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PubMed1986

Identification of 5 topographic domains of the mouse LFA-1 molecule: subunit assignment and functional involvement in lymphoid cell interactions.

We have evaluated the serologic and T cell function inhibiting properties of 10 rat mAb reactive with the mouse LFA-1 molecule. Binding inhibition studies revealed that these mAb identified five topographic domains on LFA-1, including an immunodominant epitope region (A) defined by 6 mAb (H35-89, H68-96, H85-326, H129-37, H154-595, and H155-141) and four other spatially separate epitopes each defined by a single mAb (i.e., B, H154-266; C, H129-296; D, H154-163; and E, H155-78). Immunoprecipitation studies carried out with T cell hybridoma detergent lysate containing native or dissociated alpha and beta LFA-1 subunits permitted assignment of the epitopes A, C, and D to the alpha-chain, while expression of the epitopes B and E required homologous pairing of the alpha and beta LFA-1 subunits. These anti-LFA-1 mAb did not bind to the Mac-1 positive P388D1 cells. All the six mAb directed at epitope A inhibited, in the range of 50 to 95%, the proliferative responses of alloantigen- or soluble-antigen GAT-specific T cell clones and the cytolytic activity of I-Ak-specific CTL clones. MAb reactive with the epitopes C and D also blocked these T cell responses, although to a lesser extent. No inhibition was observed with mAb specific to epitope B, whereas the epitope E-specific mAb H155-78 potentiated control T cell responses by 20 to 40%. Suboptimal amounts of anti-L3T4 mAb H129-19 were found to synergistically enhance the T cell function inhibiting properties of mAb to LFA-1 epitopes A, C, and D. These studies reveal an unexpected diversification of LFA-1 between mouse and rat species and further the functional dissection of this molecule.

Animals↗
PubMed2004

Validity of the Mindstreams computerized cognitive battery for mild cognitive impairment.

The NeuroTrax Mindstreams computerized cognitive assessment system was designed for widespread clinical and research use in detecting mild cognitive impairment (MCI). However, the capability of Mindstreams tests to discriminate the elderly with MCI from those who are cognitively healthy has yet to be evaluated. Moreover, the comparability between these tests and traditional neuropsychological tests in detecting MCI has not been examined. A two-center study was designed to assess the discriminant validity of tests in the Mindstreams Mild Impairment Battery. Participants were 30 individuals diagnosed with MCI, 29 with mild Alzheimer's disease (AD), and 39 healthy elderly. Testing was with the Mindstreams battery and traditional neuropsychological tests. Receiver operating characteristic (ROC) analysis was used to examine the ability of Mindstreams and traditional measures to discriminate those with MCI from cognitively healthy elderly. Between-group comparisons were made (Mann-Whitney U test) between MCI and healthy elderly and between MCI and mild AD groups. Mindstreams outcome parameters across multiple cognitive domains significantly discriminated between MCI and healthy elders with considerable effect sizes (p < 0.05). Measures of memory, executive function, visual spatial skills, and verbal fluency discriminated best, and discriminability was at least comparable to that of traditional neuropsychological tests in these domains. Mindstreams tests are effective in detecting MCI, providing a comprehensive profile of cognitive function. Further, the enhanced precision and ease of use of these computerized tests make the NeuroTrax system a valuable clinical tool in the identification of elders at high risk for dementia.

Aged↗
PubMed1986

Identification of three sites of proteolytic cleavage in the hinge region between the two domains of the beta 2 subunit of tryptophan synthase of Escherichia coli or Salmonella typhimurium.

