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PubMed1998

Functional analysis and extinction of different behavior problems exhibited by the same individual.

Specific extinction procedures were matched to the function of two target behaviors displayed by the same individual, with results indicating that the matched extinction procedure suppressed the behavior for which it was designed. One of the target behaviors was exposed to an irrelevant extinction procedure, which produced no beneficial effects. These results support previous research indicating the need to match extinction procedures to the function of problem behavior.

Adult↗

PubMed2004

Prospective functional analysis of trapeziectomy combined with intermetacarpal tendon stabilisation in trapeziometacarpal arthritis.

We conducted a prospective study to evaluate the functional status of patients surgically treated by trapeziectomy with intermetacarpal tendon stabilisation. Pain relief, the height of the trapezial cavity, hand impairment, and manual ability were measured in a group of 18 patients at a mean of 2.3 years after surgery. Complete relief of pain was achieved in 89% of our patients. The height of the trapezial cavity was significantly reduced. Hand strength and digital dexterity were not impaired after the operation. Manual ability was significantly improved by the trapeziectomy as demonstrated by the median VAS values of 53.5 before and 95.5 after surgery. The ABILHAND questionnaire revealed that most of the patients were satisfied with the functional results of the trapeziectomy. Based on our findings in this prospective study, it appears that trapeziectomy with intermetacarpal tendon stabilisation relieved pain in almost 90% of patients suffering from trapeziometacarpal osteoarthritis, at no functional cost.

Arthroplasty↗

PubMed1991

Structural and functional analysis of the mouse mdr1b gene promoter.

The overproduction of P-glycoprotein, an integral membrane protein thought to function as a drug efflux pump, is the hallmark of the multidrug resistance phenotype. In murine multidrug resistant J774.2 cell lines, distinct mdr genes, mdr1a and mdr1b, encode unique P-glycoprotein isoforms. To examine the transcriptional regulation of the mdr1b gene, its promoter was isolated and characterized. The transcription initiation site was mapped by primer extension, and the 5'-flanking region was sequenced. Several potential regulatory elements were identified in this region. A transient expression vector was constructed by fusion of 540 base pairs of 5'-flanking sequence and part of the first untranslated exon to the chloramphenicol acetyltransferase (CAT) gene. When transfected into monkey kidney COS-1, rat pituitary GH3 or T47D human breast cells, the mdr1b 5'-flanking sequences were capable of driving CAT expression. Transient transfection studies using deletion subclones of the mdr1b-CAT construct were done to locate potential cis-acting sequences. The studies indicate the presence of cis-acting elements in the 5'-flanking region of the mdr1b gene. The implications of these findings for expression and regulation of the mdr1b gene are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Functional analysis of the C2A domain of synaptotagmin 1: implications for calcium-regulated secretion.

Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189-192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activity in vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.

Animals↗

PubMed1998

[Functional analysis of nuclear hormone receptors].

Nuclear hormone receptors are transcription factors which regulate gene transcription by lipophilic hormones. The functional domains such as transactivation, DNA binding, ligand binding and dimerization are mapped in the distinct region of the receptors. To analyze these functions, ligand binding assay, gel shift assay, transfection study, GST pull-down method, immunoprecipitation method, two hybrid system and yeast functional assay are employed. These assays have been shown to clarify the molecular basis of the disorders which are due to mutation in the receptors.

Alleles↗

PubMed1993

Functional analysis of pre and post-knee surgery: total knee arthroplasty and ACL reconstruction.

