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At least 235 records · Page 13Linked to original sources
PubMed1989

Phenotypic and functional analysis of the cellular response in regional lymphoid tissue during an acute virus infection.

A phenotypic and functional analysis has been made of the cellular response in regional lymphoid tissue of C57BL/6J mice infected with lymphocytic choriomeningitis virus. Massive recruitment of nondividing cells occurred from 3 days after infection, with total numbers of CD8+ T lymphocytes, B220+ B cells, and Thy-1- B220- null cells being high from day 4 to day 6. In contrast, the peak counts for CD4+ T cells were recorded on day 4 and declined dramatically thereafter. Enhanced expression of IL-2R and Ly-24, both of which can be regarded as T cell activation markers, was found for both the CD4+ and the CD8+ subsets, being most prominent for the CD8+ T cells on day 6. Evidence of T cell proliferation was not recognized until days 5 and 6, coincident with enhanced responsiveness of the lymphocytes to rIL-2 and the development of virus-specific cytotoxic activity. Elimination of the CD4+ T cells by treatment of mice with mAb did not modify either the pathogenesis of lymphocytic choriomeningitis, or the expression of activation markers on the CD8+ T cells which are known to be the key effectors in this disease. Thus, the pattern of responsiveness for the CD8+ population is of recruitment to the lymph node, progressive increase in the expression of activation markers and enhanced sensitivity to rIL-2, with late proliferation and generation of cytotoxic activity. This model provides a system for the rigorous in vivo analysis of parameters influencing lymphocyte differentiation and activation in a virus infection.

Animals↗
PubMed2005

Correction of bias field in MR images using singularity function analysis.

A new approach for correcting bias field in magnetic resonance (MR) images is proposed using the mathematical model of singularity function analysis (SFA), which represents a discrete signal or its spectrum as a weighted sum of singularity functions. Through this model, an MR image's low spatial frequency components corrupted by a smoothly varying bias field are first removed, and then reconstructed from its higher spatial frequency components not polluted by bias field. The thus reconstructed image is then used to estimate bias field for final image correction. The approach does not rely on the assumption that anatomical information in MR images occurs at higher spatial frequencies than bias field. The performance of this approach is evaluated using both simulated and real clinical MR images.

Algorithms↗
PubMed2005

Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway.

Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. In VIGS, viruses engineered to carry sequences derived from plant gene transcripts activate the host's sequence-specific RNA degradation system. This mechanism targets the RNAs of the viral genome for degradation, and as the virus contains transcribed plant sequence, homologous host mRNAs are also targeted for destruction. While routinely used in some dicots, no VIGS system was known for monocot plants until the recent report of silencing in barley (Hordeum vulgare) by barley stripe mosaic virus (BSMV). Here, we report development of protocols for use of BSMV to efficiently silence genes in hexaploid wheat (Triticum aestivum). The VIGS system was first optimized in studies silencing phytoene desaturase expression. Next, we used it to assay genes functioning in leaf rust resistance mediated by Lr21, which encodes a nucleotide binding site-leucine-rich repeat class resistance gene product. We demonstrated that infection with BSMV constructs carrying a 150-bp fragment of Lr21 caused conversion of incompatible interactions to compatible, whereas infection with a control construct or one that silences phytoene desaturase had no effect on resistance or susceptibility. Additionally, silencing the RAR1, SGT1, and HSP90 genes, known to be required in many but not all nucleotide binding site-leucine-rich repeat resistance pathways in diverse plant species, resulted in conversion to compatibility, indicating that these genes are essential in Lr21-mediated resistance. These studies indicate that BSMV-VIGS is a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.

Gene Expression Regulation, Plant↗
PubMed1990

Functional analysis of Histoplasma capsulatum-reactive T-cell hybridomas.

Activation of CD4+ T cells is a crucial step in the elimination of Histoplasma capsulatum yeast cells from tissues. However, only a limited amount of information exists concerning the immunobiology of H. capsulatum-reactive T cells that are CD4+. To facilitate the analysis of the functional activities of this T-cell subpopulation, we developed a panel of 10 murine T-cell hybridomas from splenocytes of immune C57BL/6 mice. All hybridomas reacted with monoclonal anti-CD4+ antibody and released interleukin-2 after stimulation with histoplasmin. Within 3 weeks, the reactivity of hybridomas to histoplasmin declined dramatically, yet the cells responded vigorously to yeast-phase preparations that were enriched for cytosol, cell wall, or cell membrane. Of 10 hybridomas studied, only one recognized heterologous fungal antigens. Responsiveness to yeast-phase antigens was restricted by I-Ab. We mapped determinants in cytosol and cell wall or cell membrane by the technique of one-dimensional T-cell immunoblotting. The patterns of responses of hybridomas to cytosol were nearly uniform. All hybridomas responded to two immunodominant regions in cytosol with masses ranging from less than or equal to 18 to 26 kilodaltons (kDa) and 35 to 39 kDa. All hybridomas tested responded to determinants in the cell wall or cell membrane preparation with masses of 35 to 39 kDa. These hybridomas provide a useful tool for defining yeast-phase antigens that trigger T-cell activation.

