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PubMed2000

Functional analysis of eight open reading frames on chromosomes XII and XIV of Saccharomyces cerevisiae.

Deletion, together with basic functional and bioinformatic analyses has been carried out on eight novel ORFs discovered during the sequencing of the Saccharomyces cerevisiae genome. Six ORFs (YLL049w, YLL051c, YLL052c, YLL053c, YLL054c and YLL055w) located on the left arm, and one (YLR130c) on the right arm, of chromosome XII, and an eighth ORF (YNL331c) on the left arm of the chromosome XIV, have been investigated. ORFs were deleted by the SFH-PCR gene-replacement strategy. Basic functional analysis revealed no obvious phenotype for any of the eight ORFs. Bioinformatic analysis, however, revealed possible functions for seven of the ORFs on the basis of the amino acid sequence similarity of their predicted protein products to those of proteins with known functions. ORF YLL051c (FRE6) shows similarity to iron transport proteins, such as ferric reductase. YLL052c and YLL053c appear to be aquaporins. The product of YLL054c (Yll054p) is highly similar to the oleate-specific transcriptional activator protein (Pip2p), which is involved in the peroxisomal induction pathway (pip). ORF YLL055w is similar to Dal5p, allantoate permease, and may play role in allantoin transport. YLR130c (ZRT2) is a low-affinity zinc transporter protein. YNL331c is also named AAD14, which is induced by chemicals that induce oxidative stress by depleting the cell of glutathione.

Chromosomes, Fungal↗
PubMed2001

Computational methods for protein function analysis.

Two recent advances have had the greatest impact on protein function analysis so far: the complete sequences of genomes and mRNA expression level profiles. The former has spurred the development of novel techniques to study protein function: phylogenetic profiles and gene clusters. The latter has introduced a method, not based on sequence homology, that enables one to group together functionally related genes.

Animals↗
PubMed1999

The impact of functional analysis methodology on treatment choice for self-injurious and aggressive behavior.

Self-injurious behavior (SIB) and aggression have been the concern of researchers because of the serious impact these behaviors have on individuals' lives. Despite the plethora of research on the treatment of SIB and aggressive behavior, the reported findings have been inconsistent regarding the effectiveness of reinforcement-based versus punishment-based procedures. We conducted a literature review to determine whether a trend could be detected in researchers' selection of reinforcement-based procedures versus punishment-based procedures, particularly since the introduction of functional analysis to behavioral assessment. The data are consistent with predictions made in the past regarding the potential impact of functional analysis methodology. Specifically, the findings indicate that, once maintaining variables for problem behavior are identified, experimenters tend to choose reinforcement-based procedures rather than punishment-based procedures as treatment for both SIB and aggressive behavior. Results indicated an increased interest in studies on the treatment of SIB and aggressive behavior, particularly since 1988.

Aggression↗
PubMed1986

Two-color flow cytometry and functional analysis of lymphocytes cultured from human renal allografts: identification of a Leu-2+3+ subpopulation.

The phenotype of T lymphocyte subsets present in renal biopsies showing acute cellular allograft rejection in six patients on cyclosporine have been characterized in situ by immunoperoxidase staining, and after expansion in vitro in interleukin 2 (IL-2) by two-color flow cytometry, sorting, and functional analysis. After 8 to 42 days in organ culture, both Leu-3+ (CD4) and Leu-2+ (CD8) subsets were found in each culture, in a ratio that varied from 0.2 to 5.0, which was not significantly different than the results of in situ immunoperoxidase staining of the uncultured biopsy. The cultured cells were almost all Leu-4+ (CD3) T cells (89% +/- 4), which expressed the activation markers DR (82% +/- 6) and the IL 2 (CD25) receptor (15% +/- 4). The Leu-3+ cells were largely Leu-8- (90% +/- 6), whereas a minority of the Leu-2+ cells were Leu-15+ (CD11) (26% +/- 4). Only a small fraction of the Leu-2+ cells stained for Leu-7 (8% +/- 6). Functional analysis of FACS-purified Leu-2-3+ and Leu-2+3- populations indicated that both subsets proliferated in response to graft donor antigens in a mixed lymphocyte reaction (MLR) and produced IL 2. Only the Leu-2+3- population demonstrated donor-specific cytotoxic activity. A minor subpopulation in each culture were both Leu-3+ and Leu-2+ (2.0%). Leu-2+3+ cells from one biopsy were purified to homogeneity (99.8%), and were found to express the T cell antigen receptor complex Ti/CD3 (WT-31+, Leu-4+), but not the common thymocyte antigen CD1 (OKT6). The Leu-2+3+ cells neither responded in the MLR, nor showed any cytotoxic capacity. The Leu-2+3+ cells were capable of IL 2 but not interferon-gamma production. None of the purified cultures demonstrated NK activity. A subset of the purified Leu-2+3+ cells lost Leu-2+ during 1 to 3 wk in culture, and became Leu-2-3+. These studies provide evidence that the cells that infiltrate renal allografts during rejection include alloproliferative, lymphokine-producing cells of both Leu-2+ and Leu-3+ subsets. The Leu-2+3- cells are also highly cytotoxic against donor lymphocytes, indicating the presence of helper independent cytotoxic T cells. A minor population of Leu-2+3+ T cells that do not express donor specific function was also identified.

