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At least 991 records · Page 55Linked to original sources
PubMed2004

Functional analysis of HIV-1 vif genes derived from Japanese long-term nonprogressors and progressors for AIDS.

We analyzed the function of human immunodeficiency virus type 1 (HIV-1) vif gene from Japanese long-term nonprogressors (LTNPRs) and progressors (PRs) for acquired immunodeficiency syndrome (AIDS). We constructed a basic HIV-1 infectious clone, which facilitated the incorporation and evaluation of vif from infected individuals. Proviral reporter clones carrying vif from six Japanese LTNPRs and seven PRs were then generated and their in vitro growth kinetics were analyzed. The vif clones, which could confer infectivity on reporter viruses, were considered active, and the ratio of the active clones to the number of clones examined per individual was determined. For the majority of LTNPRs, there was no correlation between presence or absence of functional vif with long-term nonprogression for AIDS. There was one exception in which all the clones examined had inactive vif, suggesting a probable association of inactive vif with the nonprogression. All PRs with high viral load had a high ratio of active vif clones. Our results suggest that the presence of functional vif would influence HIV-1 infectivity and disease progression in infected individuals.

Amino Acid Sequence↗
PubMed1980

Cell-mediated immunity in herpes simplex virus-infected mice: functional analysis of lymph node cells during periods of acute and latent infection, with reference to cytotoxic and memory cells.

The functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at lease up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.

Acute Disease↗
PubMed1998

Structure-function analysis of a series of glucagon-like peptide-1 analogs.

We have used NMR in conjunction with measurements of functional bioactivity to define the receptor-binding structure of glucagon-like peptide-1 (GLP-1.) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations, to be important in the three-dimensional (3D) structure of the molecule. Identification of the receptor-bound conformation of GLP-1, because it is a flexible peptide, required constraint of the peptide backbone into a predetermined 3D structure. Constraint was achieved by the introduction of disulfide bonds and specific side chain-side chain cross-links. The biological relevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F cells and to elicit a functional response, cyclic AMP production. NMR solution structures were obtained for the most biologically relevant of these analogs. The results of this study indicated that the residues necessary for the biological activity of GLP-1 occupy approximately three equally-spaced regions of the peptide 3D structure, at the corners of an equilateral triangle whose sides are, at a minimum estimate, 12-15A.

Amino Acid Sequence↗
PubMed2000

ALGINATE LYASE: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications.

Alginate lyases, characterized as either mannuronate (EC 4.2.2.3) or guluronate lyases (EC 4.2.2.11), catalyze the degradation of alginate, a complex copolymer of alpha-L-guluronate and its C5 epimer beta-D-mannuronate. Lyases have been isolated from a wide range of organisms, including algae, marine invertebrates, and marine and terrestrial microorganisms. This review catalogs the major characteristics of these lyases, the methods for analyzing these enzymes, as well as their biological roles. Analysis of primary sequence data identifies some markedly conserved motifs that should help elucidate functional domains. Information about the three-dimensional structure of a mannuronate lyase from Sphingomonas sp., combined with various mutagenesis studies, has identified residues that are important for catalytic activity in several lyases. Characterization of alginate lyases will enhance and expand the use of these enzymes to engineer novel alginate polymers for applications in various industrial, agricultural, and medical fields. In this review, we explore both past and present applications of this important enzyme and discuss its future prospects.

Alginates↗
PubMed2002

Functional analysis of whole cell currents from hair cells of the turtle posterior crista.

