[Studies on cardiac stress insufficiency using radiocardiographic function analysis].
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Cardiac Output↗
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[Study of cardiac dynamics by use of the radiocardiographic functional analysis (RCF) method].
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Cardiac Volume↗
The identification of high-school dropouts identified as learning disabled: evaluating the utility of a discriminant analysis function.
This study examined the utility of a linear discriminant function to distinguish between students identified as learning disabled (LD) who had either been released from high school under codes suggestive of school dropout (n = 213) or graduation (n = 92). The discriminant function was comprised of six variables--student ethnicity, reading ability, family intactness, family socioeconomic status, school transfers, and school-initiated interruptions. The analysis determined that differences between the LD dropout sample and LD graduate sample were sufficient to allow for a discrimination between the groups. On the basis of group differences the discriminant function that was constructed correctly classified 83% of the school dropouts and 46% of the school graduates, for an overall 73% accuracy rate. Those factors contributing most to the function were the number of district-initiated interruptions, school transfers, and family intactness. Based on the findings, implications for school districts and future research are noted.
Achievement↗
Functional analysis of human MutSalpha and MutSbeta complexes in yeast.
Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA. The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast. Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding. The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.
Alleles↗
Genetic, genomic, and functional analysis of the granule lattice proteins in Tetrahymena secretory granules.
In some cells, the polypeptides stored in dense core secretory granules condense as ordered arrays. In ciliates such as Tetrahymena thermophila, the resulting crystals function as projectiles, expanding upon exocytosis. Isolation of granule contents previously defined five Granule lattice (Grl) proteins as abundant core constituents, whereas a functional screen identified a sixth family member. We have now expanded this screen to identify the nonredundant components required for projectile assembly. The results, further supported by gene disruption experiments, indicate that six Grl proteins define the core structure. Both in vivo and in vitro data indicate that core assembly begins in the endoplasmic reticulum with formation of specific hetero-oligomeric Grl proprotein complexes. Four additional GRL-like genes were found in the T. thermophila genome. Grl2p and Grl6p are targeted to granules, but the transcripts are present at low levels and neither is essential for core assembly. The DeltaGRL6 cells nonetheless showed a subtle change in granule morphology and a marked reduction in granule accumulation. Epistasis analysis suggests this results from accelerated loss of DeltaGRL6 granules, rather than from decreased synthesis. Our results not only provide insight into the organization of Grl-based granule cores but also imply that the functions of Grl proteins extend beyond core assembly.
Amino Acid Sequence↗
Cloning and functional analysis of a beta-tubulin gene from a benomyl resistant mutant of Aspergillus parasiticus.
A genomic DNA library prepared from a benomyl resistant strain of Aspergillus parasiticus was screened with a Neurospora crassa beta-tubulin gene probe. A unique A. parasiticus genomic DNA fragment, thought to carry a mutant beta-tubulin gene (benr), was isolated. Two plasmids, pYT1 and pYTPYRG, carrying the putative benr gene or benr plus a second selectable marker (pyrG), respectively, were used to transform a benomyl sensitive strain of A. parasiticus (CS10) to determine if benr conferred benomyl resistance (BenR). BenR colonies were obtained with pYTPYRG, pYT1 or pYT1 cotransformed with pPG3J which carries a functional pyrG gene. No BenR colonies were obtained without added DNA or with pPG3J only (controls). Southern hybridization analysis of BenR and BenS transformants suggested that plasmid integration occurred most frequently at the chromosomal bens locus, however evidence for gene conversion and heterologous recombination was also observed. The predicted amino acid sequence of benr displayed a high degree of identity (> 93%) with other fungal beta-tubulin genes which confer benomyl resistance. Sequence analysis together with the genetic data suggested that benr encodes a functional mutant beta-tubulin.
Aspergillus↗
BEACH family of proteins: phylogenetic and functional analysis of six Dictyostelium BEACH proteins.
