Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Functional analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources
PubMed1989

Transfer function analysis of autonomic regulation. I. Canine atrial rate response.

We present a useful technique for analyzing the various functional components that comprise the cardiovascular control network. Our approach entails the imposition of a signal with broad frequency content as an input excitation and the computation of a system transfer function using spectral estimation techniques. In this paper, we outline the analytical methods involved and demonstrate the utility of our approach in studying the dynamic behavior of the canine cardiac pacemaker. In particular, we applied frequency-modulated pulse trains to either the right vagus or the cardiac sympathetic nerve and computed transfer functions between nerve stimulation rate and the resulting atrial rate. We found that the sinoatrial node (and associated automatic tissue) responds as a low-pass filter to fluctuations in either sympathetic or parasympathetic tone. For sympathetic fluctuations, however, the filter has a much lower corner frequency than for vagal fluctuations and is coupled with a roughly 1.7-s pure delay. We further found that the filter characteristics, including the location of the corner frequency and rate of roll-off, depend significantly on the mean level of sympathetic or vagal tone imposed.

Algorithms↗
PubMed2004

Computational identification of transcription factor binding sites by functional analysis of sets of genes sharing overrepresented upstream motifs.

BACKGROUND: Transcriptional regulation is a key mechanism in the functioning of the cell, and is mostly effected through transcription factors binding to specific recognition motifs located upstream of the coding region of the regulated gene. The computational identification of such motifs is made easier by the fact that they often appear several times in the upstream region of the regulated genes, so that the number of occurrences of relevant motifs is often significantly larger than expected by pure chance. RESULTS: To exploit this fact, we construct sets of genes characterized by the statistical overrepresentation of a certain motif in their upstream regions. Then we study the functional characterization of these sets by analyzing their annotation to Gene Ontology terms. For the sets showing a statistically significant specific functional characterization, we conjecture that the upstream motif characterizing the set is a binding site for a transcription factor involved in the regulation of the genes in the set. CONCLUSIONS: The method we propose is able to identify many known binding sites in S. cerevisiae and new candidate targets of regulation by known transcription factors. Its application to less well studied organisms is likely to be valuable in the exploration of their regulatory interaction network.

5' Flanking Region↗
PubMed2004

Analogue functional analysis of movements associated with tardive dyskinesia.

We studied whether movements associated with tardive dyskinesia (TD) served operant functions in 2 men with developmental disabilities. We found that TD-related movements occurred more frequently in the alone and attention conditions and less frequently in control and demand conditions. Our findings suggest that TD-related movements may not be maintained by social reinforcers and that decreases in TD movements are possibly a result of engagement in activities that are incompatible with TD movements.

Antipsychotic Agents↗
PubMed2006

[Isolation and function analysis of rat CD4+ CD25+ regulatory T cells].

AIM: To investigate the isolation method and to analyze the function of rat CD4(+) CD25(+) regulatory T cells. METHODS: Lymphocytes were isolated from the rat spleens and then CD4(+) CD25(+) T cells were sorted by magnetic bead cell sorting (MACS) system. The purity and Foxp3 expression of CD4(+) CD25(+) T cells were analyzed by flow cytometry(FCM) and RT-PCR, respectively. The suppressive effect of CD4(+) CD25(+) T cells on the proliferation of CD4(+) CD25(-) T cells was analyzed by mixed lymphocyte reaction. IL-2, IFN-gamma and IL-10 levels in culture supernatant were detected by ELISA. RESULTS: The purity of CD4(+) CD25(+) T cells sorted by MACS was 86%-93%. The CD4(+) CD25(+) T cells could specifically express the Foxp3 gene as compared with CD4(+) CD25(-) T cells. In vitro CD4(+) CD25(+) T cells could suppress the proliferation of CD4(+) CD25(-) T cells and IFN-gamma, IL-2 production, but they themselves could secrete IL-10. CONCLUSION: We established an effective procedure for enrichment of CD4(+) CD25(+) regulatory T cells by MACS with satisfactory cell purity, viability and function. CD4(+) CD25(+) T cells can suppress CD4(+) CD25(-) T cells and specifically express Foxp3 gene.

Animals↗
PubMed1990

The mouse ribosomal protein L7 gene. Its primary structure and functional analysis of the promoter region.

The expressed gene coding for mouse ribosomal protein L7 (rpL7) was structurally and functionally characterized. It consists of seven exons, spans 3107 base pairs, and its coding sequence initiates within exon 1. The primary structure of mouse rpL7 (270 amino acids), as inferred from the nucleotide sequence of the exons of the gene, and from the cDNA, is 12 residues longer than the rat counterpart. The rpL7 gene shares common structural features with most other mammalian ribosomal protein genes analyzed thus far. These include the lack of a canonical TATA box and a major transcription initiation site at a cytidine residue embedded in a stretch of 14 pyrimidines, flanked by C + G-rich regions. Transient expression assays revealed that the promoter region of rpL7 gene bears several regulatory elements, both upstream to the capsite and within the transcribed portion of the gene. One internal regulatory element was assigned to the first intron and a second one to a 20-base pair region spanning the first exon-intron junction. The activity of a deletion mutant of rpL32 gene, lacking its internal elements can be rescued by insertion, in the sense orientation, of the corresponding elements from the rpL7 gene. The unique spatial organization of the regulatory elements in rpL7 gene, as well as in other murine ribosomal protein genes examined thus far, might indicate that this common architecture is involved in the mechanism coordinating their expression.

Amino Acid Sequence↗
PubMed1986

[Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties].

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.

Chromosome Mapping↗
PubMed1998

Organotypic brain slice cultures for functional analysis of alcohol-related disorders: novel versus conventional preparations.

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N-methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.