This paper examines the biomechanics of total knee arthroplasty as a treatment for arthritis and anterior cruciate ligament (ACL) reconstruction for repair of torn anterior cruciate ligaments of the knee. These are two of the most frequent reconstructive procedures for the knee joint. Functional testing of patients while performing various activities of daily living was used to study the relationship between the intrinsic biomechanics of the knee and function. The results of the study of patients following total knee replacement demonstrated a dynamic interaction between the posterior cruciate ligament and quadriceps function during stairclimbing. The study of patients with ACL-deficient knees demonstrated that loss of the anterior cruciate ligament can cause the avoidance of quadriceps contraction during activities when the knee is near full extension. Other studies demonstrated a relationship between tibiofemoral joint mechanics and patellofemoral mechanics. In addition, the importance of combined ligamentous laxity with higher than normal adduction moments during gait was examined in relationship to progressive degenerative changes to the medial compartment of the knee. In summary, functional testing such as gait analysis has proven to be an important basic research tool as well as extremely effective for clinical testing of new procedures and devices.

Anterior Cruciate Ligament↗

PubMed2002

Bioinformatics classification and functional analysis of PhoH homologs.

PhoH protein is a putative ATPase belonging to the phosphate regulon in Escherichia coli. EC-PhoH homologs are present in different organisms, but it is not clear if they are functionally related, besides nothing is known about their regulation. To distinguish true functional orthologs of EC-PhoH in different classes of bacteria and to identify their functional role in bacterial metabolic network we performed phylogenetic analysis of these proteins and comparative study of position and regulation of the related genes. Three groups of proteins were identified. Proteins of the first group (BS-PhoH orthologs) are present in most of bacteria and are proposed to be functionally linked to phospholipid metabolism and RNA modification. Proteins of the second group (BS-YlaK orthologs) are present in most of aerobes and Actinobacterial YlaK orthologs are shown to be members of a fatty acid beta-oxidation regulons. EC-PhoH orthologs are classified in a third group, specific for Enterobacteria. Functional role of PhoH homologs in the lipid and RNA metabolism and proposed interrelation of PhoH paralogs in one organism are discussed.

Bacteria↗

PubMed2000

Nephrogenic diabetes insipidus: functional analysis of new AVPR2 mutations identified in Italian families.

The aim of this study was to identify loss-of-function mutations of the V2 vasopressin receptor gene (AVPR2) in Italian patients affected by X-linked nephrogenic diabetes insipidus (NDI). Mutations were found in 15 of the 18 unrelated families investigated: nine of these mutations were previously unknown, including two affecting residues located in regions known to be important for determining the pharmacologic properties of the receptor, which were therefore functionally investigated. The first (A84D) involves a residue located near an aspartic acid (D85) that is highly conserved in all G protein-coupled receptors and that is believed to play a role in the process of their isomerization into functionally active and inactive states. The present study indicates that this mutation not only affects receptor folding in such a way as to lead to its retention inside the intracellular compartments but, as expected, also has profound effects on its binding and coupling properties. The second was a mutation of a tryptophan located at the beginning of the first extracellular loop (W99R) that greatly impaired the binding properties of the receptor and had a minor effect on its intracellular routing. Molecular analysis of the first extracellular loop bearing this mutation suggests that this residue plays a fundamental role in stabilizing the peptide/receptor interactions responsible for the high-affinity binding of agonists to the V2 receptor.

Adolescent↗

PubMed2002

[Functional analysis of the human T-type calcium channel alpha 1H subunit gene in cellular proliferation].

An increase in the functional expression of the T-type calcium (Ca) channel previously has been proposed to be associated with vascular smooth muscle cell proliferation although direct evidence for the channel causing these effects remains to be demonstrated. In this study, we provide evidence that stable over-expression of the alpha 1H subunit of the T-type Ca channel (CACNA1H) in HEK-293 cells confers a significant growth advantage. Over-expression of the alpha 1H subunit of the T-channel was confirmed in stable transfects by RT-PCR analysis using specific primer pairs and also by electrophysiology. Growth curve assays showed population doubling time for alpha 1H stable transfects was 14.0 +/- 0.4 h, whereas control cultures had a population doubling time of 22.1 +/- 1.1 h (n = 3, P < 0.05). In addition, total cell numbers significantly increased in stable transfects at all time points investigated from 48-120 h after plating (5 x 10(3) cells/well) compared with control cultures. Consistent with these findings flow cytometry showed that 53.9% of control cells were in G1, 34.5% in S and 11.6% in G2/M whereas alpha 1H transfectants displayed 45.6% of cells in G1, 44.6% in S and 9.8% in G2/M. Finally, the Western blotting results verified that the levels of protein expression of CDK2, cyclin A and cyclin E were relatively high in alpha 1H transfectants compared to control cultures. Our results demonstrate that the T-type Ca channel provides a growth advantage to HEK-293 cells when stably expressed and that it probably exerts these effects via control of the G1/S cell cycle mechanism.

