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At least 523 records · Page 29Linked to original sources
PubMed1996

An exploratory stepwise discriminant function analysis of malingered and nondistorted responses to the Neuropsychological Symptom Inventory.

Clinicians are often asked to evaluate a patient's potential for malingering during a neuropsychological examination. This problem is particularly crucial when financial gain may hinge on the results of the assessment. Response distortion may occur even when a head injury has been verified by radiological means (e.g., MRI). Research in the identification of malingering has yet to produce results that are effective in the clinical setting. The present study addressed several shortcomings of past research with the use of a self-report symptom inventory. The responses of 242 subjects told to distort symptomology were compared to 128 nonmalingerers. A discriminant function analysis of these data produced a 90% hit rate overall with 2.3% false positives. The results were discussed in terms of clinical utility.

Cognition Disorders↗
PubMed2006

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus.

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050-850 bp), Box 2 (723-606 bp), and Box 3 (224-168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.

Bacterial Proteins↗
PubMed2002

Full-length sequence and functional analysis of hepatitis B virus genome in a virus carrier: a case report suggesting the impact of pre-S and core promoter mutations on the progression of the disease.

In chronic hepatitis B virus (HBV) infection, the quiescent immunotolerant phase evolves into the immunoactive phase. The aim of the present study was to clarify the virological alterations relevant to progression. Serial serum samples obtained from a patient with HBV during long-term follow-up were analysed by sequencing of the full-length HBV-DNA using polymerase chain reaction (PCR). In addition, PCR products of HBV genome from each serum sample were transfected into HuH-7 human hepatoma cells for the functional analysis of the transfected viral genomes. Based on the HBV-DNA sequence analysis, the patient had the genotype C virus, and the mutant HBV with common core promoter mutations (T(1762)A(1764)) and deletion of the pre-S region responsible for large surface protein transcription emerged before the onset of hepatitis. When the vigorous host immune response developed (indicated by the flare-up of hepatitis), the mutant HBV containing common core promoter mutations and another pre-S deletion causing lack of the surface protein promoter became predominant. The HBV-DNA sequences, other than pre-S and core promoter regions were identical to the wild-type sequence throughout the study. Transfection of PCR products containing the mutant HBV sequences resulted in increased amounts of intracellular replicative intermediates but the decreased secretion of HBsAg and HBeAg into culture media, suggesting accumulation of nonenveloped viral core particles within the cells. These results indicate that pre-S deletion and core promoter mutations may participate cooperatively in progression of the disease.

Adult↗
PubMed1989

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240.

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.

Amino Acid Sequence↗
PubMed1999

Functional analysis of the p57KIP2 gene mutation in Beckwith-Wiedemann syndrome.

p57KIP2 is a potent tight-binding inhibitor of several G1 cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57KIP2 in patients with BWS that were transmitted from the patients' carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57KIP2 would have existed, which might have caused the disorders in BWS patients.

Animals↗
PubMed1988

Cloning of authentic human epidermal growth factor as a bacterial secretory protein and its initial structure-function analysis by site-directed mutagenesis.

A synthetic chimeric gene, coding for the human epidermal growth factor fused to the signal peptide of Escherichia coli alkaline phosphatase, was cloned into E. coli under the transcriptional control of the trp-lac (tac) promoter. Following induction with isopropylthiogalactoside, the secretion of the correctly processed protein product into the bacterial periplasm was detected and quantitated by its specific binding to the epidermal growth factor receptor. The purified protein was identical to authentic human epidermal growth factor in size, amino acid composition, primary sequence, receptor binding, and stimulation of receptor protein-tyrosine kinase activity. Based on interspecies homologies, structural considerations, and reported studies with peptide fragments, structure-function analysis was initiated with alterations of targeted amino acid residues by oligonucleotide-directed mutagenesis. The receptor binding affinity of each mutant, relative to the wild type, was measured by both radioreceptor competition and receptor tyrosine kinase stimulation assays. In general, the values obtained by the two methods were in agreement for each species of epidermal growth factor and followed the order: wild type greater than Glu24----Gly greater than Asp27----Gly much greater than Pro7----Thr greater than Tyr29----Gly greater than Leu47----His. The relatively low values obtained with the last two mutants suggest that Tyr29 and Leu47 may be important for the biological activity of human epidermal growth factor.