Controlled currents were used to study possible functions of voltage-sensitive, outwardly rectifying conductances. Results were interpreted with linearized Hodgkin-Huxley theory. Because of their more hyperpolarized resting potentials and lower impedances, type I hair cells require larger currents to be depolarized to a given voltage than do type II hair cells. "Fast" type II cells, so-called because of the fast activation of their outward currents, show slightly underdamped responses to current steps with resonant (best) frequencies of 40-85 Hz, well above the bandwidth of natural head movements. Reflecting their slower activation kinetics, type I and "slow" type II cells have best frequencies of 15-30 Hz and are poorly tuned, being critically damped or overdamped. Linearized theory identified the factors responsible for tuning quality. Our fast type II hair cells show only modestly underdamped responses because their steady-state I-V curves are not particularly steep. The even poorer tuning of our type I and slow type II cells can be attributed to their slow activation kinetics and large conductances. To study how ionic currents shape response dynamics, we superimposed sinusoidal currents of 0.1-100 Hz on a small depolarizing steady current intended to simulate resting conditions in vivo. The steady current resulted in a slow inactivation, most pronounced in fast type II cells and least pronounced in type I cells. Because of inactivation, fast type II cells have nearly passive response dynamics with low-frequency gains of 500-1,000 Momega. In contrast, type I and slow type II cells show active components in the vestibular bandwidth and low-frequency gains of 20-100 and 100-500 Momega, respectively. As there are no differences in the responses to sinusoidal currents for fast type II cells from the torus and planum, voltage-sensitive currents are unlikely to be responsible for the large differences in gains and response dynamics of afferents innervating these two regions of the peripheral zone. The low impedances and active components of type I cells may be related to the low gains and modestly phasic response dynamics of calyx-bearing afferents.

Animals↗
PubMed1981

Immunological properties of Fc receptor on lymphocytes. 9. Functional analysis of FcR gamma+ and FcR gamma- lymphocytes in IgE antibody responses.

The present study was undertaken to clarify the function of FcR gamma+ and FcR gamma- lymphocytes in anti-DNP IgE antibody responses. The helper activity was observed predominantly in FcR gamma- T cells, but poorly in FcR gamma+ T cells, as in the case of IgG antibody responses, indicating that the absence of FcR gamma on T cell surface is a common surface characteristic to helper T cells, and that there is no close relationship between the determination of class specificity of Ig and T cell subsets divided by the presence or absence of FcR gamma. Furthermore, IgE antibody responses were more dependent upon helper T cells than IgG antibody responses. The data presented in this paper also demonstrated that the precursors of anti-DNP IgE antibody-forming cells were equally contained in both FcR gamma+ and FcR gamma- B cells, when they were transferred into recipients with helper T cells. This is a striking contrast to IgM/IgG antibody responses, in which only FcR gamma- B cells could differentiate into antibody-forming cells. Moreover, the precursor activity of FcR gamma+ and FcR gamma- B cells for IgE antibody-forming cells was not affected by the treatment of cells with suppressive B cell factor released from FcR gamma+ B cells after binding of antigen-antibody (IgG) complexes and known to suppress the proliferation of FcR gamma- IgM/IgG antibody-forming cell precursors. Thus, IgE B cells seem to be more heterogeneous than IgM/IgG B cells with respect to the FcR gamma marker and insensitive to the regulatory effect of FcR gamma+ B cells. IgE B cells are regulated differently from IgG B cells in B-B cell interactions.

Animals↗
PubMed2002

Domain-oriented functional analysis based on expression profiling.

BACKGROUND: Co-regulation of genes may imply involvement in similar biological processes or related function. Many clusters of co-regulated genes have been identified using microarray experiments. In this study, we examined co-regulated gene families using large-scale cDNA microarray experiments on the human transcriptome. RESULTS: We present a simple model, which, for each probe pair, distills expression changes into binary digits and summarizes the expression of multiple members of a gene family as the Family Regulation Ratio. The set of Family Regulation Ratios for each protein family across multiple experiments is called a Family Regulation Profile. We analyzed these Family Regulation Profiles using Pearson Correlation Coefficients and derived a network diagram portraying relationships between the Family Regulation Profiles of gene families that are well represented on the microarrays. Our strategy was cross-validated with two randomly chosen data subsets and was proven to be a reliable approach. CONCLUSION: This work will help us to understand and identify the functional relationships between gene families and the regulatory pathways in which each family is involved. Concepts presented here may be useful for objective clustering of protein functions and deriving a comprehensive protein interaction map. Functional genomic approaches such as this may also be applicable to the elucidation of complex genetic regulatory networks.

