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At least 1,261 records · Page 70Linked to original sources
PubMed2003

Sodium loading changes urinary protein excretion: a proteomic analysis.

Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 +/- 1.74 vs. 1.70 +/- 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1alpha, diphor-1, chaperone heat shock protein 72, vacuolar H(+)-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and alpha-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.

Amino Acid Sequence↗
PubMed2006

Anatomical phenotyping in the brain and skull of a mutant mouse by magnetic resonance imaging and computed tomography.

Since genetically modified mice have become more common in biomedical research as models of human disease, a need has also grown for efficient and quantitative methods to assess mouse phenotype. One powerful means of phenotyping is characterization of anatomy in mutant vs. normal populations. Anatomical phenotyping requires visualization of structures in situ, quantification of complex shape differences between mouse populations, and detection of subtle or diffuse abnormalities during high-throughput survey work. These aims can be achieved with imaging techniques adapted from clinical radiology, such as magnetic resonance imaging and computed tomography. These imaging technologies provide an excellent nondestructive method for visualization of anatomy in live individuals or specimens. The computer-based analysis of these images then allows thorough anatomical characterizations. We present an automated method for analyzing multiple-image data sets. This method uses image registration to identify corresponding anatomy between control and mutant groups. Within- and between-group shape differences are used to map regions of significantly differing anatomy. These regions are highlighted and represented quantitatively by displacements and volume changes. This methodology is demonstrated for a partially characterized mouse mutation generated by N-ethyl-N-nitrosourea mutagenesis that is a putative model of the human syndrome oculodentodigital dysplasia, caused by point mutations in the gene encoding connexin 43.

Animals↗
PubMedDate not supplied

The origin of the molecular diversity and functional anchoring of cholinesterases.

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain. AChE subunits of type H ('hydrophobic') produce GPI-anchored dimers, but also secreted molecules; they are mostly expressed in blood cells. Subunits of type T ('tailed') exist for both AChE and BChE. They represent the enzyme forms expressed in brain and muscle. These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins, ColQ and PRiMA. Mutations in the four-helix bundle (FHB) zone of the catalytic domain indicate that subunits of type H and T use the same interaction for dimerization. On the other hand, the C-terminal T peptide is necessary for tetramerization. Four T peptides, organized as amphiphilic alpha helices, can assemble around proline-rich motifs of ColQ or PRiMA. The association of AChE(T) or BChE subunits with ColQ produces collagen-tailed molecules, which are inserted in the extracellular matrix, e.g. in the basal lamina of neuromuscular junctions. Their association with PRiMA produces membrane-bound tetramers which constitute the predominant form of cholinesterases in the mammalian brain; in muscles, the level of PRiMA-anchored tetramers is regulated by exercise, but their functional significance remains unknown. In brain and muscles, the hydrolysis of acetylcholine by cholinesterases, in different contexts, and their possible noncatalytic functions clearly depend on their localization by ColQ or PRiMA.

Acetylcholinesterase↗
PubMed2006

Inconsistencies between human genetic cytolocations and those derived using genomic sequence.

One result of the publishing of the human genome sequence is the ability to define objects through their position on the consensus sequence. While this has simplified the process of creating order maps for genes on a chromosome, it has created discrepancies between the published cytolocations of human genes, as presented through genetic references, and those locations derived computationally from the genomic sequence. For the 6,830 records with HUGO gene symbols shared between the online version of Mendelian Inheritance in Man and Ensembl, 18% of the records have a discrepancy of at least one cytogenetic band between the datasets. Discordance between data sets at this frequency would have a significant impact on the utility of datasets created by the amalgamation of numerous biological databases.

Base Sequence↗
PubMed2004

Transduction of the N-terminal fragments of MYPT1 enhances myofilament Ca2+ sensitivity in an intact coronary artery.

OBJECTIVE: The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions. METHODS AND RESULTS: Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 micromol/L TAT-MYPT1(1-374), a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca2+ under 118 mmol/L K+ depolarization, with no augmentation of the [Ca2+]i elevation. The deletion of the Tat peptide, MYPT1(1-374), abolished the augmenting effect. TAT-MYPT1(1-296) demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1(1-171), TAT-MYPT1(39-374), TAT-MYPT1(39-296), and TAT-MYPT1(297-374) had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca2+ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1(1-374) compared with the control. CONCLUSIONS: Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Actin Cytoskeleton↗
PubMed2004

Targeting to C-terminal myosin heavy chain may explain mechanotransduction involving focal adhesion kinase in cardiac myocytes.