The beta 2 subunit of tryptophan synthase is composed of two independently folding domains connected by a hinge segment of the polypeptide that is particularly susceptible to limited proteolysis by trypsin [Högberg-Raibaud, A., & Goldberg, M. (1977) Biochemistry 16, 4014-4019]. Since tryptic cleavage in the hinge region inactivates the beta 2 subunit, the spatial relationship between the two domains is important for enzyme activity. However, it was not previously known whether inactivation results from cleavage of the chain or from the loss of internal fragment(s) subsequent to cleavage at two or more sites. We now report comparative studies of limited proteolysis by three proteinases: trypsin and endoproteinases Lys-C and Arg-C. Our key finding that endoproteinase Arg-C inactivates the beta 2 subunit by cleavage at a single site (Arg-275) demonstrates the important role of the hinge peptide for enzymatic activity. We have also identified the sites of cleavage and the time course of proteolysis by trypsin at Arg-275, Lys-283, and Lys-272 and by endoproteinase Lys-C at Lys-283 and Lys-272. Sodium dodecyl sulfate gel electrophoresis, Edman degradation, and carboxypeptidases B and Y have been used to identify the several fragments and peptides produced. Our finding that the beta 2 subunit and F1 fragments have a heterogeneous amino terminus (Met-1 or Thr-2) indicates that the amino-terminal methionine is incompletely removed during posttranslational modification. Our results show that Edman degradation can be effectively used with a protein of known sequence to analyze proteolytic digests that have at least four different amino-terminal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1996

Correlation-peak imaging.

Identification and quantitation in conventional 1H spectroscopic imaging in vivo is often hampered by the small chemical-shift range. To improve the spectral resolution of spectroscopic imaging, homonuclear two-dimensional correlation spectroscopy has been combined with phase encoding of the spatial dimensions. From the theoretical description of the coherence-transfer signal in the Fourier-transform domain, a comprehensive acquisition and processing strategy is presented that includes optimization of the width and the position of the acquisition windows, matched filtering of the signal envelope, and graphical presentation of the cross peak of interest. The procedure has been applied to image the spatial distribution of the correlation peaks from specific spin systems in the hypocotyl of castor bean (Ricinus communis) seedlings. Despite the overlap of many resonances, correlation-peak imaging made it possible to observe a number of proton resonances, such as those of sucrose, beta-glucose, glutamine/glutamate, lysine, and arginine.

Ricinus communis↗
PubMed1998

Imprinting in Prader-Willi and Angelman syndromes.

Imprinted genes are marked in the germline and retain molecular memory of their parental origin, resulting in allelic expression differences during development. Abnormalities in imprinted inheritance occur in several genetic diseases and cancer, and are exemplified by the diverse genetic defects involving chromosome 15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss of function of multiple paternally expressed genes, while mutations in a single gene, UBE3A, which is subject to spatially restricted imprinting, occur in some AS patients. Identification of mutations in the imprinting process in PWS and AS has led to a definition of an imprinting center (IC), involving the promoter (in PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates initiation of imprint switching for all genes in a 2 Mb imprinted domain during gametogenesis. Imprinting mutations define a novel mechanism of genetic disease because they have no direct effect in the affected patient but, rather, it is the parental germline effect of an IC mutation that leads to disease in the offspring.

Angelman Syndrome↗
PubMed2003

Validity of a novel computerized cognitive battery for mild cognitive impairment.

BACKGROUND: The NeuroTrax Mindstreams computerized cognitive assessment system was designed for widespread clinical and research use in detecting mild cognitive impairment (MCI). However, the capability of Mindstreams tests to discriminate elderly with MCI from those who are cognitively healthy has yet to be evaluated. Moreover, the comparability between these tests and traditional neuropsychological tests in detecting MCI has not been examined. METHODS: A 2-center study was designed to assess discriminant validity of tests in the Mindstreams Mild Impairment Battery. Participants were 30 individuals diagnosed with MCI, 29 with mild Alzheimer's disease (AD), and 39 healthy elderly. Testing was with the Mindstreams battery and traditional neuropsychological tests. Receiver operating characteristic (ROC) analysis was used to examine the ability of Mindstreams and traditional measures to discriminate those with MCI from cognitively healthy elderly. Between-group comparisons were made (Mann-Whitney U test) between MCI and healthy elderly and between MCI and mild AD groups. RESULTS: Mindstreams outcome parameters across multiple cognitive domains significantly discriminated among MCI and healthy elderly with considerable effect sizes (p < 0.05). Measures of memory, executive function, visual spatial skills, and verbal fluency discriminated best, and discriminability was at least comparable to that of traditional neuropsychological tests in these domains. CONCLUSIONS: Mindstreams tests are effective in detecting MCI, providing a comprehensive profile of cognitive function. Further, the enhanced precision and ease of use of these computerized tests make the NeuroTrax system a valuable clinical tool in the identification of elderly at high risk for dementia.

