shower model and on the range of distance from the shower core selected. Thus the method has uncovered further deficiencies in our understanding of shower modeling that must be resolved before the mass composition can be inferred from (sec theta)(max).

Expression and cellular localization of the voltage-gated calcium channel α2δ3 in the rodent retina

High-voltage-activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes, including the influx of extracellular Ca2+, neurotransmitter release, gene transcription, and synaptic plasticity. These channels consist of a primary α1 pore-forming subunit, which is associated with an extracellular α2δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α2δ3 subunit in the mouse and rat retina is unknown. In this study using RT-PCR, a single band at ∼305 bp corresponding to the predicted size of the α2δ3 subunit fragment was found in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123-kDa band. Immunohistochemistry with an affinity-purified antibody to the α2δ3 subunit revealed immunoreactive cell bodies in the ganglion cell layer and inner nuclear layer and immunoreactive processes in the inner plexiform layer and the outer plexiform layer. α2δ3 immunoreactivity was localized to multiple cell types, including ganglion, amacrine, and bipolar cells and photoreceptors, but not horizontal cells. The expression of the α2δ3 calcium channel subunit to multiple cell types suggests that this subunit participates widely in Ca-channel-mediated signaling in the retina.

Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas,. including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16. (C) 2004 Elsevier Inc. All rights reserved.

APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

Noninvasive localization of petroleum-derived spray oil in plants with chemical shift selective magnetic resonance imaging

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to detect petroleum-derived spray oils (PDSOs) in citrus seedlings and trees. The NMR spectrum of the phantom containing 10% (v/v) of a nC24 agricultural mineral oil (AMO) showed the resonance of the water protons at delta = 5 ppm, while the resonance of the oil protons at delta = 1.3 to 1.7 ppm. The peak resolution and the chemical shift difference of more than 3.3 ppm between water and oil protons effectively differentiated water and the oil. Chemical shift selective imaging (CSSI) was performed to localize the AMO within the stems of Citrus trifoliata L. seedlings after the application of a 4% (v/v) spray. The chemical shift selective images of the oil were acquired by excitation at delta = 1.5 ppm by averaging over 400 transients in each phase-encoding step. Oil was mainly detected in the outer cortex of stems within 10 d of spray application; some oil was also observed in the inner vascular bundle and pith of the stems at this point. CSSI was also applied to investigate the persistence of oil deposits in sprayed mature Washington navel orange (Citrus x aurantium L.) trees in an orchard. The trees were treated with either fourteen 0.25%, fourteen 0.5%, four 1.75%, or single 7% sprays of a nC23 horticultural mineral oil (HMO) 12 to 16 months before examination of plant tissues by CSSI, and were still showing symptoms of chronic phytotoxicity largely manifested as reduced yield. The oil deposits were detected in stems of sprayed flushes and unsprayed flushes produced 4 to 5 months after the last spray was applied, suggesting a potential movement of the oil via phloem and a correlation of the persistence of oil deposit in plants and the phytotoxicity. The results demonstrate that MRI is an effective method to probe the uptake and localization of PDSOs and other xenobiotics in vivo in plants noninvasively and nondestructively.

Subcellular localization determines MAP kinase signal output

The Raf-MEK-ERK MAP kinase cascade transmits signals from activated receptors into the cell to regulate proliferation and differentiation. The cascade is controlled by the Ras GTPase, which recruits Raf from the cytosol to the plasma membrane for activation. In turn, MEK, ERK, and scaffold proteins translocate to the plasma membrane for activation. Here, we examine the input-output properties of the Raf-MEK-ERK MAP kinase module in mammalian cells activated in different cellular contexts. We show that the MAP kinase module operates as a molecular switch in vivo but that the input sensitivity of the module is determined by subcellular location. Signal output from the module is sensitive to low-level input only when it is activated at the plasma membrane. This is because the threshold for activation is low at the plasma membrane, whereas the threshold for activation is high in the cytosol. Thus, the circuit configuration of the module at the plasma membrane generates maximal outputs from low-level analog inputs, allowing cells to process and respond appropriately to physiological stimuli. These results reveal the engineering logic behind the recruitment of elements of the module from the cytosol to the membrane for activation.

