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At least 883 records · Page 49Linked to original sources
PubMed2003

Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference.

Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes. mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission. To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi). Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression. In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified. In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited. This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.

Actin Cytoskeleton↗
PubMed2006

An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila.

Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain- and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit.

Animals↗
PubMed1988

Conservation of short patches of amino acid sequence amongst proteins with a common function but evolutionarily distinct origins: implications for cloning genes and for structure-function analysis.

Small patches of identical amino acid sequences commonly occur in proteins that have the same function but are derived from evolutionarily distant organisms. Reverse translation of such patches into degenerate pools of oligonucleotides provide useful hybridization probes for cloning the gene for the corresponding protein from other organisms. Since the conserved patches of identical amino acid sequence are probably important for the protein's biological function, they are preferred targets for reverse genetic studies aimed at defining structure-function relationships.

Amino Acid Sequence↗
PubMed1996

Functional analysis of the Enterococcus faecalis plasmid pAD1-encoded stability determinant par.

The molecular organization and functional characteristics of the PAD1 replicon-encoded par stability determinant were examined. par encodes two convergently transcribed RNAS of approximately 210 and 65 nucleotides designated RNA I and RNA II, respectively. The sequence of RNA II is largely complementary to RNA I, suggesting that RNA II could regulate RNA I function as an anti-sense RNA. Results of functional studies are consistent with a role for par as a post-segregational killing system, the first to be identified in Gram-positive bacteria, with RNA I encoding the toxin and RNA II the antidote. These results include: (i) destabilization of par-containing replicons in the presence of a second complete par or the RNA II coding sequence in the same cell; (ii) par-dependent stabilization of a highly unstable vector at the expense of host-cell growth rate; and (iii) protection of cells from the toxic effects of overexpression of RNA I by RNA II supplied in trans.

Base Sequence↗
PubMed1984

Functional analysis of B-L (Ia-like) antigen-bearing chicken peripheral blood cells.

The functional of B-L (Ia-equivalent)-positive (B-L+) adn -negative (B-L-) chicken peripheral blood lymphocytes (PBL) was studied in vitro and in vivo. The PBL were first stained in direct immunofluorescence tests with a fluorescein isothiocyanate-labelled anti-B-L alloantiserum and then separated by means of a fluorescence-activated cell sorter. In agreement with our previous findings, B-L- cells showed functional properties of T lymphocytes, responding to concanavalin A and phytohaemagglutinin-P in vitro and inducing a graft-versus-host (GVH) reaction when injected into allogeneic embryos. Sorted B-L+ gave no responses in any of these assays. Neither B-L+ nor B-L- cells, when tested alone, responded significantly to pokeweed mitogen, but mixtures of the two restored the responsiveness to that of the original unsorted suspension. Of the B-L+ PBL, 10% were T cells, which may account for the low GVH reactivity given by this population.

Animals↗
PubMed1995

Functional analysis of a new polymorphism in the human TNF alpha gene promoter.

In this paper the functional relevance of a TNFA promoter polymorphism, a G/A polymorphic sequence at position -238, was tested by analysing its influence on TNF alpha production upon in vitro stimulation of monocytes from 78 healthy, unrelated individuals by lipopolysaccharide (LPS) or after allogenic stimulation in a panel of 32 healthy individuals. All TNFA-A positive individuals were either DR3 or DR7 positive, confirming the previously reported strong linkage disequilibrium of the TNFA-A allele with the two extended haplotypes (B18, F1C30, DR3) and (B57, SC61, DR7). No individuals homozygous for the TNFA-A allele were present in the panel. The mean level of TNF alpha production was not significantly different in TNFA-G/G homozygous and in TNFA-A/G heterozygous individuals after LPS stimulation of monocytes (P = 0.35) or after allogenic stimulation (P = 0.7). After LPS and allogenic stimulation DR3 positive individuals had a higher mean TNF production. This could not be further differentiated by typing for TNF -283.

Adolescent↗
PubMed2006

Functional analysis of luxS in Staphylococcus aureus reveals a role in metabolism but not quorum sensing.