Journal Article↗
PubMed1990

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding.

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.

Amino Acid Sequence↗
PubMed2000

Identification and spatial distribution of the mRNA encoding an egg envelope component of the Cyprinid zebrafish, Danio rerio, homologous to the mammalian ZP3 (ZPC).

Using degenerate reverse transcription polymerase chain reaction (RT-PCR) techniques we have isolated a cDNA encoding a putative component of the zebrafish Danio rerio egg chorion, homologous to the mammalian ZP3 (ZPC). The predicted protein (zfZPC) has a calculated molecular mass of 58.4 kDa and contains a signal peptide (located in the N-terminal region) composed of 11 hydrophobic amino acid residues followed by a signal peptide cleavage site. The zfZPC contains the ZP domain, a characteristic amino acid sequence shared by all ZP proteins of the mammalian zona pellucida and of both amphibian and bird egg envelope components. The zfZPC also exhibits certain unique features including five N-terminal Q-rich tandem repeats presumably involved in the hardening of the chorion after the fertilization of the egg and a long C-terminal tail containing two potential sites of N-linked type glycosylation. RT-PCR and in situ hybridization revealed a restricted pattern of tissue distribution: the gene encoding zfZPC is transcribed only in the growing oocyte of sexually mature female fish.

Amino Acid Sequence↗
PubMed2005

NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors.

DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.

Anti-Infective Agents↗
PubMed2006

Identification of mitogen-activated protein kinase docking sites in enzymes that metabolize phosphatidylinositols and inositol phosphates.

BACKGROUND: Reversible interactions between the components of cellular signaling pathways allow for the formation and dissociation of multimolecular complexes with spatial and temporal resolution and, thus, are an important means of integrating multiple signals into a coordinated cellular response. Several mechanisms that underlie these interactions have been identified, including the recognition of specific docking sites, termed a D-domain and FXFP motif, on proteins that bind mitogen-activated protein kinases (MAPKs). We recently found that phosphatidylinositol-specific phospholipase C-gamma1 (PLC-gamma1) directly binds to extracellular signal-regulated kinase 2 (ERK2), a MAPK, via a D-domain-dependent mechanism. In addition, we identified D-domain sequences in several other PLC isozymes. In the present studies we sought to determine whether MAPK docking sequences could be recognized in other enzymes that metabolize phosphatidylinositols (PIs), as well as in enzymes that metabolize inositol phosphates (IPs). RESULTS: We found that several, but not all, of these enzymes contain identifiable D-domain sequences. Further, we found a high degree of conservation of these sequences and their location in human and mouse proteins; notable exceptions were PI 3-kinase C2-gamma, PI 4-kinase type IIbeta, and inositol polyphosphate 1-phosphatase. CONCLUSION: The results indicate that there may be extensive crosstalk between MAPK signaling and signaling pathways that are regulated by cellular levels of PIs or IPs.

Journal Article↗
PubMed2002

The dorsal-ventral axis of the neural retina is divided into multiple domains of restricted gene expression which exhibit features of lineage compartments.

The neural retina is a complex sensory structure designed to receive, integrate, and transmit visual information. An important aspect of retinal development is the establishment of pattern along the dorsal-ventral (D-V) and anterior-posterior (A-P) axes. The recent identification and functional characterization of a dorsal-specific and a ventral-specific transcription factor suggested that the D-V axis is divided into two domains. This study characterizes the expression patterns of these and other D-V markers, and establishes that the retina is subdivided into at least four domains of gene expression along this axis. The composition and spatial relation of these expression domains alters our model of D-V patterning, suggesting more complexity in the way that the retina is patterned than was previously recognized. As domains of gene expression within developing tissues sometimes comprise compartments whose borders are not crossed by clonally related cells, we performed a retroviral lineage study. A strong preference for cells to remain in their original domain of gene expression was observed, suggesting that these borders comprise developmental compartments.

Animals↗
PubMed2005

A novel hook-related protein family and the characterization of hook-related protein 1.

