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A comprehensive proteome map of growing Bacillus subtilis cells.

The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B. subtilis genes, 2515 were actively transcribed in cells grown under standard conditions. From these genes 1544 proteins should be covered by our standard gel system pI 4-7. Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region. Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis. Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots. By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications. In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. The vegetative proteome containing 876 proteins in total is now ready for physiological applications. Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.

Bacillus subtilis↗

Developmental changes in the metabolic protein profiles of wheat endosperm.

A combined two-dimensional gel electrophoresis-mass spectrometry approach was utilized to identify over 250 proteins of wheat (Triticum aestivum L., cv. Butte 86) starchy endosperm that participate in 13 biochemical processes: ATP interconversion reactions, carbohydrate metabolism, cell division, cytoskeleton, lipid metabolism, nitrogen metabolism, protein synthesis/assembly, protein turnover, signal transduction, protein storage, stress/defense, transcription/translation, and transport. Endosperm protein populations were compared at early (10 days post-anthesis, dpa) and late (36 dpa) stages of grain development. Analysis of protein number and spot volume revealed that carbohydrate metabolism, transcription/translation, and protein synthesis/assembly were the principal endosperm functions at 10 dpa followed by nitrogen metabolism, protein turnover, cytoskeleton, cell division, signal transduction, and lipid metabolism. Carbohydrate metabolism and protein synthesis/assembly were also major functions at 36 dpa, but stress/defense and storage were predominant. The results provide insight into biochemical events taking place during wheat grain development and highlight the value of proteomics in characterizing complex biochemical processes. Further, the proteome maps will facilitate future studies addressing the effects of genetic and environmental factors on the development and quality of wheat grain.

Electrophoresis, Gel, Two-Dimensional↗

Plant proteome analysis: a 2004-2006 update.

Since the appearance of the review entitled "Plant Proteome Analysis" in Proteomics in February 2004 (Cánovas, F. M., Dumas-Gaudot, E., Recorbert, G., Jorrín, J. et al., Proteomics 2004, 4, 285-298), about 200 original articles focusing on plant proteomics have been published. Although this represents less than 1% of the global proteomics output during this period, it nevertheless reflects an increase in activity over the period 1999-2004. These papers concern the proteome of at least 35 plant species but have concentrated mainly on thale cress (Arabidopsis thaliana) and rice (Oryza sativa). The scientific objectives have ranged from a proteomic analysis of organs, tissues, cell suspensions, or subcellular fractions to the study of plant development and response to various stresses. A number of contributions have covered PTMs and protein interactions. The dominant analytical platform has been 2-DE coupled to MS, but "second generation" techniques such as DIGE, multidimensional protein identification technology, isotope-coded affinity tags, and stable isotope labeling by amino acids in cell culture have begun to make an impact. This review aims to provide an update of the contribution of proteomics to plant biology during the period 2004-2006, and is divided into six sections: introduction, subcellular proteomes, plant development, responses to biotic and abiotic stresses, PTMs, and protein interactions. The conclusions summarize a view of the major pitfalls and challenges of plant proteomics.

Arabidopsis↗

Sentence comprehension in Alzheimer's disease.

We asked 22 patients with Alzheimer's disease (AD) to respond to simple probes of sentences where we manipulated grammatical factors, semantic factors, and cognitive resource demands associated with a sentence. The results demonstrated limitations in the cognitive resources needed to appreciate atypical syntactic-thematic mapping relations and difficulty processing selection restrictions associated with a verb. By comparison, comprehension in AD was not influenced by the active or passive voice of a sentence. These findings are consistent with the hypothesis that impaired sentence comprehension in AD is multifactorial in nature, including difficulty processing cognitive resource and semantic aspects of sentences.

Aged↗

Trypanosoma brucei: inheritance of kinetoplast DNA maxicircles in a genetic cross and their segregation during vegetative growth.

The inheritance of maxicircle DNA was determined using a polymorphic EcoRI restriction site in the maxicircle variable region. In 11 hybrid progeny from a genetic cross of two stocks of Trypanosoma brucei, 7 progeny apparently inherited maxicircles uniparentally from either parent, in agreement with the results from previous studies, but in 4 progeny inheritance was biparental. Three subclones from 2 of these 4 progeny were made, and in these the maxicircles of only one parental type were detected. These data are considered in terms of a simple model whereby half the maxicircle genomes are inherited from each parent into progeny at meiosis with subsequent stochastic segregation at each mitotic division. This model generates a quantitative prediction as to the period of time required for fixation of inheritance to a uniparental pattern which provides a reasonable fit to the experimental data. These results provide an explanation as to why previous studies have shown that maxicircles are (apparently) inherited uniparentally: the kinetoplast is a unitary organelle inherited faithfully at cell division, but the maxicircles that it contains are best considered as a population that divides stochastically. Consideration of the model also explains why maxicircle populations are homogeneous.