The function of AI-2 in many bacteria and the physiological role of LuxS, the enzyme responsible for its production, remain matters of debate. Here, we show that in Staphylococcus aureus the luxS gene forms a monocistronic transcriptional unit under the control of a sigma(70)-dependent promoter. The gene was transcribed throughout growth under a variety of conditions, including intracellular growth in MAC-T cells. AI-2 was produced in rich media under aerobic and anaerobic conditions, peaking during the transition to stationary phase, but was hardly detectable in a sulfur-limited defined medium. In the presence of glucose or under anaerobic conditions, cultures retained considerable AI-2 activity after entry into stationary phase. Inactivation of luxS in various S. aureus strains did not affect virulence-associated traits, such as production of hemolysins and extracellular proteases, biofilm formation, and the agr signaling system. Conversely, AI-2 production remained unchanged in an agr mutant. However, luxS mutants grown in a sulfur-limited defined medium exhibited a growth defect. When grown together with the wild type in mixed culture, luxS mutants of various S. aureus strains showed reduced ability to compete for growth under these conditions. In contrast, a complemented luxS mutant grew as well as the parent strain, suggesting that the observed growth defect was of an intracellular nature and had not been caused by either second-site mutations or the lack of a diffusible factor. However, the LuxS/AI-2 system does not appear to contribute to the overall fitness of S. aureus RN6390B during intracellular growth in epithelial cells: the wild type and a luxS mutant showed very similar growth patterns after their internalization by MAC-T cells.

Aerobiosis↗
PubMed2002

5-HT(2A) receptors: location and functional analysis in intestines of wild-type and 5-HT(2A) knockout mice.

The distribution and function of the 5-hydroxytryptamine (5-HT(2A)) receptor were investigated in the intestines of wild-type (5-HT(2A) +/+) and knockout (5-HT(2A) -/-) mice. In 5-HT(2A) +/+ mice, rats, and guinea pigs, 5-HT(2A) receptor immunoreactivity was found on circular and longitudinal smooth muscle cells, neurons, enterocytes, and Paneth cells. Muscular 5-HT(2A) receptors were concentrated in caveolae; neuronal 5-HT(2A) receptors were found intracellularly and on the plasma membranes of nerve cell bodies and axons. Neuronal 5-HT(2A) immunoreactivity was detected as early as E14 in ganglia, intravillus nerves, and the deep muscle plexus. The 5-HT(2A) -/- colon did not express 5-HT(2A) receptors and did not contract in response to exogenous 5-HT. 5-HT(2A) -/- enterocytes were smaller, Paneth cells fewer, and muscle layers thinner (and showed degeneration) compared with those of 5-HT(2A) +/+ littermates. The 5-HT(2A) receptor may thus be required for the maintenance and/or development of enteric neuroeffectors and other enteric functions, although gastrointestinal and colonic transit times in 5-HT(2A) -/- and +/+ mice did not differ significantly.

Animals↗
PubMed2003

Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy.

The three-dimensional optical transfer function is derived for analyzing the imaging performance in fiber-optical two-photon fluorescence microscopy. Two types of fiber-optical geometry are considered: The first involves a single-mode fiber for delivering a laser beam for illumination, and the second is based on the use of a single-mode fiber coupler for both illumination delivery and signal collection. It is found that in the former case the transverse and axial cutoff spatial frequencies of the three-dimensional optical transfer function are the same as those in conventional two-photon fluorescence microscopy without the use of a pinhole.However, the transverse and axial cutoff spatial frequencies in the latter case are 1.7 times as large as those in the former case. Accordingly, this feature leads to an enhanced optical sectioning effect when a fiber coupler is used, which is consistent with our recent experimental observation.

Journal Article↗
PubMed1997

Functional analysis and treatment of social-communicative behavior of adolescents with developmental disabilities.

This investigation used functional analyses to identify the social variables that maintained the inappropriate social-communicative behaviors of 2 adolescent students with mental retardation. Analyses were performed in the students' classrooms with the assistance of peers and teachers. The results of these assessments were used to identify appropriate, functionally equivalent behaviors that the students were taught to self-monitor. Findings showed substantial decreases in inappropriate social responding and increases in the use of appropriate social skills.

Adolescent↗
PubMed1999

Effects of session duration on functional analysis outcomes.

We examined the extent to which variations in session duration affected the outcomes of functional analyses. Forty-six individuals, all diagnosed with mental retardation and referred for assessment and treatment of self-injurious or aggressive behavior, participated in functional analyses, consisting of repeated exposure to multiple test conditions during 15-min sessions. For each set of assessment data, new data sets based on session durations of 10 and 5 min were prepared by deleting data from the last 5 and 10 min, respectively, of each session. Each graph (N = 138) was then reviewed individually by graduate students who had previous experience conducting and interpreting functional analyses, but who were blind to both participant identity and session duration. Interpretations of behavioral function based on the 10- and 5-min data sets were then compared with those based on the 15-min data sets. All of the 10-min data sets yielded interpretations identical to those based on 15-min data sets. Interpretations based on the 5-min and 15-min data sets yielded three discrepancies, all of which were the result of increased response rates toward the latter parts of sessions. These results suggest that the efficiency of assessment might be improved with little or no loss in clarity by simply reducing the duration of assessment sessions.