Alkaline Phosphatase↗
PubMed2003

Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI.

Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.

Adenosine Triphosphatases↗
PubMed2001

Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type 1/nerve growth factor receptor mutations.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare genetic disease characterized by absence of reaction to noxious stimuli and anhidrosis. The genetic bases of CIPA have remained long unknown. A few years ago, point mutations affecting both coding and noncoding regions of the neurotrophic tyrosine receptor kinase type 1 (NTRK1)/nerve growth factor receptor gene have been detected in CIPA patients, demonstrating the implication of the nerve growth factor/NTRK1 pathway in the pathogenesis of the disease. We have previously shown that two CIPA mutations, the G571R and the R774P, inactivate the NTRK1 receptor by interfering with the autophosphorylation process. We have extended our functional analysis to seven additional NTRK1 mutations associated with CIPA recently reported by others. Through a combination of biochemical and biological assays, we have identified polymorphisms and pathogenic mutations. In addition to the identification of residues important for NTRK1 activity, our analysis suggests the existence of two novel pathogenic mechanisms in CIPA: one based on the NTRK1 receptor processing and the other acting through the reduction of the receptor activity.

Amino Acid Substitution↗
PubMed1998

Four addictions: the MMPI and discriminant function analysis.

Over the past twenty years many MMPI studies of substance abuse have investigated the complex relationship between personality profile and drug of choice. This work has repeatedly established that alcoholics, heroin, cocaine and polydrug addicts share 4-2/2-4 (Psychopathy and Depression) or 4-8/8-4 (Psychopathy and Thought Disorder) MMPI profiles, but that the substance abuse populations differ in the plane of severity in that general profile. The alcoholics occupy the least disturbed sector, the polydrug abusers the most disturbed level and the heroin and cocaine addicts positions of moderate disturbance. The vast majority of studies, however, cite only group means to buttress their conclusions. Our work probed more deeply into the data using Discriminant Function Analysis. With this methodology we discovered important differences between the groups, previously hidden, which may carry differential treatment implications.

Adult↗
PubMed2007

Functional analysis of Survivin in spindle assembly in Xenopus egg extracts.

Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.

Animals↗
PubMed2006

Is a novel I214M substitution in the NEFL gene a cause of Charcot-Marie-Tooth disease? Functional analysis using cell culture models.

Recent studies have shown that mutations in neurofilament light subunit gene (NEFL) cause Charcot-Marie-Tooth (CMT) disease. Since the first description of the Gln333Pro mutation in the NEFL gene, 10 pathogenic mutations in the NEFL gene have been reported in patients affected with CMT disease. We report a novel I214M amino acid substitution in the NEFL gene in two unrelated patients affected with CMT. Because the I214M amino acid substitution in the NEFL protein was not detected in a CMT affected brother of the proband, its pathogenic effect became unclear. In order to determine whether this amino acid substitution is a benign polymorphism or causative of the disease, we performed a functional analysis of the mutant I214M neurofilament protein (NFL). Transfections of the mutant protein in cultured cells revealed an increased tendency to form highly compacted filamentous structures but no other alterations of neurofilament assembly or transport were observed. Furthermore, the sibling of one of the patients was also affected with CMT but did not have the I214M substitution. These data suggest that this I214M substitution in the NEFL gene was not a direct cause of the disease but could be a polymorphism or possibly a modifier of the CMT phenotype.

Adolescent↗
PubMed2001

Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeast for protein purification and functional analysis.