Animals↗

Plasmodium falciparum: gp195 tripeptide repeat-specific monoclonal antibody inhibits parasite growth in vitro.

Seven monoclonal antibodies (mAbs) were produced to the precursor of the merozoite surface antigens (MSA-1 or gp195) using the Plasmodium falciparum Uganda-Palo Alto isolate. Three mAbs (CE2, DB8, and EB2) reacted with epitopes on the 83-kDa N-terminal processing fragment by immunoprecipitation of radiolabeled proteins and in immunoblots of native and recombinant proteins. Three other mAbs (BC9, AG5, and AD9) reacted with epitopes on the 42- and 19-kDa C-terminal processing fragments while one remaining mAb (24A1.7) reacted with only 150- and 110-kDa intermediate processing fragments. Epitopes were mapped to either conserved or dimorphic regions of the expressed protein when parasite isolates with known MSA-1 alleles were examined by indirect immunofluorescence. Moreover, one mAb (CE2), specific for the variable tripeptide repeat region SAQ(SGT)5, was growth inhibitory for P. falciparum in vitro. Growth inhibition by the mAb was concentration dependent and its parasite-neutralizing properties were not enhanced when used in combination with other gp195-specific mAbs. These results may be useful in the elucidation of biological variation of field isolates and in the definition of immunologically relevant epitopes in a gp195-based malaria vaccine.

Amino Acid Sequence↗

BAC trimming: minimizing clone overlaps.

Bacterial vectors containing large inserts of genomic DNA are now the standard substrates for large-scale genomic sequencing. Long overlaps between some clones lead to considerable redundant effort. A method for deleting defined regions from bacterial artificial chromosome (BAC) inserts, using homologous recombination, was applied to minimize the overlap between successive BAC clones. This procedure, called trimming, was carried out in the recA(-) BAC host. We have precisely deleted up to 70 kb of DNA from BACs that were to be sequenced. This method requires minimal prior characterization of the clones: collections of BAC end sequences or STS-based maps will accelerate the process. BAC trimming will be useful in both small and large genome sequencing projects and will be of particular utility for gap closure in finishing phases.

Arabidopsis↗

Signal source separation in the analysis of neural activity in brain.

This research examines the spatial and temporal characteristics of the responses to stimulation of the barrel cortex in anesthetized rats using optical imaging with particular emphasis on methods of analysis which reduce the effects of low-frequency oscillations on localization of the activated cortical region. Image sequences were captured using a light source with a narrow bandwidth of wavelength (590 +/- 2 nm). On each trial image data were collected at 15 Hz and stored over a 12-s period starting 8 s before stimulation onset. Stimulation was for 1 s using an oscillating whisker vibrator (approximately 1-mm deflection at 5 Hz). For each subject a total of 30 experimental trials were collected and averaged. There was an interstimulus interval of 26 s. The trial-averaged data were analyzed using two related signal source separation algorithms. Both algorithms use a weak model of the expected temporal response as a filter to exclude contributions from low-frequency baseline oscillations which we call the V-signal. We found that both algorithms successfully separated most of the effects of the V-signal from the response to the stimulation. The performance of the algorithms compared favorably with the performance of related algorithms without weak constraints and the "ratio of means" strategy used by C. H. Chen-Bee et al. (1996b, J. Neurosci. Methods 68:28-37; C. H. Chen-Bee et al., 2000, J. Neurosci. Methods 97:157-173).

Algorithms↗

Functional brain mapping of monkey tool use.