Focal adhesion kinase (Fak) has been implicated as a signaling molecule involved in the early response of cardiac myocytes to mechanical stress. The mechanism of Fak activation by mechanical stimuli is not clear. In this study, we report the load-induced Fak activation and its association with myosin heavy chain in cardiac myocytes. Pressure overload lasting from 3 to 60 minutes was shown to induce Fak phosphorylation at Tyr-397, -576/7, -861, and -925 as detected by phosphospecific antibodies. This was paralleled by increases of Fak/Src association and Src activity (Tyr-418 phosphorylation). Yeast two-hybrid screening of an adult rat cDNA library revealed an interaction between Fak and C-terminal coiled-coil region of alpha-myosin heavy chain. This was confirmed by pulldown assay with GST-C-terminal myosin fragment and native Fak from rat left ventricle. Such interaction was confirmed by coimmunoprecipitation assay with anti-Fak and anti-heavy chain cardiac myosin antibodies, confocal microscopy of double-labeled isolated cardiac myocytes and immunoelectron microscopy with anti-Fak antibody. Fak activation by mechanical stress was accompanied by a reduction of Fak/myosin heavy chain association and its relocation at subcellular sites such as costameres, Z-discs, and nuclei. Thus, our present data identify Fak interaction with C-terminal region of myosin heavy chain adding comprehensive data on Fak activation by mechanical stress and mechanotransduction in cardiac myocytes.

Amino Acid Motifs↗
PubMed1998

Weight-value convergence of the SOM algorithm for discrete input.

Some insights on the convergence of the weight values of the self-organizing map (SOM) to a stationary state in the case of discrete input are provided. The convergence result is obtained by applying the Robbins-Monro algorithm and is applicable to input-output maps of any dimension.

Algorithms↗
PubMed2004

Development and the Baldwin effect.

Baldwin's classic hypothesis states that behavioral plasticity can speed evolution by (a) smoothing the fitness landscape and (b) indirect genetic assimilation of acquired characteristics. This latter phase demands a strong correlation between genotype and phenotype space. But the natural world shows signs of this correlation at only a very coarse level, since the intervening developmental process greatly complicates the mapping from genetics to physiology and ethology. Hence, development appears to preclude a strong Baldwin effect. However, by adding a simple developmental mechanism to Hinton and Nowlan's classic model of the Baldwin effect, and by allowing evolution to determine the proper balance between direct and indirect mapping of genome to phenotype, this research reveals several different effects of development on the Baldwin effect, some promoting and others inhibiting. Perhaps the most interesting result is an evolved cooperation between direct blueprints and indirect developmental recipes in searching for unstructured and partially structured target patterns in large, needle-in-the-haystack fitness landscapes.

Adaptation, Physiological↗
PubMed2003

Cone photoreceptor betagamma-transducin: posttranslational modification and interaction with phosducin.

PURPOSE: To characterize the structure of cone betagamma-transducin (Tbeta3gamma8) and its interaction with phosducin (pdc). METHODS: The Tgamma8 subunit of Tbeta3gamma8 was isolated by column chromatography for peptide mapping with mass spectrometry. Tbeta3gamma8 was compared with rod betagamma-transducin (Tbeta1gamma1) in terms of the electrophoretic mobility, pdc binding affinity, and the effects of phosphorylation and methylation, and then the correlation to the crystal structures and functional domains of Tbeta1gamma1 was determined. RESULTS: The mature Tgamma8 is a 65-amino-acid peptide encoded by the Ggamma8 gene with an acetylated and a farnesylated-methylated N- and C-terminus, respectively. Purified Tbeta3gamma8 is similar to Tbeta1gamma1 in that (1) both are heterogeneous, containing methylated and demethylated Tgamma subunits; (2) each demethylated dimer migrates faster than its methylated counterpart during native gel electrophoresis, and the methylation-associated mobility differential is masked by pdc binding; and (3) both dimers bind pdc with the same affinity, and the affinity is reduced threefold by PKA phosphorylation of pdc and twofold by demethylation at the C-terminus of Tgamma. Tbeta3gamma8 differs from Tbeta1gamma1 in exhibiting lower intrinsic electrophoretic mobility, and the difference is unaffected by either pdc binding or the status of Tgamma methylation. CONCLUSIONS: Tbeta3gamma8 is identical with Tbeta1gamma1 in Tgamma isoprenylation, the spatial organization, and the mode of pdc binding, indicating that its interaction with pdc does not play an important role in the specialization of cones. Changes in Tbetagamma characteristics by Tgamma methylation reveal conformational changes on a surface domain that is essential for Tbetagamma functions and support a regulatory role for reversible methylation.

