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PubMed1986

Methods for the identification of evoked response components in the frequency and combined time/frequency domains.

Two prominent frequency components designated f1 and f2 have been identified in the visual evoked response to the transient presentation of sinusoidal luminance gratings in the range of 0.5-8 c/deg. The components occur at temporal frequencies below the alpha band, with the f1 frequency being roughly half that of the f2 frequency. The f1 component is largest at low spatial frequencies with f2 becoming progressively dominant as spatial frequency is increased. The frequency and amplitude of f1 and f2 change substantially over the time course of the response. This has been studied by calculating the temporal frequency spectrum of the transient evoked potential over successive short-time epochs running through the response. Using this technique, the response is shown to consist of narrow-band frequency peaks or 'formants' emerging at different times after stimulus onset. These formants occur at frequencies other than those of the spontaneous EEG and undergo changes in frequency and amplitude over the time course of the response. Two spectrum analysis techniques were employed: the Discrete Fourier Transform and Linear Predictive Coding. Frequency components were successfully identified in single-trial responses using the LPC technique.

Electroencephalography↗
PubMed2005

A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma.

Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.

Animals↗
PubMed1997

The use of antibodies to study the architecture and developmental regulation of plant cell walls.

This review covers the generation and use of antibodies to defined components of plant and algal cell walls and how these have contributed to our understanding of the spatial and developmental regulation of cell walls. Particular emphasis is placed upon the generation and characterization of monoclonal antibodies to matrix polysaccharides, extensins, and arabinogalactan-proteins of higher plants, and algal polysaccharides and glycoproteins. Immunolocalization studies are discussed in relation to the identification of molecular domains within cell walls, cell adhesion, cell differentiation, and the establishment of plant interactions with other organisms.

Antibodies, Monoclonal↗
PubMed2001

Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps.

Calcium pumps of the plasma membrane (also known as plasma membrane Ca(2+)-ATPases or PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells. Together with Na(+)/Ca(2+) exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca(2+) concentration. Like the Ca(2+) pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca(2+) from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca(2+) pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca(2+) signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA "knockout" mice further support the concept of specific, nonredundant roles for each Ca(2+) pump isoform in cellular Ca(2+) regulation.

Alternative Splicing↗
PubMed2004

Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-alpha using computer modeling and deletion mutant technique.

The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-alpha were cloned, and according to the translated sequence of amino acids, the spatial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-alpha was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-alpha. hTNF-alpha molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92-157 of hTNF-alpha, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF-a 141-146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-alpha were specially recognized by Z12 antibody.

Animals↗
PubMed2006

Identification of a promoter element within the zebrafish colXalpha1 gene responsive to runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2alphaA2.

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Amino Acid Sequence↗
PubMed2003

Identification of an enhancer region in the mouse ephA8 locus directing expression to the anterior region of the dorsal mesencephalon.

Eph receptors and ephrins are dynamically expressed in a wide range of regions of the vertebrate during embryogenesis. The dorsal mesencephalon appears to be segmented into two broad regions demarcated by the mutually exclusive expression of EphA receptors and ephrinA ligands. It is of considerable interest to elucidate how these expression domains are established in the development of the mesencephalon. In this study, we used a transgenic approach to define the cis-acting DNA regulatory elements involved in the anterior mesencephalon-specific expression of the mouse ephA8 gene. Our analyses of the temporal and spatial expression patterns of various ephA8/lacZ gene fusions in transgenic mice revealed that the 10-kb genomic DNA 5' immediately upstream of the ephA8 coding sequence is capable of directing lacZ expression in an ephA8-specific manner. Further deletion analyses of the ephA8 genomic region led to the identification of a 1-kb enhancer region, which directs expression in the embryo to the anterior region of the developing midbrain. This ephA8-specific regulatory DNA sequences can now be used in biochemical analyses to identify proteins modulating the anterior differentiation of the optic tectum, and in functional analyses to direct the expression of other developmentally important genes to this region.

5' Flanking Region↗
PubMed1996

Evolution of mouse T-box genes by tandem duplication and cluster dispersion.

The T-box genes comprise an ancient family of putative transcription factors conserved across species as divergent as Mus musculus and Caenorhabditis elegans. All T-box gene products are characterized by a novel 174-186-amino acid DNA binding domain called the T-box that was first discovered in the polypeptide products of the mouse T locus and the Drosophila melanogaster optomotor-blind gene. Earlier studies allowed the identification of five mouse T-box genes, T, Tbx1-3, and Tbr1, that all map to different chromosomal locations and are expressed in unique temporal and spatial patterns during embryogenesis. Here, we report the discovery of three new members of the mouse T-box gene family, named Tbx4, Tbx5, and Tbx6. Two of these newly discovered genes, Tbx4 and Tbx5, were found to be tightly linked to previously identified T-box genes. Combined results from phylogenetic, linkage, and physical mapping studies provide a picture for the evolution of a T-box subfamily by unequal crossing over to form a two-gene cluster that was duplicated and dispersed to two chromosomal locations. This analysis suggests that Tbx4 and Tbx5 are cognate genes that diverged apart from a common ancestral gene during early vertebrate evolution.

