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PubMed1996

Functional analysis of YCL09C: evidence for a role as the regulatory subunit of acetolactate synthase.

We have analysed the function of the open reading frame (ORF) YCL09C. The deletion of this ORF from chromosome III does not affect the physiology of the corresponding yeast strain enough to give a distinct phenotype. Nevertheless a computational analysis reveals high homology between this ORF and the enterobacterial genes encoding the regulatory subunit of acetolactate synthase. We have therefore tested the possibility that yc109cp is the regulatory subunit of yeast acetolactate synthase by in vitro enzymatic analysis. The acetolactate synthase was previously shown to be retroinhibited by its final product valine. In Escherichia coli this retro-control is assured by the regulatory subunit. Using a yeast strain carrying a complete deletion of YCL09C, we have observed the loss of such retro-inhibition. These results together with the computational predictions show that YCL09C encodes the regulatory subunit of yeast acetolactate synthase.

Acetoin↗
PubMed2002

[Functional analysis of the alpha1G subunit of the T-type calcium channel in cellular proliferation].

Increases of functional T-type calcium channel (T-channel) expression have been associated with cellular proliferation although evidence for this remains controversial. In the present study, we have used a variety of cellular, molecular and electrophysiological techniques to test the hypothesis that T-type channels play a causal role in the signaling pathway leading to proliferation. The results showed that stable over-expression of alpha1G T-channel subunit in HEK-293 cells conferred a significant growth advantage. Thus, cell population doubling time was reduced to 13.7 +/- 0.3 h in alpha1G transfectants, compared to control cultures (22.1 +/- 1.1 h) and flow cytometry analysis showed that this was due to a reduction in the number of alpha1G transfectants residing in the G0/G1 phases of the cell cycle compared to controls. The selective T-type calcium channel blocker, mibefradil, induced a dose-dependent inhibition of proliferation in alpha1G tansfectants. Furthermore, the Western blotting results proved that the level of protein expression of CDK2, cyclin A and cyclin E was high in alpha1G transfectants compared to control cultures. Our results demonstrate that the T-type calcium channel provides a significant growth advantage to HEK-293 cells that might occur via effects on the G1/S cell cycle mechanism.

Blotting, Western↗
PubMed1991

Functional analysis of a liver-specific enhancer of the hepatitis B virus.

The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liver-specific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAACAGTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.

CCAAT-Enhancer-Binding Proteins↗
PubMed2003

Functional analysis of tight junctions.

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.

Cytological Techniques↗
PubMedDate not supplied

Functional analysis of a CArG-like promoter element in cardiac myosin light chain 2 gene.

We have examined the functional properties of a putative regulatory sequence, CCAAAAGTGG, (element A) in chicken cardiac myosin light chain 2 (MLC2) gene promoter by deletion/substitution mutagenesis and transcriptional analysis of RNA by S1 nuclease mapping. The results indicate that the element A sequence, which resembles the evolutionarily conserved A/T-rich CArG box, plays a role in defining the transcription initiation site in MLC2 gene. This is accomplished via repression of a potential transcription initiation at site -40 and promoting the initiation at +1. One of the two other dA-dT-rich sequences (element C), located proximal to initiation site (+1), serves as the basal promoter while the distal A/T rich element B participates in tissue specific transcription of the gene.

Animals↗
PubMed1989

Normalized power spectrum density function analysis on spike trains. I. Mathematical method and its evaluation.

The mathematical descriptions of normalized power spectrum density function (NPSDF) for rapidly estimating quantitatively the characteristics of frequency domain of neural spike trains were created and the efficient analytical method was thus established. All the tests and practical applications showed that the method reflects the frequency domain accurately and can satisfy most cases of the analysis in both frequency resolution and calculation speed.

Animals↗
PubMed1999

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Cell Differentiation↗
PubMed2006

Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.).

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6-phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.

Chromosome Mapping↗
PubMed2002

Race/ethnicity and depressive symptoms in community-dwelling young adults: a differential item functioning analysis.

To examine variations in the manifestation of depressive symptomatology across racial/ethnic groups, analyses of differential item functioning (DIF) on the Center for Epidemiologic Studies Depression Scale (CES-D) were separately conducted for representative samples of young adults in the following groups: African-Americans (n = 434), Hispanics born in the US (n = 493), and Hispanics born outside the US (n = 395). Non-Hispanic whites (n = 463) were employed as the reference group in all analyses. The effects of gender and age were controlled. DIF analyses indicated that: (1) about half of the CES-D items functioned differently among non-Hispanic whites compared to each of the other racial/ethnic groups; (2) the manifestation of symptoms seemed to be similar for both Hispanic groups, except for low positive affect; (3) African-Americans tended to favor somatic symptoms over affective (depressive) symptoms; (4) Immigrant Hispanics appeared to inhibit the expression of positive affect, and thus more high scorers on the total CES-D were observed within this subgroup. In contrast, no differences were observed when only negative items were considered. The use of positive affect items might artifactually induce spurious differences among people who were born outside the United States or North America.