The human intestinal peptide transporter hPEPT1 has been expressed in the yeast Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-dehydrogenase gene. A myc-epitope fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 for ease of detection and purification. Yeast cells transformed with tagged hPEPT1 exhibited 30-fold increased dipeptide uptake compared to control cells with a substrate specificity and pH dependence similar to the native transporter. The tagged hPEPT1 protein was detected in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membrane protein. These studies demonstrate that tagged hPEPT1 can be expressed functionally in P. pastoris with unaltered phenotypical characteristics allowing the yeast cells to be used for functional analysis such as screening for compounds utilizing the peptide transporter for absorption in the human intestine. Moreover, recombinant hPEPT1 can now easily be detected for further purification purposes using immobilized metal-affinity chromatography.

Anti-Bacterial Agents↗
PubMed1989

Structure-function analysis of angiotensin-converting enzyme inhibitors as modifiers of radiation-induced pulmonary endothelial dysfunction in rats.

We have previously demonstrated that thiol-containing collagen antagonists (penicillamine) and angiotensin-converting enzyme (ACE) inhibitors (Captopril and CL242817) ameliorate endothelial dysfunction in irradiated rat lung. The purpose of the present study was to determine whether the non-thiol ACE inhibitor CGS13945 also modifies radiation-induced pulmonary endothelial dysfunction in rats sacrificed 2 months after a single dose (0-30 Gy) of 60 Co gamma rays to the right hemithorax. The CGS13945 was administered in the feed continuously after irradiation at a regimen of 30 mg (kg body weight)-1 day-1. Four markers of lung endothelial function were monitored: ACE activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Right lung ACE and PLA activities decreased with increasing radiation dose, and CGS13945 significantly ameliorated both responses. Dose-reduction factors (DRF) for the inhibitor were 1.80 for ACE activity and 1.41 for PLA activity (p less than 0.05). In contrast, lung PGI2 and TXA2 production increased with increasing radiation dose, and CGS13945 did not influence either response significantly. Thus the ACE inhibitor CGS13945 modifies radiation-induced pulmonary endothelial dysfunction in rats, indicating that the presence of a thiol group is not essential for therapeutic efficacy in this class of compounds. On the other hand, CGS13945 exhibits a differential sparing of radiation-induced pulmonary endothelial dysfunction, as does penicillamine. A structure-function analysis of the present and previous data indicates that all of the ACE inhibitors tested (Captopril, CL242817 and CGS13945) spare the radiation-induced suppression in lung ACE and PLA activity; all of the thiol compounds tested (penicillamine, Captopril and CL242817) spare the radiation-induced elevation in lung PGI2 and TXA2 production; and the thiol ACE inhibitors (Captopril and CL242817) spare all four endothelial responses. These data confirm a novel and potentially important application for ACE inhibitors as modifiers of radiation-induced lung injury, and suggest that there are at least two components to their mechanism of therapeutic action in this model.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2005

Functional analysis of a reproductive organ predominant expressing promoter in cotton plants.

Transgenic Bt insect-resistant cotton plants have high insect resistance in the early stage of development, but relatively low resistance in the late stage. Substituting a reproductive organ-specific promoter for the CaMV35S promoter presently being used could be an ideal solution. For the first time, the promoter sequence of ADP-ribosylation factor 1 (arf1) gene was isolated from Gossypium hirsutumY18 by means of inverse PCR. The sequencing result discovered the unique structure of the arf1 promoter, including four promoter-specific elements, the initiator, TATA box, CAAT box and GC box, and also an intron in 5'-untranslation region. Four plant expression vectors were constructed for functional analysis of the promoter. Based on the pBl121 plant expression vector, four truncated arf1 promoters took the place of the CaMV35S promoter. These vectors were different only in their promoter regions. They were introduced into cotton plants via pollen tube pathway. Histochemical GUS staining and fluorescence quantitative analyses were performed to examine the expression patterns of the GUS gene driven by the 4 arf1 truncated promoters in transgenic cotton plants respectively. The results showed that the arf1 promoter was a typical reproductive organ-specific promoter. Hopefully, the arf1 promoter can be a regulatory element for designing cotton reproductive organs with desired characteristics.

ADP-Ribosylation Factor 1↗
PubMed2004

Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G-protein signaling-1.