Alcoholism↗
PubMed2006

Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes.

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.

Acclimatization↗
PubMed1997

cDNA cloning and functional analysis of p44.5 and p55, two regulatory subunits of the 26S proteasome.

We have employed cDNA cloning to deduce the complete primary structures of p44.5 and p55, two subunits of PA700, a 700-kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consist of 422 and 456 amino acids with calculated molecular masses of 47463 and 52903, and isoelectric points of 6.06 and 7.56, respectively. Computer-assisted homology analysis revealed high sequence similarities of p44.5 and p55 with yeast proteins whose functions are yet unknown. Disruption of the yeast genes, termed NAS4 and NAS5 (non-ATPase subunits 4 and 5), resulted in lethality, indicating that each of the two subunits is essential for proliferation of yeast cells.

Amino Acid Sequence↗
PubMed1990

[Morphological and functional analysis of dento-orofacial complex in monozygotic twins with Duchenne type muscular dystrophy].

The form of dento-orofacial complex and masticatory muscle function of monozygotic twins with Duchenne type muscular dystrophy were investigated. They had no environmental difference. Morphological analysis were performed on the dental casts and cephalograms. EMG recordings were derived from the bipolar surface electrodes on the masseter muscle and the anterior belly of digastric muscle on the left side. Each consisted of the data for three years. Results obtained are as follows: 1) Based on the average data, these patients showed an elongated dental arch in the maxilla and mandible, which might be caused by enlarged tongues. There were little difference in the tooth and dental arch sizes between them. 2) Cephalometric findings indicated that the elder brother showed a clockwise rotation of the mandible with larger gonial angle than the younger brother. Both of them showed a larger gonial angle based on the mean values. 3) Analysis of EMG recordings revealed an elongated silent period induced by teeth tapping and chin tapping, and a variable masticatory rhythm compared with that of normal sample. Moreover an annually increased imbalance between masseter muscle and digastric muscle was evident, which were parallel to the change of the blood creatine kinase value. Differences in the form and function of orofacial complex between them might be caused by their polygene heredity and the large size of DMD gene (XP 21).

Adolescent↗
PubMed2003

A gateway cloning vector set for high-throughput functional analysis of genes in planta.

The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the beta-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.

Arabidopsis↗
PubMed2003

Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system.

Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)-independent growth.

Animals↗
PubMed1996

Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo.

Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were created. We defined a region within the middle of GCN5 required for interaction with ADA2 in vitro. We identified regions of ADA2 required for function in vivo and determined whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 in vitro. Two regions were crucial for ADA2 function in vivo, the amino terminus and a middle region. Immunoprecipitation analysis showed that the amino terminus of ADA2 was required for interaction with GCN5, while a region in the middle of ADA2 was necessary for interaction with ADA3. Deletions of the region that was required for interaction with ADA3 abolished dependence of lexA-ADA2 transcriptional activity on ADA3. Moreover, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 was demonstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interaction with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiologically relevant adaptor complex.

Base Sequence↗
PubMed1998

Structure-function analysis of the trypanosomatid spliced leader RNA.

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA.

Animals↗
PubMed2002

Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases.

Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (-)-beta-pinene and gamma-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn(2+) as bivalent metal ion cofactor resulted in higher activity than Mg(2+), with an optimum concentration of 0.6 mm. K(m) values ranged from 0.7 to 3.1 microm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.

Amino Acid Sequence↗
PubMed1992

Phenotypic and functional analysis of mucosal lymph node T cell subpopulations proximal and distal to chronic staphylococcal antigen challenge.

We investigated the functional and subset surface marker characteristics of supramammary lymph node T cell populations at sites proximal and distal to the mammary region of goats repeatedly injected with heat-treated Staphylococcus aureus antigen (HK-SAC). Flow cytometric studies showed quantitative differences in CD4+ and CD8+ T cell subsets among large and small lymphocyte populations in ipsilateral and contralateral supramammary lymph nodes of these animals. Although ipsilateral (draining) lymph nodes were enriched with CD4+ and CD8+ T cells, CD4/CD8 ratios were comparatively lower than those of contralateral (non-draining) lymph nodes (2.30 vs 2.60, respectively). Cell size analysis by flow cytometry showed that nearly 70% of the lymphocytes in ipsilateral nodes were of large cell phenotype with CD4/CD8 ratios of 2.52. In contrast, there were only 56.1% large lymphocytes in contralateral lymph nodes but with similar CD4/CD8 ratios of 2.55. The number of large lymphocytes in corresponding nodes of uninoculated control animals was significantly lower (50%) with much lower CD4/CD8 ratios (2.08). Alloantigenic responses of both ipsilateral and contralateral lymph node T cells were greater than those of uninoculated controls. Antigen-specific proliferation studies showed that ipsilateral lymph node T cells greatly enhanced both primed and non-primed lymph node B cell responses to HK-SAC, whereas those from contralateral lymph nodes were less stimulatory. In contrast, contralateral lymph node T cells had greater enhancing effects on PWM-induced polyclonal B cell responses. These studies indicate that repeated local infection with bacterial antigen induce changes in the numbers, ratios and antigen-specific and non-specific responses among ipsilateral (draining) and distal contralateral (non-draining) lymph node T cell populations in mucosal-associated immune systems such as the mammary gland.

Animals↗
PubMed1983

Implications of functional analysis.

This brief paper is intended to amplify three points which are contained in Slade (1982) and Owens & Ashcroft (1982). They are that (i) functional relationships must be demonstrated, not merely claimed; (ii) functional relationships are likely to be complex, and clinicians should be wary of superficial analysis; and (iii) there is a distinction between discriminative stimuli and setting conditions which has important implications for clinical practice.

Behavior↗
PubMed1998

LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs.

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.

Animals↗