Antibodies, Monoclonal↗
PubMed1997

Metal-ion-mediated allosteric triggering of yeast pyruvate kinase. 1. A multidimensional kinetic linked-function analysis.

Regulation of the glycolytic pathway is considered to be primarily achieved by the carbon metabolites resulting from glucose metabolism [e.g., fructose 1,6-diphosphate (FDP), phosphoenolpyruvate (PEP), and citrate] and by the ATP charge of the cell. The divalent cations (e.g., Mg2+ and Mn2+) have not been considered as having regulatory roles in glycolysis, although they are involved in almost every enzyme-catalyzed reaction in the pathway. Using a kinetic linked-function analysis of steady-state kinetic data for the interactions of PEP, FDP, and Mn2+ with yeast pyruvate kinase (YPK), we have found that the divalent metal is the principal trigger of the allosteric responses observed with this enzyme. The interaction of Mn2+ to YPK enhances the interaction of FDP by -1.6 kcal/mol and the interaction of PEP by -2.8 kcal/mol. The simultaneous interaction of all three of these ligands to YPK is favored by -4.3 kcal/mol over the sum of their independent binding free energies. Surprisingly, the binding of the allosteric activator FDP does not directly influence the binding of the substrate PEP since a coupling free energy near zero was calculated for these two ligands. Thus, communication between the PEP and FDP sites occurs structurally through the metal by an allosteric relay mechanism. These conclusions are supported by results of a thermodynamic linked-function analysis of direct binding data for the interactions of PEP, FDP, and Mn2+ with YPK [Mesecar, A. D., & Nowak, T. (1997) Biochemistry (following paper in this series)]. Our findings raise important questions as to the possible roles of divalent metals in modulating multiligand interactions with YPK and in the regulation of the glycolytic pathway.

Adenosine Diphosphate↗
PubMed1995

Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro.

Similarities between the physical structures of avian and mammalian major histocompatibility complex (MHC) class I glycoproteins have been proposed based on comparative alignment of their amino acid sequences. To investigate the physical structure of the chicken class I glycoprotein, we cloned the cDNA representing the BFIV locus of the B21 haplotype. A unique, chimeric class I glycoprotein was constructed by incorporating an epitope tag (FLAG) at the N terminus. Monoclonal antibodies to the FLAG epitope served to monitor cell-surface expression for functional analysis of the BFIV21 class I glycoprotein. The chimeric class I glycoprotein was expressed in target cells using an avian leukosis virus (ALV)-derived retrovirus vector (RCASBP). The presence of the FLAG epitope did not interfere with either alloantibody recognition or cytotoxic T lymphocyte interaction. Functional analysis employing site-directed mutagenesis identified BF amino acid residues forming serologic epitopes as well as residues important in antigen presentation to ALV-induced cytotoxic T lymphocytes. BF residues 78 and 81, corresponding to HLA 79 and 82, form an antibody epitope with a slight effect on ALV antigen presentation, consistent with their predicted orientation based on the HLA-A2 crystal structure. Alignment of the BFIV21 sequence with previously published BFIV sequences revealed polymorphisms at position 34 (HLA 34), a monomorphic residues in HLA and H-2. Residue 34 is located in pocket B and is predicted to contact the main-chain carbon of peptides bound in HLA-A2. A site-directed substitution in BFIV residue 34 dramatically alters ALV antigen presentation by the BFIV21 class I glycoprotein. These data indicate that the physical molecular structure of the chicken MHC class I glycoprotein is similar to HLA.

Amino Acid Sequence↗
PubMed1994

Functional analysis of human bronchial mucosal T cells extracted with interleukin-2.