Animals↗
PubMed2004

[Methods for preparation and functional analysis of islet beta-cells].

Regulation of blood glucose is a fundamental homeostasis in the body. Insulin is released from pancreatic beta-cells in response to changes in blood glucose, the defect of which leads to impaired insulin secretion and diabetes mellitus. Pancreatic beta-cells that release insulin occupy approximately 70% of the islet cells, while alpha, delta, PP-cells are also present in islets. Therefore, analysis of beta-cells is a direct approach for studying mechanisms of insulin secretion. Insulin secretion is regulated by cytosolic Ca(2+) in beta-cells, and its concentration and localization can be measured in real time by fluorescence imaging using indicators. Glucose metabolism is assessed by measurements of NAD(P)H by its autofluorescence. Furthermore, beta-cells are equipped with ion channels, which transduce glucose-evoked metabolic signals to electric signals. Electrophysiological analysis by patch clamp techniques detects the activity of various ion channels and membrane potential. Several beta-cell lines including HIT, MIN6, INS1, RIN, and beta TC are used; however, they do not necessarily retain normal responsiveness to glucose. Therefore, analysis of physiologic functions of beta-cells requires the use of acutely isolated or primary cultured beta-cells from normal animals. The methods for preparation of islets and single beta-cells using collagenase can be applied to a variety of animals of small to large sizes, which can produce islets and beta-cells with physiological responsiveness to glucose. These primary cultured beta-cells can be used for elucidating signal transduction mechanisms, and evaluating effects of drugs, providing excellent tools for physiological, pharmacological, and disease-oriented studies.

Animals↗
PubMed1999

Functional analysis of antigen-specific T lymphocytes by serial measurement of gene expression in peripheral blood mononuclear cells and tumor specimens.

The cloning of cancer Ags recognized by T cells has provided potentially new tools to enhance immunity against metastatic cancer. The biological monitoring of effective immunization has, however, remained a dilemma. We describe here a sensitive molecular quantitation methodology that allows analysis of in vivo immune response to vaccination. Metastatic melanoma patients were immunized with a synthetically modified peptide epitope (209-2M) from the melanoma self-Ag gp100. Using serial gene expression analysis, we report functional evidence of vaccine-induced CTL reactivity in fresh cells obtained directly from the peripheral blood of postimmunized patients. Further, we demonstrate in vivo localization of vaccine-induced immune response within the tumor microenvironment. The results of these molecular assays provide direct evidence that peptide immunization in humans can result in tumor-specific CTL that localize to metastatic sites.

Biopsy, Needle↗
PubMed1992

Functional analysis of HIV-1 reverse transcriptase amino acids involved in resistance to multiple nonnucleoside inhibitors.

Several novel, structurally distinct classes of specific human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) nonnucleoside inhibitors have been described recently. These include the pyridinone derivatives L-697,639, L-697,661, and L-696,229 as well as BI-RG-587 and the tetrahydroimidazo[4,5,1-j,k]-benzodiazepin-2(1H)-one and -thione compounds. Previous studies have implicated involvement of the RT amino acid residues at positions 103, 181, and 188 in the activity of the compounds. Accordingly, HIV-1 RT mutants containing a series of amino acid substitutions at these positions were constructed. The relative resistance of purified mutant enzymes to each of the inhibitors was assessed. This analysis established the functional equivalence of the three inhibitor classes and provided evidence for the interaction of the 103 site with the 181/188 region. Amino acid substitutions at these positions were also found to influence RT sensitivity to inhibition by phosphonoformate, thereby suggesting a close association between this pyrophosphate analog's binding site in RT and the binding site of the nonnucleoside inhibitors. In addition, aromatic stacking of the amino acid side groups at residues 181 and 188 was suggested to be required for inhibitor activity.

Amino Acid Sequence↗
PubMed2004

[Construction and functional analysis of the pepB gene disruptant in Aspergillus niger].

An integration plasmid pMW1-pepB for the pepB gene disruption in Aspergillus was constructed. The plasmid contained the pepB gene upstream (P) 1.4kb and downstream (T) 1.3kb homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. P and T DNA fragments were synthesized by PCR from Aspergillus niger chromosomal DNA. The integration plasmid was digested with the Hpa I restriction enzyme, the resultant 4.2kb linear fragment was introduced into the Aspergillus niger strain GICC2773 which expressing the glucoamylase/laccase fusion protein by PEG-mediated transformation. 62 Hygromycin resistance transformants were screened, and from them one strain named pepB29 was identified to be the pepB disruptant by PCR analysis. Data of functional assay of the pepB29 strain indicated that the disruption of the pepB gene secreted reduced acid proteolytic activity, and improved the heterologous protein laccase production.