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook-related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C-terminal HkRP domain. The central region of each protein is comprised of an extensive coiled-coil domain, and the N-terminus contains a putative microtubule-binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule-associated. While endogenous HkRP1 has no distinct organelle association, expression of the C-terminal membrane-binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C-terminal HkRP1 domain causes an accumulation of internal membranes with an electron-dense coat. Co-localization studies show a concomitant redistribution of the early endosome marker sorting-nexin 1 but not the early endosome antigen-1 (EEA1). The steady-state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C-terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin-1 positive membranes from early endosome subdomains.

Amino Acid Sequence↗
PubMed2000

Spatial frequencies in short-term memory for faces: a test of three frequency-dependent hypotheses.

Spatial frequency manipulations have been shown to have significant utility in ascertaining the various types of information that might be important in object identification and recognition tasks. This utility suggests that, given some mapping between ranges of spatial frequencies and different types of psychological information, it should be possible to examine the roles of these different types of psychological information by way of spatial frequency manipulations. One potential problem, however, is that there is no well-specified, unambiguous mapping between the distinctions in the frequency domain and the distinctions in the informational domain. Three experiments provide tests of three general hypotheses regarding the ways in which different spatial frequencies might map to different information in facial perception and memory tasks: (1) the low-frequency dominance hypothesis, which proposes that low-frequency information should be superior (to high-frequency information) as a cue to perception and memory; (2) the distinct informational roles hypothesis, which holds that high spatial frequencies should carry featural information while low spatial frequencies should carry information about the configuration of those features; and (3) the task-dependent information hypothesis, which suggests that high-frequency information should be best suited to discrimination tasks while low-frequency information should be best suited for recognition tasks. Results generally contradict the first two of these hypotheses, while providing support for the third. Implications with regard to the various issues related to the mapping between spatial frequencies and the informational content of faces, as well as the need to consider important interactions among perceptual and memory processes, are discussed.

Adult↗
PubMed1993

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.

Amino Acid Sequence↗
PubMed1998

Topology of the calmodulin-melittin complex.

The topology of the Ca2+-calmodulin-melittin ternary complex has been investigated by a combined strategy which integrates limited proteolysis and cross-linking experiments with mass spectrometric methodologies. The rationale behind the methods is that the interface regions of two interacting proteins are accessible to the solvent in the isolated molecules, whereas they become protected following the formation of the complex. Therefore, when limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential peptide maps are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly controlled conditions lead to the identification of spatially closed amino acid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify covalently linked residues. Our results show that melittin interacts with calmodulin by adopting a parallel orientation, i.e. the N and C-terminal halves of the peptide are anchored to the amino and carboxy-terminal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model of the complex was designed and refined on the basis of the experimental results, supporting the above conclusions. This finding reveals a further dimension to the already remarkable capability of calmodulin in binding different protein substrates, providing this protein with the capability of regulating an even larger number of enzymes.

Amino Acid Sequence↗
PubMed1996

The role of gap junction membrane channels in development.

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.

Animals↗
PubMed2004

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation.

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.

Animals↗
PubMed1998

Identification and characterization of LMO4, an LMO gene with a novel pattern of expression during embryogenesis.

LMO4 is a novel member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors. LMO1, LMO2, and LMO4 have distinct expression patterns in adult tissue, and we demonstrate that nuclear retention of LMO proteins is enhanced by the nuclear LIM interactor (NLI). In situ hybridization to early mouse embryos of 8-14.5 days revealed a complex pattern of LMO4 expression spatially overlapping with NLI and LHX genes. LMO4 expression in somite is repressed in mice mutant for the segment polarity gene Mesp2 and expanded in Splotch mutants. During jaw and limb outgrowth, LMO4 and LMO2 expression define mesenchyme that is uncommitted to regional fates. Although both LMO2 and LMO4 are activated in thymic blast cells, only LMO4 is expressed in mature T cells. Mesenchymal and thymic blast cell expression patterns of LMO4 and LMO2 are consistent with the suggestion that LMO genes inhibit differentiation.

Adaptor Proteins, Signal Transducing↗