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At least 451 records · Page 25Linked to original sources
PubMed2000

Sequence variants and functional analysis of human papillomavirus type 16 E5 gene in clinical specimens.

Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process.

Base Sequence↗
PubMed1986

Prediction of coronary events following myocardial infarction using a discriminant function analysis.

This study was undertaken to derive an index for predicting coronary events in the first year after a myocardial infarction in "low-risk" patients enrolling in a Cardiac Rehabilitation Program. Data from 145 consecutive patients were analysed. The events were classified as follows: angina requiring further therapy, re-infarction and coronary death. Seventy patients had events: Angina--52, Re-infarction--8, Coronary Death--10. A discriminant function analysis was performed to predict such events using data available at the time of discharge from hospital. The following were significant predictors: (1) previous infarction/angina, (2) radiological evidence of cardiomegaly or lung congestion in the Coronary Care Unit, (3) Non-Q wave infarction and (4, 5 and 6) angina, atrial arrhythmias and a decrease in R wave amplitude in V5 during a pre-discharge exercise test. The jack-knife method classified correctly 71.2% of those with events and 72.6% of those without events. In patients with discriminant scores greater than +0.2, 82% developed events.

Adult↗
PubMed2005

Structure-function analysis of the three domains of RuvB DNA motor protein.

RuvB protein forms two hexameric rings that bind to the RuvA tetramer at DNA Holliday junctions. The RuvAB complex utilizes the energy of ATP hydrolysis to promote branch migration of Holliday junctions. The crystal structure of RuvB from Thermus thermophilus (Tth) HB8 showed that each RuvB monomer has three domains (N, M, and C). This study is a structure-function analysis of the three domains of RuvB. The results show that domain N is involved in RuvA-RuvB and RuvB-RuvB subunit interactions, domains N and M are required for ATP hydrolysis and ATP binding-induced hexamer formation, and domain C plays an essential role in DNA binding. The side chain of Arg-318 is essential for DNA binding and may directly interact with DNA. The data also provide evidence that coordinated ATP-dependent interactions between domains N, M, and C play an essential role during formation of the RuvAB Holliday junction ternary complex.

Adenosine Triphosphatases↗
PubMed1995

Characterization and functional analysis of the rat endothelin-1 promoter.

To define the molecular mechanisms of endothelin-1 (ET-1) gene regulation, we cloned, sequenced, and characterized the rat ET-1 promoter. A sequence consisting of the first 1329 bp of the rat ET-1 promoter was investigated in greater detail. Sequence analysis identified putative binding sites for a number of transcriptional factors that may be involved in ET-1 gene regulation. Several of these factors have been proposed earlier to be involved in cell-specific gene regulation and may be responsible for directing ET-1 expression in vivo. For functional analysis of the ET-1 promoter, we generated a reporter gene construct using luciferase as reporter gene under control of the promoter fragment isolated. The construct was transfected transiently into bovine aortic endothelial cells, and luciferase expression was evaluated. The results indicated that the promoter segment used showed high expression in endothelial cells comparable to that induced by viral promoters. Since ET-1 is regulated by a number of vasoactive substances, we studied the effect of angiotensin II on endothelin transcription. We could demonstrate a dose-dependent transcriptional activation of ET-1 transcription by angiotensin.

Angiotensin II↗
PubMed1999

Sensitivity, functional analysis, and behavior genetics: a response to Freeman et al.

Freeman et al. (1998) asserted that sensitivity theory is circular, unsupported by empirical evidence, and represents an "either/or" decision in regard to applied behavior analysis. We reply by showing that sensitivities are objectively measured and that our theory permits testable predictions. Further, we briefly summarize the results of recent studies that support our theory, including investigations that link sensitivities to genes and challenging behavior. Finally, we reject the idea that sensitivity theory and functional analysis represent an "either/or" viewpoint and call again for research to integrate these approaches.

Behavioral Symptoms↗
PubMed1997

Language production and serial order: a functional analysis and a model.

In speech production, previously spoken and upcoming words can impinge on the word currently being said, resulting in perseverations (e.g., "beef needle soup") and anticipations (e.g., "cuff of coffee"). These errors reveal the extent to which the language-production system is focused on the past, the present, and the future and therefore are informative about how the system deals with serial order. This article offers a functional analysis of serial order in language and develops a general formal model. The centerpiece of the model is a prediction that the fraction of serial-order errors that are anticipatory, as opposed to perseveratory, can be closely predicted by overall error rate. The lower the error rate, the more anticipatory the errors are, regardless of the factors influencing error rate. The model is successfully applied to experimental and natural error data dealing with the effects of practice, speech rate, individual differences, age, and brain damage.

Adult↗
PubMed2003

Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel "cell-wall invertase-like" specific 6-FEH from sugar beet (Beta vulgaris L.).

About 15% of flowering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans. The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levan-type fructans (beta-2,6) are the best substrates for the enzyme, while inulin-type fructans (beta-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and beta-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.

Amino Acid Sequence↗
PubMed2006

Functional analysis of mutations in TGIF associated with holoprosencephaly.

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.

Animals↗
PubMed1976

Discriminant function analysis of factors related to myocardial infarction in male patients on antihypertensive therapy.