Functional analysis of T cells from the bronchial mucosa has been limited by difficulties in extracting T cells from this tissue. Because interleukin-2 (IL-2) is chemotactic for T cells, we determined whether this cytokine could be used to extract T cells from human bronchial wall (BW). Fresh tissue was obtained from 21 patients undergoing surgery for malignancy. Within the BW, 95% of T cells stained for the memory/activation marker CD45RO. When BW sections were incubated with IL-2 for 24 h, 88 to 91% of T cells emigrated into the culture medium. Compared with autologous blood T cells (also exposed to IL-2), these BW T cells expressed CD2 at a greater intensity and showed a fourfold reduction in cloning efficiency in response to phytohemagglutinin, and T-cell clones derived from the BW population displayed a tendency for higher interferon-gamma production. Furthermore, we were also able to extract and clone T cells from bronchoscopic biopsies in four subjects, suggesting that this method will provide a new avenue for examining T-cell function in airway inflammatory diseases.

Aged↗
PubMed1981

About the possibilities of registration of drug influence by means of radiocardiographic function analysis.

Changes in cardiac dynamics were analyzed by means of radiocardiographic function analysis under an infusion of epinephrine using the method of RAB. Typical changes in heart rate, stroke volume and blood pressure were observed after the application of epinephrine. Compared to examinations under physical exercise in case of healthy control subjects there was no typical decrease of enddiastolic volume dependent on the heart rate. Ejection time and ejection velocity was placed within the variation compared to values of a control group under physical exercise but there was a steeper increase of the curve under the effect of epinephrine. Under the influence of Solcoseryl no significant change could be observed in this series of tests compared to the basic values. The effect of epinephrine was changed by Solcoseryl in the way of an essentially smaller rise in heart rate and of a definite prolongation in diastolic time. A change in ejection time under the influence of epinephrine after the application of Solcoseryl could not be found. This allows the conclusion, that under the influence of Solcoseryl and after the administration of epinephrine better conditions for the metabolic restitution of heart muscle are given and consequently a direct effect of Solcoseryl on the heart muscle can be assumed.

Actihaemyl↗
PubMed1994

Discriminant function analysis of body surface potential maps in acute myocardial infarction.

Using a newly developed 64-electrode portable mapping device, QRS and ST-T isointegral maps were compared in 194 control subjects and 101 patients. One hundred ninety-four control subjects (mean age, 48 years; 120 men) with no history of cardiac disease were selected randomly and mapped. One hundred one patients (mean age, 62 years; 77 men) were mapped at presentation of chest pain suggestive of first myocardial infarction (MI); all patients had classic 12-lead electrocardiographic findings--46 with anterior and 55 with inferior MI. The diagnosis was confirmed in all cases by a significant rise in serial cardiac enzymes. The mean delay between onset of chest pain to map recording was 163 minutes. Of the 101 patients, 78 were first mapped outside the hospital. Applying discriminant function analysis to the isointegral measurements made on the control subjects and on the first map of MI patients achieved a correct classification of 97% of the control subjects (189 of 194) and 72% of the anterior (33 of 46) and 76% of the inferior (42 of 55) MI groups. This preliminary study suggests that discriminant function analysis, based on isointegral maps, not only provides a method of separating control subjects from MI patients but that it can also differentiate between types of infarct. Further studies are required to improve the predictive values of discriminant function and to extend the methodology to assess both the site and size of MI.

Adult↗
PubMed1996

Functional analysis of a p21WAF1,CIP1,SDI1 mutant (Arg94 --> Trp) identified in a human breast carcinoma. Evidence that the mutation impairs the ability of p21 to inhibit cyclin-dependent kinases.

Human p21 (also known as WAF1, CIP1, or SDI1) is a dual inhibitor of cyclin dependent kinases (CDKs) and the replication factor PCNA, which plays a role as a downstream mediator of the cell-cycle arrest induced by the tumor suppressor p53. To determine whether inactivation of downstream targets of p53 might contribute to cellular transformation, we have examined the integrity of the p21 gene in 36 invasive ductal breast carcinomas. Direct sequence analysis of the polymerase chain reaction-amplified p21 gene revealed a C to T transition in codon 94 that caused the substitution of a tryptophan for an arginine in a tumor specimen. This mutation was not detected in normal DNA extracted from the same patient nor in a polymerase chain reaction-restriction fragment length polymorphism of 50 unrelated individuals, indicating that it corresponds to a tumor-specific alteration. Functional analysis of the p21(R94W) protein produced in different eukaryotic and prokaryotic expression systems revealed that this mutation impaired the ability of p21 to inhibit CDKs. By contrast, the R94W mutant was unaltered in its ability to promote cyclin-CDK association as well as in its ability to bind proliferating cell nuclear antigen, thus leaving its putative functions as kinase activator or as inhibitor of replicative DNA synthesis intact. On the basis of these functional analysis, we propose that the Arg residue at position 94 is important for the CDK inhibitory role of p21.