Amino Acid Sequence↗
PubMed1999

Yeast transcriptional regulator Leu3p. Self-masking, specificity of masking, and evidence for regulation by the intracellular level of Leu3p.

Recent work suggests that the masking of the activation domain (AD) of yeast transactivator Leu3p, observed in the absence of the metabolic signal alpha-isopropylmalate, is an intramolecular event. Much of the evidence came from the construction and analysis of a mutant form of Leu3p (Leu3-dd) whose AD is permanently masked (Wang, D., Hu, Y., Zheng, F., Zhou, K., and Kohlhaw, G. B. (1997) J. Biol. Chem. 272, 19383-19392). In a modified two-hybrid experiment, the ADs of both wild type Leu3p and Leu3-dd were shown to interact with the remainder of the Leu3 protein, in an alpha-isopropylmalate-dependent manner. The finding that masking and unmasking proceed apparently normally when full-length Leu3p is expressed in mammalian cells is also consistent with the notion of intramolecular masking. Here we report on the identification of nine missense mutations (all of them suppressors of the Leu3-dd phenotype) that cause permanent unmasking of Leu3p. The nine mutations map to three short segments located within a 140-residue-long region of the C-terminal part of the middle region of Leu3p. These segments may be part of a spatial trap for the AD. We also performed "domain swaps" between Leu3p and Cha4p, a serine/threonine-responsive activator that, like Leu3p, belongs to the family of Zn(II)2Cys6 proteins. We show that AD masking and response to the appropriate metabolic signal only occur when a given AD remains attached to its own middle region; middle region swapping results in constitutively active proteins. Finally, we show that the extent to which Leu3p regulates reporter gene expression depends on the intracellular concentration of Leu3p. The possible physiological significance of this observation is discussed in light of the known regulation of Leu3p by Gcn4p.

Amino Acid Substitution↗
PubMed2006

Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia.

Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full-length gene was cloned from rat brain and its spatial and temporal expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up-regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system and thalamus, as well as in layers 3 and 5 in the cortex of the unchallenged brain. When overexpressed in HEK 293 cells, Npas4 appears as a protein of approximately 100 kDa. In brain samples, however, in addition to the 100 kDa band a specific 200 kDa immunoreactive band was also detected. Ischemic challenge lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury.

Animals↗
PubMed2001

Episodic memory for object location versus episodic memory for object identity: do they rely on distinct encoding processes?

Three experiments were conducted to determine whether encoding processes that support episodic memory for object location are distinct from those that support memory for object identity. Guided by transfer-appropriate processing notions, we examined with an incidental learning paradigm whether an attentional focus on object location at encoding promotes subsequent recovery of object location, whereas a focus on object identity promotes recovery of object identity. We found that judging spatial relationships at encoding selectively supports recovery of object location, provided the test assesses memory for these relationships (rather than absolute location); our results also showed that judging physical and semantic object attributes promotes recovery of object identity preferentially. Contrasting with these domain-specific effects was evidence that identification processes involved in object naming boost memory for object identity as well as for absolute object location. Object identification at encoding may support memory performance in both domains by triggering the binding of identity and location information through a mechanism of object-based attentional selection.

Adult↗
PubMed1994

Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding.

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.

Amino Acid Sequence↗
PubMed1989

Frequency domain analysis of contrast echocardiographic images.

Sequences of echocontrastographic images of the dog's myocardium are analysed in the frequency domain for the identification of underperfused areas. Owing to the fact that echo images are contaminated by noise and artifacts, we have applied filtering techniques based on Fourier's methods in sequences of raw frames recorded during perfusion with a contrast agent. Thus we have been able to assess correctly the spatial and temporal distribution of the contrast, i.e. the agent kinetics which parallel the distribution of coronary blood flow, in terms of the time necessary for the flow gradient to reach its maximum value, and to describe such a distribution by a functional image obtained by means of an original procedure.

Algorithms↗
PubMed2003

Protein structural alignment for detection of maximally conserved regions.