Aspartic Acid Endopeptidases↗
PubMed2005

[Construction and functional analysis of a novel eukaryotic expression plasmid of recombinant immunotoxin DT390-Rantes].

OBJECTIVE: To construct a novel eukaryotic expression plasmid for recombinant immunotoxin DT390-Rantes and perform preliminary analysis of its function. METHODS: The gene fragment coding for Rantes was obtained from the liver tissues of C57BL/6 mice using RT-PCR, and inserted into the eukaryotic expression plasmid SRalpha containing DT390 gene to construct the recombinant plasmid DT390-Rantes-SRalpha, which was transformed into E. coli JM109, followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR, restriction endonuclease digestion and DNA sequencing. The recombinance plasmid DT390-Rantes-SRalpha was transfected into NIH3T3 cells and its expression was observed by immunofluorescence detection. The activity of the expressed DT390-Rantes in vitro was evaluated by MTT assay. RESULTS: The gene fragment of Rantes was correctly inserted into the eukaryotic expression plasmid SRalpha as verified by restriction endonuclease digestion and DNA sequencing, and could be expressed in NIH3T3 cells. MTT assay confirmed that the expression product DT390-Rantes could kill activated T cells in vitro. CONCLUSIONS: The recombinant eukaryotic expression plasmid DT390-Rantes-SRalpha is successfully constructed and expressed in eukaryotic cells. The expressed product can specifically kill activated T cells in vitro.

Animals↗
PubMed2001

Yeast--a panacea for the structure-function analysis of membrane proteins?

In recent years, the scientific community has begun to realise that the structure-function analysis of membrane proteins has lagged considerably behind that of their soluble counterparts. A boom in the field of membrane protein biology has resulted in the tailoring of techniques for the cloning, expression, purification and characterisation of these somewhat intractable proteins and most notably in the optimisation of several alternative host systems for this purpose. This Review Article summarises the use of yeast as a host. Compared with other hosts, it is clear that yeast combines the advantages of eukaryotes with the ease of handling of prokaryotes. Moreover, this organism provides membrane protein biologists with a panacea for structure-function analyses, not least because the tools of yeast genetics are at their disposal.

Cloning, Molecular↗
PubMed1988

Discriminant function analysis to calculate a Crohn's activity group scale to predict future inactive or active disease.

A discriminant function analysis was performed on several demographic, anthropometric, clinical, and laboratory data of 685 observations performed over 12 months on 137 patients with Crohn's disease. A Crohn's activity group scale (CAGS) was calculated. The CAGS has two advantages over the usual Crohn's disease activity index: it is objective, but more important is the fact that the values, when calculated longitudinally, have predictive value. Thus, calculation of CAGS is useful for counseling purposes and may also be useful in the design of future trials assessing therapy for Crohn's disease by allowing prerandomization stratification of patients with high or low probability of future recurrences of symptomatic disease activity.

Crohn Disease↗
PubMed1989

Immunohistochemical and in vitro functional analysis of pineal-germinoma infiltrating lymphocytes: report of a case.

The present study describes the phenotypic and functional analysis of tumor-infiltrating lymphocytes isolated from a germinoma located in the pineal region. Tumor-infiltrating lymphocytes were separated from the germinoma and cultured in medium containing IL-2 (1000 U/ml). An immunohistochemical analysis of frozen sections revealed that 90% of the germinoma-infiltrating lymphocytes were CD3-positive T cells expressing CD4, CD8, and HLA Class I and Class II antigens, but were negative for CD16, CD20, CD23, CD25 and CD14 antigens. After in vitro cultivation in the presence of high concentrations of IL-2, the lymphocytes proliferated for 2 weeks, showing marked DNA synthesis. In addition, the lymphocytes could lyse NK-resistant allogeneic target cells. These results provide evidence for a potential role of germinoma-infiltrating lymphocytes in vivo, and suggest that the lymphocytes may control the growth of autochthonous tumor cells by killing those that are not restricted to the major histocompatibility complex.

Adult↗
PubMed1997

Functional analysis of Rrp7p, an essential yeast protein involved in pre-rRNA processing and ribosome assembly.

During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted. RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter. Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits. A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27. When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells. Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion. We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.

Alleles↗
PubMed2002

Functional analysis of self-injurious behavior in an adult with Lesch-Nyhan syndrome.