The prion protein gene: Identifying regulatory signals using marsupial sequence

The function of the prion protein gene (PRNP) and its normal product PrPC is elusive. We used comparative genomics as a strategy to understand the normal function of PRNP. As the reliability of comparisons increases with the number of species and increased evolutionary distance, we isolated and sequenced a 66.5 kb BAC containing the PRNP gene from a distantly related mammal, the model Australian marsupial Macropus eugenii (tammar wallaby). Marsupials are separated from eutherians such as human and mouse by roughly 180 million years of independent evolution. We found that tammar PRNP, like human PRNP, has two exons. Prion proteins encoded by the tammar wallaby and a distantly related marsupial, Monodelphis domestica (Brazilian opossum) PRNP contain proximal PrP repeats with a distinct, marsupial-specific composition and a variable number. Comparisons of tammar wallaby PRNP with PRNPs from human, mouse, bovine and ovine allowed us to identify non-coding gene regions conserved across the marsupial-eutherian evolutionary distance, which are candidates for regulatory regions. In the PRNP 3' UTR we found a conserved signal for nuclear-specific polyadenylation and the putative cytoplasmic polyadenylation element (CPE), indicating that post-transcriptional control of PRNP mRNA activity is important. Phylogenetic footprinting revealed conserved potential binding sites for the MZF-1 transcription factor in both upstream promoter and intron/intron 1, and for the MEF2, MyTI, Oct-1 and NFAT transcription factors in the intron(s). The presence of a conserved NFAT-binding site and CPE indicates involvement of PrPC in signal transduction and synaptic plasticity. (c) 2004 Elsevier B.V. All rights reserved.

Prediction of subcellular localization using sequence-biased recurrent networks

Motivation: Targeting peptides direct nascent proteins to their specific subcellular compartment. Knowledge of targeting signals enables informed drug design and reliable annotation of gene products. However, due to the low similarity of such sequences and the dynamical nature of the sorting process, the computational prediction of subcellular localization of proteins is challenging. Results: We contrast the use of feed forward models as employed by the popular TargetP/SignalP predictors with a sequence-biased recurrent network model. The models are evaluated in terms of performance at the residue level and at the sequence level, and demonstrate that recurrent networks improve the overall prediction performance. Compared to the original results reported for TargetP, an ensemble of the tested models increases the accuracy by 6 and 5% on non-plant and plant data, respectively.

Orphan nuclear receptors: therapeutic opportunities in skeletal muscle

Orphan nuclear receptors: therapeutic opportunities in skeletal muscle. Am J Physiol Cell Physiol 291: C203-C217, 2006; doi: 10.1152/ajpcell. 00476.2005.-Nuclear hormone receptors (NRs) are ligand-dependent transcription factors that bind DNA and translate physiological signals into gene regulation. The therapeutic utility of NRs is underscored by the diversity of drugs created to manage dysfunctional hormone signaling in the context of reproductive biology, inflammation, dermatology, cancer, and metabolic disease. For example, drugs that target nuclear receptors generate over $10 billion in annual sales. Almost two decades ago, gene products were identified that belonged to the NR superfamily on the basis of DNA and protein sequence identity. However, the endogenous and synthetic small molecules that modulate their action were not known, and they were denoted orphan NRs. Many of the remaining orphan NRs are highly enriched in energy-demanding major mass tissues, including skeletal muscle, brown and white adipose, brain, liver, and kidney. This review focuses on recently adopted and orphan NR function in skeletal muscle, a tissue that accounts for similar to 35% of the total body mass and energy expenditure, and is a major site of fatty acid and glucose utilization. Moreover, this lean tissue is involved in cholesterol efflux and secretes that control energy expenditure and adiposity. Consequently, muscle has a significant role in insulin sensitivity, the blood lipid profile, and energy balance. Accordingly, skeletal muscle plays a considerable role in the progression of dyslipidemia, diabetes, and obesity. These are risk factors for cardiovascular disease, which is the the foremost cause of global mortality (> 16.7 million deaths in 2003). Therefore, it is not surprising that orphan NRs and skeletal muscle are emerging as therapeutic candidates in the battle against dyslipidemia, diabetes, obesity, and cardiovascular disease.

Evaluation and comparison of mammalian subcellular localization prediction methods

Background: Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results: In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, and lysosome). The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion: No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE dataset and variable performance on individual subcellular localizations was observed. Proteins localized to the secretory pathway were the most difficult to predict, while nuclear and extracellular proteins were predicted with the highest sensitivity.