Adult↗
PubMed2002

A primary culture system for functional analysis of C. elegans neurons and muscle cells.

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.

Animals↗
PubMed1998

Relief of urinary tract obstruction in tuberculosis to improve renal function. Analysis of predictive factors.

OBJECTIVE: To assess the effect of the relief of obstruction on the ultimate function of the affected renal unit in urinary tuberculosis, and to identify predictors of functional recoverability. PATIENTS AND METHODS: Of a total of 82 patients with urinary tuberculosis presenting over 7 years, 38 with evidence of upper tract obstruction were analysed. All patients were assessed with pre-operative excretory urography, urinary mycobacterial cultures and serum chemistry. Patients with subnormally functioning kidneys were assessed with baseline renal scans. Preliminary intervention in the form of JJ stenting or percutaneous nephrostomy (PCN) was carried out in patients with reasonable renal function. Function was reassessed after 4 weeks to detect evidence of improvement and factors which could affect the outcome were determined. RESULTS: Thirty-eight patients had documented upper tract obstruction, of whom six had bilateral obstruction (total of 44 renal units). Ten renal units were not functioning at presentation, with a mean (SD) glomerular filtration rate (GFR) of 3.0 (5.73) mL/min, and no preliminary intervention was performed. In the remaining 34, preliminary intervention was carried out before definitive surgery (JJ stenting in 14, PCN in 15 and PCN followed by antegrade JJ stenting in five); 21 of these renal units were salvaged but 13 were lost despite overcoming the obstruction. Three of the 13 units deteriorated from having acceptable pre-treatment GFRs to becoming non-functional. Good renal cortical thickness, a low grade of renal involvement (Semb 1 or 2), the presence of more distal disease in the form of ureteric stricture and a GFR of > 15 mL/min were good predictors of renal recovery after diversion. CONCLUSIONS: The loss of some renal units seems inevitable in patients with urinary tuberculosis, despite advances in chemotherapy. Having pre-operative predictors of renal recovery may ensure optimal patient selection, thereby reducing the number of procedures and economic burden on the patient who does not require intervention.

Adult↗
PubMed2001

Functional analysis of the sheep Wilson disease protein (sATP7B) in CHO cells.

In this study we investigated the function of the sheep orthologue of ATP7B (sATP7B), the protein affected in the human copper toxicosis disorder Wilson disease. Two forms of sATP7B are found in the sheep, a 'normal' form and one with an alternate N terminus, both of which were expressed in CHO-K1 cells. Cells expressing either form of sATP7B were more resistant to copper than the parental CHO-K1 cells. Subcellular localisation studies showed that both forms of sATP7B were similarly located in the trans-Golgi network (TGN). When the extracellular copper concentration was increased, each form of sATP7B redistributed to a punctate, vesicular compartment that extended throughout the cytoplasm. Both forms of sATP7B recycled to the perinuclear location within one hour when the cells were subsequently incubated in basal medium. After treatment of cells with bafilomycin A1 sATP7B accumulated in cytoplasmic vesicles, implying that ATP7B continuously recycles via the endocytic pathway. These results suggest that both forms of sATP7B are functional copper-transport proteins and that the intracellular location and trafficking of the sheep protein within the cell also appears normal.

Adenosine Triphosphatases↗
PubMed1983

Deltoid ligament. Functional analysis of the medial collateral ligamentous apparatus of the ankle joint.

On 34 osteoligamentous ankle preparations the function of the various components of the deltoid ligament has been elucidated by tracing mobility patterns after successive transection of the components in varying sequence. The anterior and posterior talofibular ligaments were included in the study to investigate the interaction between these structures and the deltoid ligament. The tibiocalcaneal and the intermediate tibiotalar ligaments control abduction of the talus. The anterior tibiotalar and talofibular ligaments control plantar flexion, while dorsiflexion is inhibited by the posterior tibiotalar and talofibular ligaments, and partly by the anterior talofibular ligament as well. In combination, the anterior and intermediate tibiotalar ligaments control external rotation, while the intermediate and posterior tibiotalar ligaments control both external and, together with the anterior talofibular ligament, internal rotation of the talus. Isolated, neither the anterior nor the posterior tibiotalar ligament appears to play any major role in ankle stability.

Ankle Joint↗
PubMed1989

Phenotypic and functional analysis of circulating phagocytic cells in IgA nephropathy patients.