Adult↗
PubMedDate not supplied

[A comparative structure-function analysis and molecular mechanism of action of endonucleases from Serratia marcescens and Physarum polycephalum].

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water-magnesium complex involved in their catalytic acts. In the enzyme-substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme-substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction. The English version of the paper.

Amino Acid Sequence↗
PubMed1989

[Practice-relevant, functional analysis for the maxillary-orthopedic consultation].

The author presents a mirror of maxillary-orthopedic, functional analyses for assessment of motions and positions of the lower jaw bone, for examinations of the jaw joint and for registration of the so-called attendant function. The pertinent paper of documentation enables an ergonomically favourable utilization for basic, progress and final examinations, also in an extensive number of patients.

Humans↗
PubMed1996

Functional analysis of a genetic defect of copper transport (Menkes disease) in different cell lines.

To define the function of the Cu-transporting ATPase in Menkes disease, Menkes and normal fibroblasts were incubated with 67Cu before and after brief exposure to -SH reagents, p-chloromercuribenzoate (PCMB) and dithiothreitol (DTT). Accumulation and retention were compared among these cells, BeWo cells, and rat C6 glioma cells similarly treated. The Michaelis constant for influx of 67Cu into normal and Menkes fibroblasts was practically the same (0.21 +/- 0.07 vs. 0.24 +/- 0.06 microM). The PCMB treatment stimulated 67Cu accumulation in C6 cells, inhibited accumulation in normal and Menkes fibroblasts, and did not affect BeWo cells. DTT stimulated 67Cu uptake in all cells but BeWo cells. DTT treatment after PCMB further enhanced 67Cu accumulation in normal fibroblasts and C6 cells but had no enhancing effect on Menkes fibroblasts or BeWo cells. Menkes fibroblasts and BeWo cells released 67Cu at rates considerably slower than normal fibroblasts (0.06 and 0.09 vs. 0.22%/min, respectively). The PCMB blocked 67Cu release from normal fibroblasts but did not affect Menkes fibroblasts or BeWo cells. Reverse transcription-polymerase chain reaction analysis of total RNA from BeWo cells failed to show a predicted 943-base pair fragment representing a partial transcript of the Menkes factor. The fragment was present in extracts from normal fibroblasts. We conclude that the mechanism underlying Cu homeostasis varies among different cell types. As exemplified by BeWo and Menkes cells, failure to efflux Cu ions may be linked with the failure to express a functional Cu-transporting ATPase, namely, the Menkes protein.

Animals↗
PubMed2004

Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes.

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.

Amino Acid Motifs↗
PubMed1993

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.

Acyl Carrier Protein↗
PubMed2004

Identification and functional analysis of 'hypothetical' genes expressed in Haemophilus influenzae.

The progress in genome sequencing has led to a rapid accumulation in GenBank submissions of uncharacterized 'hypothetical' genes. These genes, which have not been experimentally characterized and whose functions cannot be deduced from simple sequence comparisons alone, now comprise a significant fraction of the public databases. Expression analyses of Haemophilus influenzae cells using a combination of transcriptomic and proteomic approaches resulted in confident identification of 54 'hypothetical' genes that were expressed in cells under normal growth conditions. In an attempt to understand the functions of these proteins, we used a variety of publicly available analysis tools. Close homologs in other species were detected for each of the 54 'hypothetical' genes. For 16 of them, exact functional assignments could be found in one or more public databases. Additionally, we were able to suggest general functional characterization for 27 more genes (comprising approximately 80% total). Findings from this analysis include the identification of a pyruvate-formate lyase-like operon, likely to be expressed not only in H.influenzae but also in several other bacteria. Further, we also observed three genes that are likely to participate in the transport and/or metabolism of sialic acid, an important component of the H.influenzae lipo-oligosaccharide. Accurate functional annotation of uncharacterized genes calls for an integrative approach, combining expression studies with extensive computational analysis and curation, followed by eventual experimental verification of the computational predictions.

Amino Acid Sequence↗