An algorithm for comparison of homologous protein structures and for study of conformational changes in proteins, has been developed. The method is based on identification of pieces of the two molecules that have similar shapes, as determined by the local conformation of the polypeptide chain. Pieces that superpose within a specified tolerance are assembled into domains based on similar transformations for superposition. The result is sets of pieces that represent conserved structural elements and conserved spatial relationships between structural elements within the proteins being compared. A similarity criterion based on maximum distance rather than on root mean square deviation reduces bias by outliers. The utility of the method is demonstrated by using examples from the protein kinase family.

Algorithms↗
PubMed1997

Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein.

P-glycoprotein, the multidrug resistance transporter, is phosphorylated in vivo and the major phosphorylation domain has been identified as the linker region (amino acids 629-686). The linker region is a highly charged segment of the transporter in which the negative and positive amino acid side chains are spatially segregated. Both of these charged domains contain several consensus phosphorylation sites for protein kinases. Three of the consensus phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein are utilized in vivo and have been identified as serines 665, 669, and 681. Mutagenesis of all the consensus basic-directed kinase phosphorylation sites in the linker region of human MDR1 P-glycoprotein did not alter the ability of the mutated transporter to confer the multidrug resistance phenotype in stably transfected cell lines. These studies would suggest that phosphorylation/dephosphorylation within the basic domain of the linker region is not directly involved in regulation of drug transporter activity. We now report that the linker region of mdr1b P-glycoprotein is also phosphorylated in vivo within the acidic domain (amino acids 631-658). These sites have been mapped using casein kinase II, a prototypic acidic-directed kinase, and a recombinant mdr1b linker region peptide (amino acids 621-687). Electrospray mass spectrometry demonstrated that casein kinase II could introduce up to five phosphates into the recombinant peptide. Two-dimensional phosphopeptide mapping indicated that all the phosphates were contained in a tryptic peptide consisting of amino acids 631-658. Phosphopeptide mapping of in vivo labeled P-glycoprotein, isolated from either J7.V1-1, a murine vinblastine-resistant cell line, or HeLa cells stably transfected with mdr1b P-glycoprotein cDNA, revealed that this tryptic peptide was phosphorylated in both proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Production and characterization of murine mAbs to the extracellular domain of human neu oncogene product GP185HER2.

The oncogene HER-2/neu encodes a transmembrane glycoprotein of 185 kDa (gp185HER-2) with tyrosine-kinase activity. Gene amplification and high levels of expression of gp185HER-2 have been found to correlate with poor clinical outcome in breast and ovarian carcinomas. Employing a somatic cell hybrid fusion protocol, which yields a high frequency production of hybridomas, we have analyzed the extent of the murine immune response to the gp 185 extracellular domain. In a single fusion experiment, using as immunogen NIH 3T3 cells expressing high levels of a transfected human HER-2 gene, we have generated mAbs, mainly of IgG1 isotype, displaying high affinity (10(7)-10(10) mol/L) to gp 185. Analysis of the epitope specificity has allowed the identification of five distinct groups of spatially related epitopes, each provided with different immunodominancy, and all resistant to formalin fixation. The use of inhibitor of N-linked glycosylation tunicamicyn has demonstrated that the mAbs bind to epitopes localized in the protein core of gp185HER-2. Because recent reports have shown that gp185HER-2 has a restricted expression in normal tissues and is homogenously detectable in metastatic foci of gp 185 + primary tumors, antibodies to this macromolecule, in addition to their prognostic value, may represent reagents for in vitro and in vivo diagnostic applications, as well as for the development of therapeutic strategies.

Animals↗
PubMed1996

Two muscle-specific LIM proteins in Drosophila.

The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.

Amino Acid Sequence↗
PubMed2002

Identification of cis-DNA regions controlling Bmp4 expression during tooth morphogenesis in vivo.

Epithelial-mesenchymal interactions are required for tooth formation. Bone morphogenetic protein 4 (Bmp4) is a crucial signaling molecule during this process. For better understanding of the role of the Bmp4 gene during tooth development, we studied the mechanisms that control its temporal and spatial expression during development. Using a transgenic approach, we determined that the domains which controlled Bmp4 expression in epithelium-derived ameloblasts were located in the region between 0.26 kb and 1.1 kb of the murine Bmp4 promoter. In contrast, the domains controlling Bmp4 expression in mesenchyme-derived odontoblasts and pulp cells existed in other regions of the Bmp4 gene. We have also demonstrated that the domains controlling Bmp4 expression in primordial tooth cells differ from those controlling Bmp4 expression in mature tooth tissues. The determination of unique domains by controlling the expression of the Bmp4 gene suggests that different transcriptional factors regulate the Bmp4 level at different stages during tooth morphogenesis.

Ameloblasts↗