Arginine↗
PubMed2003

Structure of gene flanking regions and functional analysis of sequences upstream of the rat hsp70.1 stress gene.

We present structural and comparative analysis of the flanking regions of the rat hsp70.1 stress gene. Several repetitive sequences, microsatellites and short interspersed repetitive elements (SINEs) were found, as well as a significant gap in the 3' UTR, as compared to the orthologous mouse gene. We also show that the complex microsatellite region composed of partially overlapping inverted repeat and long homopurine-homopyrimidine sequence, which is localized 1.8 kbp upstream of the transcription start site, is capable to adopt non-B DNA structures (an H-DNA and a cruciform structure) in vitro. Functional analysis performed with the use of various fragments of the 5'end flanking regions ligated to the chloramphenicol acetyltransferase (CAT) reporter gene revealed a crucial role of cooperation between heat shock element (HSE) regulatory sequences, while none of the three HSEs alone is able to drive efficient heat induced transcription of the reporter gene. We also found that the microsatellite region does not influence transcription by itself, however, it abolishes the effect of the adjacent putative silencing element. To our knowledge, this is a first extensive structural and functional analysis of the promoter region of the mammalian heat inducible hsp70i gene localized distally to the hsp70-related spermatid-specific gene in the major histocompatibility complex III.

3' Flanking Region↗
PubMed2001

Salvaged limbs after tibial shaft fractures with extensive soft-tissue injury: a biopsychosocial function analysis.

BACKGROUND: The purpose of this study was to perform a biopsychosocial function analysis of the outcome in patients with a salvaged leg after tibial shaft fractures associated with extensive soft-tissue injury. METHODS: A retrospective review of 18 consecutive patients with tibial shaft fractures and extensive soft-tissue damage with a mean Injury Severity Score of 31 was undertaken. All patients were assessed for their physical function, psychological status, and general function. RESULTS: The range of motion and torque of the ankle joint were severely affected. The knee joint was affected, but only with regard to the torque during concentric muscular activities. Nottingham Health Profile scores showed that the general and social function for these patients were significantly affected. Five of 18 patients suffered from depression. CONCLUSION: Salvage procedures should be advocated if possible, but careful selection of patients on the basis of mental and social factors is crucial for the outcome.

Adaptation, Psychological↗
PubMed2001

Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.

The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.

Antigens, Viral↗
PubMed1996

Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei.

Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter. An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants. A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter. Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression. Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression. This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi. Removal of the glucose repressor site did not affect sophorose induction. Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box.

Base Sequence↗
PubMed2002

Recombinational DNA Repair in Cancer and Normal Cells: The Challenge of Functional Analysis.

A major goal of current cancer research is to understand the functional consequences of mutations in recombinational DNA repair genes. The introduction of artificial recombination substrates into living cells has evolved into a powerful tool to perform functional analysis of DNA double strand break (DSB) repair. Here, we review the principles and practice of current plasmid assays with regard to the two major DSB repair pathways, homologous recombination and nonhomologous end-joining. A spectrum of assay types is available to assess repair in a wide variety of cell lines. However, several technical challenges still need to be overcome. Understanding the alterations of DSB repair in cancers will ultimately provide a rational basis for drug design that may selectively sensitize tumor cells to ionizing radiation and chemotherapy, thereby achieving therapeutic gain.

Journal Article↗
PubMed1995

Functional analysis and treatment of escape-maintained aggression correlated with sleep deprivation.

Functional analysis identified the consequences that maintained aggressive behavior and the relationship between these consequences and sleep deprivation for an individual with severe mental retardation. Results showed that aggression was maintained by negative reinforcement contingencies (escape from demand) and that aggression was more severe when sleep deprivation was present. A multicomponent intervention resulted in reductions of aggression for up to 7 months.

Adult↗
PubMed1997

Functional analysis of episodic self-injury correlated with recurrent otitis media.

A functional analysis examined the consequences that maintained episodic self-injury and the relationship between those consequences and otitis media for a child with moderate developmental disabilities. Results indicated that self-injury occurred only during periods of otitis media. Otitis media may have served as an establishing operation related to escape from ambient noise.

Adult↗