Blotting, Western↗

PubMed1998

Functional analysis of monocyte subsets in surgical sepsis.

BACKGROUND: Sepsis still remains a major cause of death after surgery. Impaired monocyte (Mphi) function and disruption of monocyte (Mphi/T-cell interaction were shown to be crucial for the development of septic complications in these patients. It was the objective of the study to assess more insights in Mphi behavior in surgical sepsis by means of analysis of Fc receptor- and human leukocyte antigen DR (HLA-DR) expression in Mphi subsets, and to evaluate the kinetics of these changes. METHODS: In a prospective study, 20 septic patients and 10 healthy control subjects were included. Peripheral Mphi were isolated on consecutive days after onset of sepsis, and FcR positive (FcR+) and negative (FcR-) subsets were separated by rosetting with antibody-coated human erythrocytes. Cell surface receptor expression and in vitro cytokine production were used to determine the clinical importance of these subsets. RESULTS: A significant monocytosis (3.5-fold; p < 0.01) and suppression of HLA-DR receptor expression (35%, p < 0.01) which correlate with sepsis severity and outcome could be demonstrated. There was a significant increase of FcR+ subsets in sepsis compared with control subjects (60% vs. 24%; p < 0.05). In vitro stimulation of Mphi subsets and peripheral blood mononuclear cells revealed suppressed interferon-gamma synthesis (p < 0.05 up to 0.01) from day 1 to day 5, elevated neopterin release (p < 0.05) on day 14 and increased synthesis rates of proinflammatory cytokines (e.g., interleukin-1beta); p < 0.05) predominantly in FcR+ subsets. CONCLUSIONS: Sepsis results in a significant monocytosis and suppression of HLA-DR receptor expression, which are correlating with sepsis severity and outcome. A significant shift toward FcR+ Mphi subsets can be found. This subpopulation resembles the previously described "angry macrophage" that is characterized by high proinflammatory cytokine synthesis and suppressed antigen presentation and that contributes to a disruption of adequate Mphi/T-cell interaction, rendering the host anergic toward opportunistic infections. The extend of HLA-DR suppression and the shift toward FcR+ Mphi might characterize a high risk patient subpopulation, which could benefit from immunomodulatory strategies.

Adolescent↗

PubMed2006

Functional analysis of type II thioesterase of Streptomyces lydicus AS 4.2501.

Constructing a mutant strain of single gene disruption is the basis for the study of gene function and metabolomics. Systematic and complete genome sequencing is the basis of genetic manipulation. In the case of a little knowledge about the Streptomyces lydicus genome and the speculation that polyketide synthases (type I) might be responsible for the polyketide side chain biosynthesis of streptolydigin, a 588-bp fragment was amplified by polymerase chain reaction (PCR) according to the homology existing in the same functional genes among Streptomyces. A mutant strain of this gene was constructed by single crossover homologous recombination. The results of sequence analysis as well as the metabolite analysis of the mutant and the original strain by liquid chromatography/mass spectroscopy indicated that this fragment was part of type II thioesterase (TE) gene, which was required for streptolydigin biosynthesis like other type II TEs function in related antibiotics biosynthesis. Furthermore, targeted gene manipulation based on PCR was a powerful tool for studying gene function and metabolomics, especially when little was known about the genomic sequence of streptomyces.