A three-phase functional analysis was conducted to discover controlling variables of self-injury in a 28-year-old individual with Lesch-Nyhan syndrome. Experimental verification followed information-gathering and interpretive phases. Self-injurious responses were blocked to prevent harm to the participant; therefore, responses measured were precursors to self-injury. A multielement experimental design included four assessment conditions: social attention contingent on precursory behavior, attention contingent on behavior incompatible with precursory behavior, continuous attention and minimal attention. Highest rates of precursory behavior occurred during continuous attention and when incompatible behavior was reinforced. Social attention appeared to act as a discriminative stimulus for self-injurious behavior in this participant.

Adult↗
PubMed1985

[Morphological, ultrastructural, cytochemical and functional analysis of neutrophils deficient in myeloperoxidase].

Eighteen patients with established hereditary myeloperoxidase deficiency underwent morphological, ultrastructural, cytochemical and functional analysis in order to correlate the lack of peroxidase from phagocytes with other leucocyte activities. Cytochemical and ultrastructural findings only confirmed the peroxidase defect in neutrophil and monocyte population, whereas normal peroxidase activity was detected in eosinophil granulocytes ("Alius-Grignaschi anomaly"). Morphological analysis, as determined by both ligh and electron microscopy, showed in two patients with total MPO-deficiency a large number of neutrophils (50-60%) with nuclear abnormalities very similar to Pelger-Huet's heterozygous form (two lobed neutrophils having a typical pince-nez appearance and a nuclear chromatin coarser than that of normal PMNL). Other 2 cases displayed a 50-60% five-lobed neutrophils, as occur in congenital nuclear hypersegmentation of PMNL. These findings suggest that Alius-Grignaschi anomaly and Pelger-Huet syndrome can be found associated in the same individuals, since both these abnormalities have a genetic origin. Finally, since an impaired bactericidal and fungicidal activity was observed, no patients displayed particular susceptibility to persistent or severe infections, thus confirming the presence of MPO-independent enzymatic systems.

Blood Bactericidal Activity↗
PubMed1995

Isolation of a functional homolog of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans and functional analysis of conserved residues.

To investigate the degree of conservation of the cell cycle-specific NIMA protein kinase of Aspergillus nidulans, and to help direct its functional analysis, we cloned a homolog (designated nim-1) from Neurospora crassa. Over the catalytic domain NIM-1 is 75% identical to NIMA, but overall the identity drops to 52%. nim-1 was able to functionally complement nimA5 in A. nidulans. Mutational analysis of potential activating phosphorylation sites found in NIMA, NIM-1, and related protein kinases was performed on NIMA. Mutation of threonine 199 (conserved in all NIMA-related kinases) inhibited NIMA beta-casein kinase activity and abolished its in vivo function. This site conforms to a minimal consensus phosphorylation site for NIMA (FXXT) and is analogous to the autophosphorylation site of cyclic-AMP-dependent protein kinases. However, mutation of a unique cysteine residue found only in the catalytic site of NIMA and NIM-1 had no effect on NIMA kinase activity or function. Three temperature-sensitive alleles of nimA that cause arrest in G2 were sequenced and shown to generate three different amino acid substitutions. None of the mutations prevented accumulation of NIMA protein during G2 arrest, but all prevented the p34cdc2/cyclin B-dependent phosphorylation of NIMA normally seen during mitotic initiation even though p34cdc2/cyclin B H1 kinase activity was fully activated.

Amino Acid Sequence↗
PubMed2004

Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23.

The voltage-gated sodium channel NaV1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/NaV1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant NaV1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat NaV1.8 (rNaV1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat NaV1.8 Na+ currents were recorded (789 +/- 89 pA, n=62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNaV1.8 (mean IC50 value 12.5 microM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNaV1.8 (r=0.72, P<0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta1 and beta3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNaV1.8 is discussed. We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNaV1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNaV1.8 as novel analgesics.

Animals↗
PubMed2004

FLAGdb++: a database for the functional analysis of the Arabidopsis genome.

FLAGdb++ is dedicated to the integration and visualization of data for high-throughput functional analysis of a fully sequenced genome, as illustrated for Arabidopsis. FLAGdb++ displays the predicted or experimental data in a position-dependent way and displays correlations and relationships between different features. FLAGdb++ provides for a given genome region, summarized characteristics of experimental materials like probe lengths, locations and specificities having an impact upon the confidence we will put in the experimental results. A selected subset of the available information is linked to a locus represented on an easy-to-interpret and memorable graphical display. Data are curated, processed and formatted before their integration into FLAGdb++. FLAGdb++ contains different options for easy back and forth navigation through many loci selected at the start of a session. It includes an original two-component visualization of the data, a genome-wide and a local view, which are permanently linked and display complementary information. Density curves along the chromosomes may be displayed in parallel for suggesting correlations between different structural and functional data. FLAGdb++ is fully accessible at http://genoplante-info.infobiogen.fr/FLAGdb/.

Arabidopsis↗