When using a tool, we can perceive a psychological association between the tool and the body parts-the tool is incorporated into our "body-image." During tool use, visual response properties of bimodal (tactile and visual) neurons in the intraparietal area of the monkey's cerebral cortex were modified to include the hand-held tool. Visual properties of the monkey intraparietal neurons may represent the body-image in the brain. We explored tool use-induced activation within the intraparietal area and elsewhere in alert monkey brain using positron emission tomography (PET). Tool use-related activities compared with the control condition (simple-stick manipulation) revealed a significant increase in cerebral blood flow in the corresponding intraparietal region, basal ganglia, presupplementary motor area, premotor cortex, and cerebellum. These tool use-specific areas may participate in maintaining and updating the body-image for the precise guidance of a hand-held rake onto a distant reward.

Animals↗

Primate cytomegaloviruses encode and express an IL-10-like protein.

An open reading frame (ORF) with homology to interleukin-10 (IL-10) has been identified in rhesus cytomegalovirus (RhCMV). The IL-10-like protein is generated from a multispliced, polyadenylated early gene transcript encompassing part of the corresponding UL111A ORF of human CMV (HCMV). Immunological analyses confirm expression of the IL-10-like protein both in tissue culture and in RhCMV-infected rhesus macaques. Conserved ORFs were subsequently identified in human, baboon, and African green monkey CMV, and a fully processed transcript has been mapped in fibroblasts infected with the Towne strain of HCMV. The conservation of this previously unrecognized ORF suggests that the protein may play an essential role in primate CMV persistence and pathogenesis.

Amino Acid Sequence↗

Some new phenomena in the perception of glass patterns.

Glass patterns are dot pattern displays producing percepts of local orientation and global organization. Our observations suggest that the organizational principles involved in our perception of such patterns may be more consistent with a cortical simple-cell-like mechanisms (i.e., a king of neural summation within various feature maps) than with symbolic processing based on feature similarity.

Humans↗

Accuracy of an experimental stereotactic system for MRI-based gamma knife irradiation in the rat.

Stereotactic devices for experimental Gamma Knife irradiation and magnetic resonance imaging (MRI) have recently been developed for experimental studies using rats [6,7]. The present study examined the accuracy of these devices using the following two approaches. In the first approach, Gamma Knife irradiation was performed using the stereotactic device with targets based on a standard stereotactic atlas. Gadolinium-enhanced T1-weighted magnetic resonance imaging was performed using the MRI stereotactic device. Animals were then sacrificed after Evans blue injection, and the rat brain was sliced using an attachment to the stereotactic device. The center coordinates of the gadolinium-enhanced area from the MRI and Evans blue-stained area from the tissue sections were obtained using a computer-assisted image analysis system. These coordinates were compared with the target coordinates planned from the stereotactic atlas. In the second approach, a thermoluminescence dosimeter was implanted in the rat brain. Stereotactic MRI was performed using the stereotactic MRI device, and the coordinates of the implant were obtained. Gamma Knife irradiation was then performed at this target using the stereotactic device. The absorbed dose was measured and compared with the planning dose. These experiments demonstrated a spatial error of 0.6 mm (standard error +/- 0.07) between Gamma Knife irradiation based on a comparison of the atlas coordinates and the lesion, and a spatial error of 1.0 mm (standard error +/- 0.13) based on a comparison of the stereotactic MR images and the lesion. Gamma Knife irradiation based on MR images using the stereotactic device demonstrated a maximum error of 10% in absorbed dose at the target center. Together, the stereotactic devices for Gamma Knife irradiation and magnetic resonance imaging provide useful tools for Gamma Knife research in an animal model.

Animals↗

A vector-sum process produces curved aiming paths under rotated visual-motor mappings.

Under a 90 degrees rotation of motor space relative to visual space, human two-dimensional aiming movements frequently take the form of smooth arcs such as spirals and semi-circles. A time-independent differential equation explains this tendency in terms of a rotation-induced vector field made up, at each point in the two-dimensional space, of two input vectors. One vector represents a visual error signal and the other represents a motor error signal. A trajectory's instantaneous direction of movement at each point can be described as the resultant of the two vectors. This mathematical formulation incorporates plausible visual-motor mechanisms and, when expressed in polar coordinates, leads to a new method for analyzing the spatial properties of movements (i.e., movement paths). Plots of the angle between the resultant and the target vector (phi) against distance from the target (r, in the polar representation) summarize the arc-shaped movement paths as a simple relation that can be analyzed statistically with respect to properties such as monotonicity. The polar representation is a plausible representation of visually-guided movements, with the visual error vector functioning as an objective function relative to which behavior is optimized. We extend the model and the r, phi movement path analysis to non-90 degrees rotations, and we find that the model predicts an observed qualitative shift in behavior for rotations greater than 90 degrees. It also predicts qualitatively different path shapes observed under visual-motor reflections.