Amino Acid Sequence↗
PubMed1994

Functional analysis of the cysteine residues of activin A.

Site-directed mutagenesis and mammalian cell expression was used to analyze the function of each of the 13 cysteine residues in the human activin A beta-subunit precursor. Substitution of the four cysteine residues in the proregion with alanine residues did not affect the function of the proregion in facilitating the dimerization and secretion of activin A homodimers. A series of activin mutants were constructed in which the nine cysteine residues (amino acids 4, 11, 12, 40, 44, 80, 81, 113, and 115) in the mature 116-amino acid beta-subunit were individually altered to alanine residues. Alanine substitution at either cysteine residues 4 or 12 did not interfere with homodimer formation, but the mutant activin A molecules had reduced biological and receptor binding activity (2- to 3-fold). Activin A monomers were produced when cysteine mutants 44, 80, and 113 were expressed in tissue culture cells. Monomers of cys mutants 44 and 80 had approximately 2% of the biological and receptor binding activity of wild type activin A. Cys 113 monomers had undetectable levels of biological activity. No detectable activin monomers or dimers were secreted from cells transfected with plasmids containing cys mutants 11, 40, 81, and 115. The data presented here suggest that a low level of noncovalent dimer formation of cysteine mutant monomers 44 and 80 may explain their low level of biological activity. Therefore, dimer formation is suggested to be an essential prerequisite for high affinity receptor binding and biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Activins↗
PubMed1989

Normalized power spectrum density function analysis on spike trains. II. Rhythms of unit activities in the somatosensory cortex of cats.

The normalized power spectrum density function (NPSDF) was used to analyse unit activities in the somatosensory cortex of cats. Thirty-four units from 40 cats were recorded and analysed. Rhythmic activities of 2-5 Hz were found in every unit, the strength increasing with mean firing rate and peripheral stimulation while the frequency remained constant. The results suggested: NPSDF is a better method for studying rhythm of unit activities; 2-5 Hz exists in the unit activities of the somatosensory cortex; and peripheral input may have the action of "phase tuning" in addition to the action of time-locked driven firing.

Animals↗
PubMed1987

Functional analysis of T-cell subsets in chronic experimental alcoholism.

In order to obtain a better understanding of immune system function in chronic alcoholism, we have assessed primary B-cell responses to helper T-cell independent (TI) and dependent (TD) antigens in chronic alcoholic Sprague-Dawley male rats fed totally liquid diet containing ethanol. Pair-fed littermates received the same diet except that carbohydrates isocalorically replaced ethanol, which accounted for 36% of the total calories. The ability of alcoholic animals to mount primary in vivo splenic plaque-forming cell (PFC) responses to TI pneumococcal polysaccharide type III (SIII) was elevated throughout 50 days of observation when compared to pair-fed controls; serum antibody responses to SIII paralleled the enhanced PFC responses. Primary in vivo B-cell responses to antigen sheep red blood cells (SRBC), a TD antigen, were initially elevated but were found to be significantly suppressed 30 days after chronic ethanol consumption. The degree of immunosuppression increased with length of chronic ethanol consumption. The elevated primary splenic PFC responses to TI (SIII) may be attributed to loss of T-suppressor cell control, since alcoholic rat spleen cells did not respond to low-dose priming with SIII. We suggest that either loss of function and/or actual depletion of accessory and regulatory cells (T-suppressor and T-helper) may be responsible for irregularities in B-cell function observed during chronic alcoholism. T-cell subset enumeration using fluorescein-labelled monoclonal antibodies revealed that a sequential T-helper and T-suppressor loss occurred several days following dysfunction of these T-cell subsets in splenic populations, suggesting that a combination of numerical and dysfunctional changes in lymphocyte subpopulations may be responsible for the immunological alterations observed in chronic alcoholics.