Records of 367 male patients who began attending the Dunedin Hospital Hypertension Clinic between 1959 and 1969 were coded up to the end of 1972. Of these patients, 60 had their first myocardial infarction, or sudden cardiac death without previous evidence of myocardial infarction, while they were attending the clinic. This infarct group was compared with a control group of 120 which was chosen from the remaining patients so that the two groups would be comparable with regard to their age and year of first attendance. A stepwise discriminant function analysis showed that the basal systolic and diastolic pressures measured when patients started attending the clinic were the variables with the most significant difference. Further analysis showed that inclusion of the average casual standing systolic pressure when patients were receiving antihypertensive therapy improved the discrimination between the groups. The average casual standing diastolic pressure during antihypertensive therapy also improved the discrimination, but, curiously enough, with a negative sign in the discriminant function. Quetelet's index of obesity similarly improved the discrimination, obese subjects having less risk of infarction or sudden death. Serum cholesterol, however, was not related to prognosis.

Age Factors↗
PubMed2002

Structural and functional analysis of the human mitotic-specific ubiquitin-conjugating enzyme, UbcH10.

Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. We report here the 1.95-A crystal structure of a mutant UbcH10, in which the active site cysteine has been replaced with a serine. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH2-terminal extension in UbcH10 is disordered and that a conserved 3(10)-helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH2-terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure.

Amino Acid Motifs↗
PubMed1998

Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells.

The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.

Amino Acid Sequence↗
PubMed2000

Functional analysis of the yeast genome.

Just as Saccharomyces cerevisiae itself provides a model for so many processes essential to eukaryotic life, we anticipate that the methods and the mindset that have moved yeast biological research "beyond the genome" provide a prototype for making similar progress in other organisms. In this review I describe the experimental processes, results and utility of the current large-scale experimental approaches that use genomic data to provide a functional analysis of the yeast genome.

DNA Footprinting↗
PubMed1984

Metropolitan Forensic Anthropology Team (MFAT) studies in identification: 1. Race and sex assessment by discriminant function analysis of the postcranial skeleton.

A case study is presented to demonstrate the utility of the team approach to the identification of human remains, and to illustrate a methodological innovation developed by MFAT. Case 1 represents the first of several planned case studies, each designed to present new methodological solutions to standard problems in identification. The present case describes a test, by application, of race and sex assessment of the postcranial skeleton by discriminant function analysis.

Adult↗
PubMed2001

Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (alpha1S) expressing two complementary protein fragments.

BACKGROUND: The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of alpha1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments. RESULTS: Functional analysis of cDNA transfected dysgenic myotubes lacking alpha1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-alpha1S) expressed the N-terminal half of alpha1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-alpha1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current. DISCUSSION: A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

Amino Acid Sequence↗
PubMed1999

Functional analysis and response covariation in the assessment of personality disorders: a reply to Staats and to Bissett and Hayes.

In this reply to Bissett and Hayes (this issue) and Staats (this issue) we address critical comments in response to our initial proposal and highlight points of agreement. The overall thesis of our reply is that classification schemes based on nomothetic response covariation, such as DSM, complement, but do not substitute for, an idiographically-based functional analysis and behavioral assessment. In the context of our reply, we address the following primary concerns raised by Bissett, Hayes, and Staats: (a) we are essentially proposing the melding of two theoretically incongruent approaches, and that such a melding is inherently not viable or useful; (b) the behavior analytic approach cannot account for personality or psychological constructs; and (c) that categories based on topography do not have demonstrated treatment utility. We also discuss points of agreement with our respondents: (d) a theoretically-based descriptive classification system is required to ultimately advance clinical science, (e) the DSM personality disorder classification system, to remain viable, needs a stronger empirical base; and (f) that alternatives to DSM classification that more strongly emphasize behavioral principles are in need of development.

Behaviorism↗
PubMed2000

Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome.

Helicobacter pylori is a gram-negative bacterium, which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. The genomic sequences of two H.pylori strains, 26695 and J99, have been published recently. About two dozen potential restriction-modification (R-M) systems have been annotated in both genomes, which is far above the average number of R-M systems in other sequenced genomes. Here we describe a functional analysis of the 16 putative Type II R-M systems in the H. pylori J99 genome. To express potentially toxic endonuclease genes, a unique vector was constructed, which features repression and antisense transcription as dual control elements. To determine the methylation activities of putative DNA methyltransferases, we developed polyclonal antibodies able to detect DNA containing N6-methyladenine or N4-methylcytosine. We found that <30% of the potential Type II R-M systems in H.pylori J99 strain were fully functional, displaying both endonuclease and methyltransferase activities. Helicobacter pylori may maintain a variety of functional R-M systems, which are believed to be a primitive bacterial 'immune' system, by alternatively turning on/off a subset of numerous R-M systems.

Adenine↗
PubMed1997

Mutational and functional analysis of the neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20-27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD.

Base Sequence↗
PubMed2006

Sequence variability and functional analysis of MutS of hypermutable Pseudomonas aeruginosa cystic fibrosis isolates.

In this study, we investigated the variability of MutS among Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. Sequencing of the mutS gene of 15 hypermutable P. aeruginosa isolates obtained from different patients revealed high rates of nucleotide substitutions as compared to that of strain PAO1. Significantly more synonymous than non-synonymous nucleotide substitutions have been found, indicating that generally MutS is highly conserved. The functional analysis of MutS variants by complementation of a PAO1 mutS mutant revealed 5 isolates with a defective MutS due to frameshift mutations or amino acid substitutions. This work supports the hypothesis that the respiratory tract of CF patients represents an environment that favors the selection of highly adaptive mutator phenotypes.

Adolescent↗