Nuclear translocation of cytochrome c during apoptosis

Release of cytochrome c from mitochondria is a major event during apoptosis. Released cytochrome c has been shown to activate caspase-dependent apoptotic signals. In this report, we provide evidence for a novel role of cytochrome c in caspase-independent nuclear apoptosis. We showed that cytochrome c, released from mitochondria upon apoptosis induction, gradually accumulates in the nucleus as evidenced by both immunofluorescence and subcellular fractionation. Parallel to nuclear accumulation of cytochrome c, acetylated histone H2A, but not unmodified H2A, was released from the nucleus to the cytoplasm. Addition of purified cytochrome c to isolated nuclei recapitulated the preferential release of acetylated, but not deacetylated, histone H2A. Cytochrome c was also found to induce chromatin condensation. These results suggest that the nuclear accumulation of cytochrome c may be directly involved in the remodeling of chromatin. Our results provide evidence of a novel role for cytochrome c in inducing nuclear apoptosis.

The relative contributions of colour and luminance signals towards the visuomotor localisation of targets in human peripheral vision

We sought to determine the extent to which colour (and luminance) signals contribute towards the visuomotor localization of targets. To do so we exploited the movement-related illusory displacement a small stationary window undergoes when it has a continuously moving carrier grating behind it. We used drifting (1.0-4.2 Hz) red/green-modulated isoluminant gratings or yellow/black luminance-modulated gratings as carriers, each curtailed in space by a stationary, two-dimensional window. After each trial, the perceived location of the window was recorded with reference to an on-screen ruler (perceptual task) or the on-screen touch of a ballistic pointing movement made without visual feedback (visuomotor task). Our results showed that the perceptual displacement measures were similar for each stimulus type and weakly dependent on stimulus drift rate. However, while the visuomotor displacement measures were similar for each stimulus type at low drift rates (<4 Hz), they were significantly larger for luminance than colour stimuli at high drift rates (>4 Hz). We show that the latter cannot be attributed to differences in perceived speed between stimulus types. We assume, therefore, that our visuomotor localization judgements were more susceptible to the (carrier) motion of luminance patterns than colour patterns. We suggest that, far from being detrimental, this susceptibility may indicate the operation of mechanisms designed to counter the temporal asynchrony between perceptual experiences and the physical changes in the environment that give rise to them. We propose that perceptual localisation is equally supported by both colour and luminance signals but that visuomotor localisation is predominantly supported by luminance signals. We discuss the neural pathways that may be involved with visuomotor localization. © 2007 Springer-Verlag.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Using Coding to Improve Localization and Backscatter Communication Performance in Low-Power Sensor Networks

Backscatter communication is an emerging wireless technology that recently has gained an increase in attention from both academic and industry circles. The key innovation of the technology is the ability of ultra-low power devices to utilize nearby existing radio signals to communicate. As there is no need to generate their own energetic radio signal, the devices can benefit from a simple design, are very inexpensive and are extremely energy efficient compared with traditional wireless communication. These benefits have made backscatter communication a desirable candidate for distributed wireless sensor network applications with energy constraints.

The backscatter channel presents a unique set of challenges. Unlike a conventional one-way communication (in which the information source is also the energy source), the backscatter channel experiences strong self-interference and spread Doppler clutter that mask the information-bearing (modulated) signal scattered from the device. Both of these sources of interference arise from the scattering of the transmitted signal off of objects, both stationary and moving, in the environment. Additionally, the measurement of the location of the backscatter device is negatively affected by both the clutter and the modulation of the signal return.

This work proposes a channel coding framework for the backscatter channel consisting of a bi-static transmitter/receiver pair and a quasi-cooperative transponder. It proposes to use run-length limited coding to mitigate the background self-interference and spread-Doppler clutter with only a small decrease in communication rate. The proposed method applies to both binary phase-shift keying (BPSK) and quadrature-amplitude modulation (QAM) scheme and provides an increase in rate by up to a factor of two compared with previous methods.

Additionally, this work analyzes the use of frequency modulation and bi-phase waveform coding for the transmitted (interrogating) waveform for high precision range estimation of the transponder location. Compared to previous methods, optimal lower range sidelobes are achieved. Moreover, since both the transmitted (interrogating) waveform coding and transponder communication coding result in instantaneous phase modulation of the signal, cross-interference between localization and communication tasks exists. Phase discriminating algorithm is proposed to make it possible to separate the waveform coding from the communication coding, upon reception, and achieve localization with increased signal energy by up to 3 dB compared with previous reported results.

The joint communication-localization framework also enables a low-complexity receiver design because the same radio is used both for localization and communication.

Simulations comparing the performance of different codes corroborate the theoretical results and offer possible trade-off between information rate and clutter mitigation as well as a trade-off between choice of waveform-channel coding pairs. Experimental results from a brass-board microwave system in an indoor environment are also presented and discussed.