The beige and Chediak-Higashi syndrome (BEACH)-domain containing proteins constitute a new family of proteins found in all eukaryotes. The function of these proteins, which include the Chediak-Higashi syndrome (CHS) protein, Neurobeachin, LvsA, and FAN, is still poorly understood. To understand the diversity of this novel protein family, we analyzed a large array of BEACH-family protein sequences from several organisms. Comparison of all these sequences suggests that they can be classified into five distinct groups that may represent five distinct functional classes. In Dictyostelium we identified six proteins in this family, named LvsA-F, that belong to four of those classes. To test the function of these proteins in Dictyostelium we created disruption mutants in each of the lvs genes. Phenotypic analyses of these mutants indicate that LvsA is required for cytokinesis and osmoregulation and LvsB functions in lysosomal traffic. The LvsC-F proteins are not required for these or other processes such as growth and development. These results strongly support the concept that BEACH proteins from different classes have distinct cellular functions. Having six distinct BEACH proteins, Dictyostelium should be an excellent model system to dissect the molecular function of this interesting family of proteins.
Amino Acid Sequence↗
Functional analysis of mutant and wild-type Drosophila origin recognition complex.
The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.
Adenosine Triphosphatases↗
Domain organization and functional analysis of Thermus thermophilus MutS protein.
MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.
Adenosine Triphosphatases↗
Generating functional analysis of the dynamics of the batch minority game with random external information.
We study the dynamics of the batch minority game, with random external information, using generating functional techniques introduced by De Dominicis. The relevant control parameter in this model is the ratio alpha=p/N of the number p of possible values for the external information over the number N of trading agents. In the limit N-->infinity we calculate the location alphac of the phase transition (signaling the onset of anomalous response), and solve the statics for alpha>alphac exactly. The temporal correlations in global market fluctuations turn out not to decay to zero for infinitely widely separated times. For alpha 0 we analyze our equations in leading order in alpha, and find asymptotic solutions with diverging volatility sigma=O(alpha(-1/2)) (as regularly observed in simulations), but also asymptotic solutions with vanishing volatility sigma=O(alpha(1/2)). The former, however, are shown to emerge only if the agents' initial strategy valuations are below a specific critical value.
Journal Article↗
Functional analysis of alternative isoforms of the transcription factor PAX3 in melanocytes in vitro.
Transcription factor PAX3 has seven isoforms of which PAX3c has been studied extensively whereas the functions of the other isoforms are less well known. Here, we found that PAX3 isoforms in a stable transfection system have different biological functions in mouse melanocytes in vitro. PAX3a and PAX3b had negative effects on melanocyte proliferation but had no discernable effect on melanocyte growth in soft agar. PAX3a did not affect cell migration and apoptosis but PAX3b reduced migration and accelerated apoptosis. PAX3c and PAX3d promoted cell proliferation, migration, transformation, and survival. PAX3e reduced melanocyte growth; transformation and migration were unchanged and apoptosis was increased in vitro. PAX3g did not influence cell proliferation or apoptosis. Cells expressing PAX3g were able to grow in soft agar but migration was reduced. PAX3h increased cell proliferation, migration, survival, and transformation. These functional studies have advanced our understanding of the effects of PAX3 isoforms in melanocytes and their potential contribution in tumorigenesis.
Cell Cycle↗
The amygdala: historical and functional analysis.
The amygdala, part of the limbic system, is a small collection of neurons located in the ventral temporal lobe, and can be divided into two morphologically and functionally distinct nuclei - the corticomedial, a phylogenetically old group, and the basolateral, a phylogenetically more recent group. Both divisions of the amygdala have extensive interconnections with the hypothalamus, the corticomedial nucleus via the stria terminalis and the basolateral nucleus via the ventral amygdalohypothalamic pathway. The amygdala receives input from the orbital frontal cortex, the piriform cortex, the hypothalamus, the thalamus, and all sensory modalities as well as from the other structures of the limbic system. Early theories positing strictly emotional, olfactory, or visceral functions for the amygdala are at best incomplete. An intact amygdala seems to be essential for the successful performance of behavior patterns such as instinctive food getting and instinctive defensive reactions that are necessary for the survival of the individual organism and the species. Ablation of the amygdala produces many behavioral deficits that center around the inability to integrate changes in reinforcement contigencies into new behavior patterns.