Amino Acid Sequence↗
PubMed2007

Clinical relevance of neutral endopeptidase (NEP/CD10) in melanoma.

BACKGROUND: Overexpression of Neutral Endopeptidase (NEP) has been reported in metastatic carcinomas, implicating NEP in tumor progression and suggesting a role for NEP inhibitors in its treatment. We investigated the role of NEP expression in the clinical progression of cutaneous melanoma. METHODS: We screened 7 melanoma cell lines for NEP protein expression. NEP-specific siRNA was transfected into the lines to examine the role of gene transcription in NEP expression. Immunohistochemistry was done for 93 specimens and correlated with clinicopathologic parameters. Thirty-seven metastatic melanoma specimens were examined for NEP transcript expression using Affymetrix GeneChips. In a subset of 25 specimens for which both transcript and protein expression was available, expression ratios were used to identify genes that co-express with NEP in GeneChip analysis. RESULTS: NEP was overexpressed in 4/7 human melanoma cell lines, and siRNA knock-down of NEP transcripts led to downregulation of its protein expression. NEP protein overexpression was significantly more common in metastatic versus primary tumors (P = 0.002). Twelve of 37 (32%) metastatic tumors had increased NEP transcript expression, and an association was observed between NEP transcript upregulation and protein overexpression (P < 0.0001). Thirty-eight genes were found to significantly co-express with NEP (p < 0.005). Thirty-three genes positively correlated with NEP, including genes involved in the MAP kinase pathway, antigen processing and presentation, apoptosis, and WNT signaling pathway, and 5 genes negatively correlated with NEP, including genes of focal adhesion and the notch signaling pathways. CONCLUSION: NEP overexpression, which seems to be largely driven by increased transcription, is rare in primary melanoma and occurs late in melanoma progression. Functional studies are needed to better understand the mechanisms of NEP regulation in melanoma.

Cell Line, Tumor↗
PubMed1998

Estrogen receptor-alpha is developmentally regulated during osteoblast differentiation and contributes to selective responsiveness of gene expression.

Estrogen responsiveness of bone is a fundamental regulatory mechanism operative in skeletal homeostasis. We examined the expression of estrogen receptor-alpha (ER) messenger RNA (mRNA) in cultured rat calvarial-derived osteoblasts during progressive development of the osteoblast phenotype. Levels of ER message were compared with the expression of traditional osteoblastic markers that have been mapped throughout the differentiation process of these cells. ER transcripts, measured using semiquantitative RT-PCR analysis, were expressed at low levels in early stage proliferating osteoblasts and increased at confluence upon initial expression of bone cell phenotypic genes. A 23-fold up-regulation of ER mRNA expression coincided with the initiation of alkaline phosphatase activity (day 8). ER mRNA levels progressively increased 70-fold, reaching a maximum level on days 22-25 in fully differentiated osteoblasts when osteocalcin expression peaked, but declined precipitously by day 32 in osteocytic cells. Analysis of RNA isolated directly from rat calvaria confirmed these in vitro results and demonstrated that ER message levels become more abundant postnatally as bone becomes more mineralized. We also examined the responsiveness of osteoblasts to 17beta-estradiol (17beta-E2) at two periods of maturation: the nodule-forming stage (day 14) and the late mineralization stage (day 30). Estradiol suppressed the levels of alkaline phosphatase, osteocalcin, osteonectin, and ER mRNAs on day 14, but up-regulated these messages on day 30. In contrast, 17beta-E2 treatment regulated the steady state levels of transforming growth factor-beta1 and type I procollagen mRNAs only in the late mineralization stage, whereas histone H4 message was unaffected by the steroid at either stage of differentiation. Thus, the observed developmental expression of ER mRNA correlates with progressive osteoblast differentiation and may be a contributing factor to differential regulation of bone cell gene expression by 17beta-E2.

Animals↗
PubMed2005

Fgf signals from a novel signaling center determine axial patterning of the prospective neural retina.