Cybernetics↗

Brain volumes in healthy adults aged 40 years and over: a voxel-based morphometry study.

BACKGROUND AND AIMS: Gender and age effect on brain morphology have been extensively investigated. However, the great variety in methods applied to morphology partly explain the conflicting results of linear patterns of tissue changes and lateral asymmetry in men and women. The aim of the present study was to assess the effect of age, gender and laterality on the volumes of gray matter (GM) and white matter (WM) in a large group of healthy adults by means of voxel-based morphometry. This technique, based on observer-independent algorithms, automatically segments the 3 types of tissue and computes the amount of tissue in each single voxel. METHODS: Subjects were 229 healthy subjects of 40 years of age or older, who underwent magnetic resonance (MR) for reasons other than cognitive impairment. MR images were reoriented following the AC-PC line and, after removing the voxels below the cerebellum, were processed by Statistical Parametric Mapping (SPM99). GM and WM volumes were normalized for intracranial volume. RESULTS: Women had more fractional GM and WM volumes than men. Age was negatively correlated with both fractional GM and WM, and a gender x age interaction effect was found for WM, men having greater WM loss with advancing age. Pairwise differences between left and right GM were negative (greater GM in right hemisphere) in men, and positive (greater GM in left hemisphere) in women (-0.56+/-4.2 vs 0.99+/-4.8; p=0.019). CONCLUSIONS: These results support side-specific accelerated WM loss in men, and may help our better understanding of changes in regional brain structures associated with pathological aging.

Adult↗

Stimulation of c-Rel transcriptional activity by PKA catalytic subunit beta.

Nuclear factor kappaB (NF-kappaB) is a eukaryotic transcription factor which responds to different extracellular signals. It is involved in immune response, inflammation, and cell proliferation. Increased expression of c-Rel (or its viral homolog v-Rel), one component of the NF-kappaB factors, induces tumorigenesis in different systems. The activity of NF-kappaB can be regulated by protein kinase A (PKA) in a cAMP-independent manner. Our previous results showed that c-MYC induces the activity of PKA by inducing the transcription of the gene encoding the PKA catalytic subunit beta (PKA-Cbeta). Constitutive expression of PKA-Cbeta in Rat1a cells induces their transformation. Here we show that CREB is unlikely to be a phosphorylation target of PKA-Cbeta as characterized by different cell lines. Electrophoretic mobility shift assays showed that c-Rel is present as a significant component of the NF-kappaB factors in c-MYC overexpressing status. The transcriptional activity of c-Rel was significantly stimulated by PKA-Cbeta. Coactivators p300/CBP are at least partially responsible for the enhanced activation mediated by c-Rel and PKA-Cbeta. Interaction between c-Rel and PKA-Cbeta was demonstrated using coimmunoprecipitation assays. Immunoprecipitation-in vitro phosphorylation assays showed the direct phosphorylation of c-Rel by PKA-Cbeta. These results indicate that c-Rel is a reasonable phosphorylation target of PKA-Cbeta, and that the transcriptional activity of c-Rel is stimulated by PKA-Cbeta possibly through the interaction with p300/CBP.

Animals↗

Representation of heading direction in far and near head space.

Manipulation of objects around the head requires an accurate and stable internal representation of their locations in space, also during movements such as that of the eye or head. For far space, the representation of visual stimuli for goal-directed arm movements relies on retinal updating, if eye movements are involved. Recent neurophysiological studies led us to infer that a transformation of visual space from retinocentric to a head-centric representation may be involved for visual objects in close proximity to the head. The first aim of this study was to investigate if there is indeed such a representation for remembered visual targets of goal-directed arm movements. Participants had to point toward an initially foveated central target after an intervening saccade. Participants made errors that reflect a bias in the visuomotor transformation that depends on eye displacement rather than any head-centred variable. The second issue addressed was if pointing toward the centre of a wide-field expanding motion pattern involves a retinal updating mechanism or a transformation to a head-centric map and if that process is distance dependent. The same pattern of pointing errors in relation to gaze displacement was found independent of depth. We conclude that for goal-directed arm movements, representation of the remembered visual targets is updated in a retinal frame, a mechanism that is actively used regardless of target distance, stimulus characteristics or the requirements of the task.

Arm↗