Amygdala↗
Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.
STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.
Amino Acid Sequence↗
Functional analysis of the mismatch repair system in bladder cancer.
In bladder cancer the observed microsatellite instability indicates that mismatch repair deficiency could be a frequently involved factor in bladder cancer progression. To investigate this hypothesis we analysed extracts of seven bladder cancer cell lines and, as a novel approach, five clinical cancer samples for mismatch repair activity. We found that one cell line (T24) and three of the clinical samples had a reduced repair capacity, measured to approximately 20% or less. The T24 cell extract was unable to repair a G-G mismatch and showed reduced repair of a 2-base loop, consistent with diminished function of the MSH2-MSH6 heterodimer. The functional assay was combined with measurement for mutation frequency, microsatellite analysis, sequencing, MTT assay, immunohistochemical analysis and RT-PCR analysis of the mismatch repair genes MSH2, MSH3, MSH6, PMS1, PMS2 and MLH1. A >7-fold relative increase in mutation frequency was observed for T24 compared to a bladder cancer cell line with a fully functional mismatch repair system. Neither microsatellite instability, loss of repair nor mismatch repair gene mutations were detected. However, RT-PCR analysis of mRNA levels did detect changes in the ratio of expression of the Mut S and Mut L homologues. The T24 cell line had the lowest MSH6 expression level of the cell lines tested. Identical RT-PCR analysis of seventeen clinical samples (normal urothelium, 7; pTa low stage, 5; and pT1-4 high stage, 5) indicated a significant change in the expression ratio between MSH3/MSH6 (P< 0.004), MSH2/MSH3 (P< 0.012) and PMS2/MLH1 P< 0.005, in high stage bladder tumours compared to normal urothelium and low stage tumours. Collectively, the data suggest that imbalanced expression of mismatch repair genes could lead to partial loss of mismatch repair activity that is associated with invasive bladder cancer.
Base Pair Mismatch↗
Molecular cloning and functional analysis of (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase from Pseudomonas mendocina LZ.
An inactive (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase (PhaG(Pm)) was cloned from a newly isolated Proteobacteria Pseudomonas mendocina LZ. It is the first characterized native inactive PhaG protein. Sequence analysis indicated that there were only two sites where the amino acid sequence differed between this inactive protein and the functional PhaG(Pp) from P. putida. The differences were located at position 78 and in the region 109-113 in the amino acid sequence. Mutagenesis was carried out to investigate these two sites. A recombinant strain harboring a S78C PhaG(Pp) mutant accumulated polyhydroxyalkanoates (PHA) at 11.9% of the cellular dry weight, as compared to the 21.6% PHA produced by the recombinant harboring the wild-type PhaG(Pp). On the other hand, the changes in the amino acid region 109-113 of PhaG(Pp) to its corresponding region of PhaG(Pm) resulted in negligible PHA accumulation. This demonstrated that region 109-113 in PhaG is relatively important for transacylase activity, while position 78 just plays a supporting role for the enzyme. Furthermore, 3-D structural models of PhaG(Pp) and PhaG(Pm) developed by computational prediction revealed that the variation in amino acids at 109-113 leads to the destruction of the PhaG catalytic center, resulting in the loss of enzyme activity.
Acyltransferases↗
[Cloning and prokaryotic expression of transcriptional co-activator gene of Clonorchis sinensis and functional analysis of the expressed protein].
OBJECTIVE: To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function. METHODS: A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamH I and Sal I, the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E. coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni2+) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC. RESULTS: The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel. CONCLUSION: The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.