Axial eye patterning determines the positional code of retinal ganglion cells (RGCs), which is crucial for their topographic projection to the midbrain. Several asymmetrically expressed determinants of retinal patterning are known, but it is unclear how axial polarity is first established. We find that Fgf signals, including Fgf8, determine retinal patterning along the nasotemporal (NT) axis during early zebrafish embryogenesis: Fgf8 induces nasal and/or suppresses temporal retinal cell fates; and inhibition of all Fgf-receptor signaling leads to complete retinal temporalization and concomitant loss of all nasal fates. Misprojections of RGCs with Fgf-dependent alterations in retinal patterning to the midbrain demonstrate the importance of this early patterning process for late topographic map formation. The crucial period of Fgf-dependent patterning is at the onset of eye morphogenesis. Fgf8 expression, the restricted temporal requirement for Fgf-receptor signaling and target gene expression at this stage suggests that the telencephalic primordium is the source of Fgf8 and acts as novel signaling center for non-autonomous axial patterning of the prospective neural retina.

Animals↗
PubMed1995

Monoclonal and polyclonal antibodies detect a new type of post-translational modification of axonemal tubulin.

Polyclonal (PAT) and monoclonal (AXO 49) antibodies against Paramecium axonemal tubulin were used as probes to reveal tubulin heterogeneity. The location, the nature and the subcellular distribution of the epitopes recognized by these antibodies were, respectively, determined by means of: (i) immunoblotting on peptide maps of Paramecium, sea urchin and quail axonemal tubulins; (ii) immunoblotting on ciliate tubulin fusion peptides generated in E. coli to discriminate antibodies directed against sequential epitopes (reactive) from post-translational ones (non reactive); and (iii) immunofluorescence on Paramecium cells, using throughout an array of antibodies directed against tubulin sequences and post-translational modifications as references. AXO 49 monoclonal antibody and PAT serum were both shown to recognize epitopes located near the carboxyl-terminal end of both subunits of Paramecium axonemal tubulin, whereas the latter recognized additional epitopes in alpha-tubulin; AXO 49 and a fraction of the PAT serum proved to be unreactive over fusion proteins; both PAT and AXO 49 labelled a restricted population of very stable microtubules in Paramecium, consisting of axonemal and cortical ones, and their reactivity was sequentially detected following microtubule assembly; finally, both antibodies stained two upward spread bands in Paramecium axonemal tubulin separated by SDS-PAGE, indicating the recognition of various alpha- and beta-tubulin isoforms displaying different apparent molecular masses. These data, taken as a whole, definitely establish that PAT and AXO 49 recognize a post-translational modification occurring in axonemal microtubules of protozoa as of metazoa. This modification appears to be distinct from the previously known ones, and all the presently available evidence indicates that it corresponds to the very recently discovered polyglycylation of Paramecium axonemal alpha- and beta-tubulin.

Animals↗
PubMed2006

Three-dimensional surface contouring of macroscopic objects by means of phase-difference images.

We report a technique to determine the 3D contour of objects with dimensions of at least 4 orders of magnitude larger than the illumination optical wavelength. Our proposal is based on the numerical reconstruction of the optical wave field of digitally recorded holograms. The required modulo 2pi phase map in any contouring process is obtained by means of the direct subtraction of two phase-contrast images under different illumination angles to create a phase-difference image of a still object. Obtaining the phase-difference images is only possible by using the capability of numerical reconstruction of the complex optical field provided by digital holography. This unique characteristic leads us to a robust, reliable, and fast procedure that requires only two images. A theoretical analysis of the contouring system is shown, with verification by means of numerical and experimental results.

Journal Article↗
PubMed2004

Nucleocytoplasmic glycosylation, O-GlcNAc: identification and site mapping.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is posttranslationally added to serine and threonine residues of many nuclear and cytoplasmic proteins found in metazoans. This modification is dynamic and responsive to numerous stimuli and conditions, suggesting an important role in many regulatory pathways. Moreover, the O-GlcNAc modification seems to compete with phosphorylation for sites of attachment, indicating a reciprocal relationship with phosphorylation. This chapter includes protocols for: (1) identifying the O-GlcNAc modification on proteins through immunoblotting, lectin affinity chromatography, and galactosyltransferase labeling; and (2) identifying and enriching for the sites of attachment using the mass spectrometry-based beta-elimination followed by Michael addition with dithiothreitol (BEMAD) technique.

Acetylglucosamine↗
PubMed2007

Protein identification by peptide mass fingerprinting.

Peptide mass fingerprinting is an effective way of identifying, e.g., gel-separated proteins, by matching experimentally obtained peptide mass data against large databases. However, several factors are known to influence the quality of the resulting matches, such as proteins contaminating the sample in question, modifications altering the mass of the peptides, ionization efficiency of the individual peptides, and the degree of missed cleavage sites. Here, these factors are discussed and methods for elimination of contaminants from the dataset and prediction of various modifications are introduced. Useful tips on how to specify various search parameters and how to manually evaluate the search results are also given.

Databases, Protein↗