The model organism Arabidopsis thaliana contains a restricted set of heterotrimeric G-protein subunits, with only one canonical Galpha subunit (AtGPA1), one Gbeta subunit (AtAGB1), and two Ggamma subunits (AtAGG1 and AtGG2) identified. We have identified a novel additional component of heterotrimeric G-protein signaling in the A. thaliana genome, regulator of G-protein signaling-1 (AtRGS1). This protein has the predicted topology and structure of a G-protein-coupled receptor in that it contains seven transmembrane domains, but AtRGS1 also contains a unique C-terminal extension, namely a regulator of G-protein signaling domain (RGS box). This article describes methods for the purification and in vitro functional analysis of the RGS box of AtRGS1.

Amino Acid Sequence↗
PubMed1987

Functional analysis and quantification of the complement C3 derived anaphylatoxin C3a with a monoclonal antibody.

The C3 fragment C3a belongs to the anaphylatoxins. It has immune regulatory activity and contributes to the pathogenesis of the adult respiratory distress syndrome (ARDS). The low molecular weight (9 kD) of C3a complicates the production of antibodies to C3a. We obtained a monoclonal antibody (designated H13) to human C3a. It reacts with C3a or C3a-desArg and with native C3 but not with C5 or C5a. In immunoblot analysis it reacts with the alpha- but not with beta-chain of C3 and binds to a protein with a mol. wt of about 10 kD present in zymosan-activated sera which is only marginally detectable in nonactivated serum and absent in plasma. H13 crossreacts with the analogous proteins of rabbit, guinea pig and sheep. H13 has the capacity to bind 125I-radiolabelled C3a efficiently but fails totally to react with 125I-C5a or with other C3 alpha-chain fragments. H13 blocks C3a functional activity. It markedly inhibits C3a-induced 3H-serotonin release from platelets in vitro and similarly inhibits the C3a-induced extravasation of Evans blue into the skin in vivo. H13 does not interfere with the haemolytic activity of C3. An ELISA system was established using H13 which permits quantification of C3a in sera of polytrauma patients. The antibody H13 should facilitate further functional analysis of C3a in experimental systems. It should be useful for quantification of C3a in diagnostic assays and also for application in immunopathology.

Animals↗
PubMed1998

Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue.

A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach. The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pi) of 8.20. Alignment of the A. thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae showed good sequence homology (42-45% identity, 61-66% similarity), particularly around the active site. Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species. Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S. cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase. The level of the DNA ligase transcript was not increased in A. thaliana seedlings in response to DNA damage induced by a period of enhanced UV-B irradiation. However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells.

Amino Acid Sequence↗
PubMed2001

Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele.

The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVNef is characterized by three rare amino acid substitutions, G(140)E, V(153)L and E(177)G. It was reported previously that the expression of F12-HIVNef in the context of the highly productive NL4-3 HIV-1 strain blocks virus replication at the level of virus assembly and/or release by a mechanism depending on the presence of the CD4 intracytoplasmic tail. Here, it is reported that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-mutated in each amino acid substitution readily replicated in CD4(+) cells. Attempting to correlate possible functional alterations with antiviral effects, both F12-HIVNef and its back mutants were tested in terms of well-characterized markers of Nef expression. Both F12-HIVNef and its G(177)E back mutant did not down-regulate CD4 as the consequence of a greatly reduced rate of CD4 internalization. On the other hand, F12-HIVNef as well as the E(140)G and L(153)V back mutants failed to activate the p62 Nef-associated kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated rates of CD4 internalization and p62NAK activation, whereas at least one Nef function was restored in all of the back mutants. Infection of cells expressing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef back mutants showed that both the lack of accelerated CD4 endocytosis and an, as yet, still unidentified function are required for the F12-HIVNef inhibitory phenotype. These results provide a detailed functional analysis of the F12-HIVnef allele and support the idea that both CD4 accelerated internalization and p62NAK activation are part of the essential steps in the virus replication cycle.

Alleles↗