Amino Acid Sequence↗
Structural and functional analysis of SFT, a stimulator of Fe Transport.
Previous studies demonstrated that SFT (Stimulator of Fe Transport) facilitates both transferrin and nontransferrin-bound iron uptake in HeLa cells (Yu, J., and Wessling-Resnick, M. (1998) J. Biol. Chem. 273, 6909-6915). To further characterize the structure and function of SFT, we studied this human factor in rodent BHK cells. Kyte-Doolittle analysis suggests that SFT has six transmembrane-spanning segments. This transport protein also displays an REXXE motif resembling domains involved in iron binding by ferritin and in iron uptake mediated by the yeast transporter Ftr1. Using N- and C-terminal epitope tags, we have identified that modification of either protein terminus does not interfere with SFT function in nontransferrin-bound iron uptake. The N- and C-terminal domains are intracellularly disposed since antibodies against these epitopes fail to recognize expressed proteins unless BHK cells are solubilized with detergents. To define the topology of two large extramembranous loop domains, anti-peptide antibodies were employed; anti-loop 4 antibodies show no immunoreactivity unless cells are permeabilized but anti-loop 5 antibodies recognize and bind surface SFT. Thus, loop 4 must be intracellular while loop 5 is extracellular. These topological studies situate the putative iron-binding REXXE domain on the cytosolic face of the plasma membrane. However, 55Fe-binding studies reveal that the ability of SFT to bind and mediate transport of extracellular iron is defective in mutants with Glu --> Ala conversions in this motif. Curiously, we also find that depletion of intracellular iron by desferrioxamine impairs SFT transport and iron-binding functions. These observations lead to the speculation that the REXXE motif may play an important role in regulating SFT activity through interaction with intracellular iron and demonstrate that iron transport mediated by SFT is itself an iron-dependent process.
Animals↗
Functional analysis of fibronectin isoforms in chondrogenesis: Full-length recombinant mesenchymal fibronectin reduces spreading and promotes condensation and chondrogenesis of limb mesenchymal cells.
Fibronectin (FN), a large dimeric glycoprotein, functions primarily as a connecting molecule in the extracellular matrices of tissues by mediating both cell-matrix and matrix-matrix interactions. All members of the FN family are products of a single FN gene; heterogeneity arises from the alternative splicing of at least three regions (IIIB, IIIA, and V) during processing of a common primary transcript. During chick embryonic limb chondrogenesis, FN structure changes from B+A+ in precartilage mesenchyme to B+A- in differentiated cartilage, and exon IIIA has been shown to be necessary for the process of mesenchymal cellular condensation, a requisite event that precedes overt expression of chondrocyte phenotype. This study aims to investigate the mechanistic action of the FN isoforms in mesenchymal chondrogenesis and, in particular, to identify the specific cellular function in mesenchymal condensation mediated by the mesenchymal (B+A+) FN isoform. Full-length cDNAs corresponding to four splice variants (B+A+, B+A-, B-A+, B-A-) of FN were constructed, and expressed the corresponding proteins using a baculovirus expression vector system. Cell adhesion assays with purified proteins showed that, although the relative levels of cell attachment were approximately the same, chick limb-bud mesenchymal cells spread up to 40 % less on mesenchymal (B+A+) FN than on cartilage (B+A-) FN, (B-A+) FN, or plasma (B-A-) FN. Cellular condensation and chondrogenic differentiation were also promoted in high-density micromass cultures of limb mesenchymal cells plated onto B+A+ FN. These observations suggest that the process of mesenchymal condensation is mediated at least in part by the enhanced ability of chondrogenic mesenchymal cells to migrate and aggregate as a consequence of residing in and interacting with mesenchymal FN. Our findings are consistent with and provide a mechanistic basis for previous observations that rounding of limb mesenchymal cells precedes the onset of chondrogenesis.
Animals↗