Alcoholism↗
PubMed1978

[Statistical considerations on the evaluation of the Krogh-Poulsen clinical functional analysis].

361 patients, who five years previously were supplied with removable partial protheses, were submitted to Krogh-Poulson clinical function analyses. The test showed no suggestion of functional disturbances in only 12% of patients. Investigations showed that the test should be improved as to reliability, objectivity and validity before being used as a screening procedure in practice.

Denture Retention↗
PubMed1994

Molecular and functional analysis of porcine E-selectin reveals a potential role in xenograft rejection.

In this study, we report the molecular and functional characterization of porcine E-selectin. Incubation of porcine endothelial cells with human TNF alpha but not human IL-1 resulted in a marked increase in binding to human neutrophils. In order to confirm that this interaction was mediated by E-selectin, we isolated the full-length porcine E-selectin cDNA which contained an open reading frame encoding 485 amino acids with 75% identity to human E-selectin. Expression or recombinant porcine E-selectin in COS cells resulted in surface expression of the protein and increased binding to human neutrophils. Northern blot analysis showed that treatment of porcine endothelial cells with human TNF alpha but not human IL-1 resulted in high levels of porcine E-selectin mRNA. Taken together, our data establish that porcine E-selectin mediates adhesive interactions between porcine endothelial cells and human leukocytes that may contribute to xenograft rejection.

Amino Acid Sequence↗
PubMed2002

Functional analysis of a divergent system II protein, Ccs1, involved in c-type cytochrome biogenesis.

The Ccs1 gene, encoding a highly divergent novel component of a system II type c-type cytochrome biogenesis pathway, is encoded by the previously defined CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZalpha bacterial topological reporters were used to deduce a topological model of the Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and therefore by analogy Ccs1, possesses a large soluble lumenal domain at its C terminus that is tethered in the thylakoid membrane by three closely spaced transmembrane domains in the N-terminal portion of the protein. Molecular analysis of ccs1 alleles reveals that the entire C-terminal soluble domain is essential for Ccs1 function and that a stromal loop appears to be important in vivo, at least for maintenance of Ccs1. Site-directed mutational analysis reveals that a single histidine (His(274)) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys(199)) is shown to be non-essential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.

Alleles↗
PubMed1992

Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3.

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.

Bacterial Proteins↗
PubMed2004

Structural and functional analysis of the C-terminal cytoplasmic domain of FlhA, an integral membrane component of the type III flagellar protein export apparatus in Salmonella.

FlhA is an integral membrane component of the Salmonella type III flagellar protein export apparatus. It consists of 692 amino acid residues and has two domains: the N-terminal transmembrane domain consisting of the first 327 amino acid residues, and the C-terminal cytoplasmic domain (FlhAC) comprising the remainder. Here, we have investigated the structure and function of FlhAC. DNA sequence analysis revealed that temperature-sensitive flhA mutations, which abolish flagellar protein export at the restrictive temperature, lie in FlhAC, indicating that FlhAC plays an important role in the protein export process. Limited proteolysis of purified His-FlhAC by trypsin and V8 showed that only a small part of FlhAC near its N terminus (residues 328-351) is sensitive to proteolysis. FlhAC38K, the smallest fragment produced by V8 proteolysis, is monomeric and has a spherical shape as judged by analytical gel filtration chromatography and analytical ultracentrifugation. The far-UV CD spectrum of FlhAC38K showed that it contains considerable amounts of secondary structure. FlhA(Delta328-351) missing residues 328-351 failed to complement the flhA mutant, indicating that the proteolytically sensitive region of FlhA is important for its function. FlhA(Delta328-351) was inserted into the cytoplasmic membrane, and exerted a strong dominant negative effect on wild-type cells, suggesting that it retains the ability to interact with other export components within the cytoplasmic membrane. Overproduced FlhAC38K inhibited both motility and flagellar protein export of wild-type cells to some degree, suggesting that FlhAC38K is directly involved in the translocation reaction. Amino acid residues 328-351 of FlhA appear to be a relatively flexible linker between the transmembrane domain and FlhAC38K.