We analysed the possible mechanisms responsible for the functional abnormalities reported on phagocyte cell function in IgA nephropathy patients. The oxidative metabolism by the superoxide anion ferricytochrome C reduction test on monocytes and the chemiluminescence response on neutrophils was examined in 32 patients. Moreover the expression of cell membrane structures involved in immune response and phagocytosis (i.e. Fc receptors and HLA class II antigens) was evaluated cytofluorimetrically using specific monoclonal antibodies. Our own results did not show striking differences in oxidative potential and phenotype of patients' phagocyte cells as compared with healthy controls. However a HLA class II-associated structure was found to be quantitatively more expressed in patients' monocytes than in normal controls (P less than 0.005). These findings suggest that the defective phagocyte system function reported in IgA nephropathy patients is not simply due to a loss of cell surface receptors, nor to abnormalities in intracellular metabolic pathways implying oxygen consumption. The increase in expression of some HLA class II structures, often associated with the presence of circulating IgA immune complexes, probably reflects a protracted immunological stimulation.

Adolescent↗
PubMed1996

Radionuclide venography and its functional analysis in superior vena cava syndrome.

UNLABELLED: In addition to imaging, radionuclide venography maybe used for the functional assessment of superior vena cava (SVC) syndrome by applying the indices of transit time (TT), time of half-peak count (TH) and peak count ratio (PC ratio). METHODS: Ten healthy subjects (Group N) and 107 patients with SVC syndrome (64 symptomatic and 43 asymptomatic) were studied. Images were visually assessed for collaterals or jugular venous reflux and values of the indices were calculated. RESULTS: The 107 patients were subclassified into three groups according to the images obtained. Collateral circulation was seen in 37 patients (Group C). In 20 patients, jugular venous reflux was observed (Group J). Fifty patients who showed neither collaterals nor reflux were included in Group P. In comparison to Group N [3.6 +/- 0.56 see (sem)], TT values were significantly higher (p < 0.05) for Group J (7.13 +/- 1.16 sec) and Group C (7.00 +/- 0.87 sec). Values of TH were significantly prolonged (p < 0.05) for Group J (23.6 +/- 4.8 sec) and Group C (18.8 +/- 2.2 sec) in comparison to Group N (9.2 +/- 1.5 sec). PC ratio values were higher in all patient groups in comparison to Group N (3.4 +/- 0.57). CONCLUSION: These indices are potentially useful in the initial diagnosis and post-therapeutic evaluation of SVC syndrome. In the absence of other causes, appearance of jugular venous reflux may be considered a sign of SVC syndrome.

Adult↗
PubMed1998

Event-related magnetic fields in the auditory cortex of man during unilateral movements: a discriminant function analysis.

It is often assumed that sensorimotor coordination is a feature of the sensorimotor areas of the neocortex only. The purpose of the present study was to examine how this phenomenon is reflected in the auditory cortex of man. Ten subjects were engaged in a stimulus-reaction paradigm, in which each of two acoustical tones was associated to either of two motor reactions. Magnetic fields recorded with a 122-channel magnetometer were modelled by current dipoles. The spatial coordinates as well as the amplitudes of the dipoles were analyzed from 90 to 110 ms after stimulus onset using discriminant analysis. The results suggest that the dipole trajectory in the auditory cortex of the right hemisphere and amplitudes of the dipoles in the auditory cortex of the left hemisphere already 90-110 ms after the beginning of the stimulus could be affected not only by physical features of the stimulus, but also by the motor task required as a reaction.

Acoustic Stimulation↗
PubMed2006

A scalable method for integration and functional analysis of multiple microarray datasets.

MOTIVATION: The diverse microarray datasets that have become available over the past several years represent a rich opportunity and challenge for biological data mining. Many supervised and unsupervised methods have been developed for the analysis of individual microarray datasets. However, integrated analysis of multiple datasets can provide a broader insight into genetic regulation of specific biological pathways under a variety of conditions. RESULTS: To aid in the analysis of such large compendia of microarray experiments, we present Microarray Experiment Functional Integration Technology (MEFIT), a scalable Bayesian framework for predicting functional relationships from integrated microarray datasets. Furthermore, MEFIT predicts these functional relationships within the context of specific biological processes. All results are provided in the context of one or more specific biological functions, which can be provided by a biologist or drawn automatically from catalogs such as the Gene Ontology (GO). Using MEFIT, we integrated 40 Saccharomyces cerevisiae microarray datasets spanning 712 unique conditions. In tests based on 110 biological functions drawn from the GO biological process ontology, MEFIT provided a 5% or greater performance increase for 54 functions, with a 5% or more decrease in performance in only two functions.

Algorithms↗