Amino Acid Sequence↗
PubMed1994

Isolation and functional analysis of the promoter of the bovine serum albumin gene.

The bovine serum albumin (bSA) promoter has been cloned from bovine genomic DNA using the polymerase chain reaction. In common with other albumin promoters, this promoter functions efficiently in the differentiated rat hepatoma cell line H4II and not in the its dedifferentiated derivative, H5. Analysis of 5' deletions of the bSA promoter after transient transfection into H4II has revealed that a short construct containing the HNF1 binding site and TATA box functions efficiently but requires the presence of the more upstream sequences to achieve full activity Footprint analysis of the promoter reveals seven sites of DNA protein interaction extending from -31 to -213. One of these sites, extending from -170 to -236, whose deletion results in a four fold increase in promoter activity. This site has not previously been reported in other albumin promoters and is bound by the C/EBP-like family of proteins.

Animals↗
PubMed2004

A new computed-assisted technique for experimental sciatic nerve function analysis.

BACKGROUND: Peripheral nerve injury is a well-known experimental tool to evaluate the effect of various neurotrophic substances. Besides histological and electrophysiological techniques, nerve functional status has been assessed by means of the sciatic function index (SFI) and the static sciatic index (SSI). However, these techniques are still based on a complex and old-fashioned apparatus requiring a relatively long time of execution. This study aimed to assess a novel, rapid, computerized method for assessment of SSI in a rat model of sciatic nerve injury. MATERIAL/METHODS: Fourteen rats were used in this study. The left sciatic nerve was exposed and compressed for 2 minutes with an aneurysm clip. Functional evaluation was performed by analysing the footprints of standing rats, comparing the injured to the uninjured limb with the static sciatic index (SSI). For this purpose the rats were placed on a digital scanner and the resulting images were acquired on a personal computer. Analysis was carried out every day after surgery for 21 consecutive days. RESULTS: Two-minute compression of the sciatic nerve produced a major reduction in motor function as assessed by SSI. Improvement was observed by day 7. By day 20, SSI was completely restored. CONCLUSIONS: This technique is simple to use, quick, without expensive technical equipment. It provides good accuracy and precise quantification of the extent of functional loss and recovery after sciatic nerve injury.

Animals↗
PubMed2005

Functional analysis of organic cation transporter 3 expressed in human placenta.

The aim of this study is to investigate the placental transport mechanism of cationic compounds by comparison of the uptake of an organic cation into human placental basal membrane vesicles (BLMVs) with that into organic cation transporter 3 (OCT3)-expressing cells. Reverse transcription-polymerase chain reaction analysis demonstrated that OCT3 is the only OCT isoform expressed in the human placenta. The function of OCT3 was investigated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP(+)) into human embryonic kidney (HEK)293 cells stably expressing OCT3 (HEK/OCT3 cells). The OCT3-mediated uptake of MPP(+) was sodium- and chloride-independent and saturable, with a Michaelis constant (K(m)) of 82.5 microM. The OCT3-mediated uptake was inhibited by various cationic drugs in a concentration-dependent manner but not by anionic compounds, such as p-aminohippuric acid and captopril, or a zwitterion, carnitine. Western blotting analysis of membrane vesicles prepared from human term placenta revealed that OCT3 is expressed only in BLMVs but not in microvillous membrane vesicles. The uptake of MPP(+) into BLMVs was membrane potential-dependent and saturable, with a K(m) value of 51.8 muM, which is similar to that in HEK293/OCT3 cells. The inhibitory spectrum of various compounds on MPP(+) uptake by BLMVs was also similar to that in HEK293/OCT3 cells. These results suggest that OCT3 is expressed on the basal membrane of human trophoblast cells and plays an important role in the placental transport of cationic compounds.

Adult↗