Amino Acid Sequence↗

PubMed1996

cDNA cloning and functional analysis of the p97 subunit of the 26S proteasome, a polypeptide identical to the type-1 tumor-necrosis-factor-receptor-associated protein-2/55.11.

Molecular cloning of cDNA for a new regulatory subunit, designated p97, of the human 26S proteasome showed that the polypeptide consists of 908 amino acid residues with a calculated molecular mass of 100184 Da and an isoelectric point of 4.94. Computer analysis showed that p97 is very similar to type-1 tumor-necrosis-factor (TNF)-receptor-associated protein (TRAP)-2 and 55.11, both of which were identified recently as binding proteins of the cytoplasmic domain of type-1 TNF receptor by yeast two-hybrid screening. This finding suggests that the 26S proteasome might serve as a mediator molecule in the TNF signaling pathway in cells. Computer-assisted similarity analysis also revealed the high sequence similarity of p97 with a yeast protein whose function is yet unknown, the gene for which is here termed NAS1 (non-ATPase subunit 1). Disruption of NAS1 resulted in several phenotypes, including lethality and temperature-sensitive growth, depending on the genetic background of the cells used. The human p97 cDNA suppressed the growth defect of nas1 disruptant cells, when expressed from single-copy or multi-copy vectors, indicating that p97 is functionally equivalent to yeast Nas1p. Culturing of the temperature-sensitive nas1 cells at the restrictive temperature promoted the accumulation polyubiquitinated cellular proteins, implying that the 26S proteasome requires a functional Nas1p subunit for ubiquitin-dependent proteolysis. These results indicate that p97/Nas1p plays an important regulatory role in the function of the 26S proteasome.

Amino Acid Sequence↗

PubMed2001

Functional classification of patients with idiopathic scoliosis assessed by the Quantec system: a discriminant functional analysis to determine patient curve magnitude.

STUDY DESIGN: A stepwise discriminant analysis was used to define a spinal deformity score based on three-dimensional measurements by the Quantec spinal image system (raster stereophotograph). OBJECTIVE: To provide functional classification of spinal deformity in patients with mild idiopathic scoliosis without using radiographs. SUMMARY OF BACKGROUND DATA: Most studies classify the degree of spinal deformity in terms of coronal plane radiograph without analyzing transverse rotation. To the authors' knowledge, no studies investigating classification of spinal deformity in idiopathic scoliosis using Quantec system measurements have been documented. METHODS: In this study, 129 patients with a single curve and 119 patients with a double curve were divided into three groups according to Cobb angle: Group 1 (less than 10 degrees ), Group 2 (10-20 degrees ), and Group 3 (greater than 20 degrees ). RESULTS: The patients were assigned to the group with the highest scores after application of a stepwise discriminant analysis. The accuracy of the classification system by functional scores for the patients with a single curve was 85% for Group 1, 63.5% for Group 2, and 71.7% for Group 3. The accuracy of classification by functional scores for the patients with a double curve was 87.1% for Group 2 and 76.1% for Group 3. CONCLUSION: The back surface image study is a method for providing a quantitative assessment of mild spinal deformity, allowing evaluation of patients by integrated three-dimensional parameters with no reference to radiographs.

Adolescent↗

PubMed2000

Functional analysis of the OCA-B promoter.

OCA-B was identified as a B cell-specific coactivator that functions with either Oct-1 or Oct-2 to mediate efficient cell type-specific transcription via the octamer site (ATGCAAAT) both in vivo and in vitro. Mice lacking OCA-B exhibit normal Ag-independent B cell maturation. In contrast, Ag-dependent functions, including production of secondary Ig isotypes and germinal center formation, are greatly affected. To better understand OCA-B expression and, ultimately, the defects observed in the OCA-B knockout mice, we have cloned the OCA-B promoter and examined its function in both transformed and primary B cells. We show here that the OCA-B promoter is developmentally regulated, with activity increasing throughout B cell differentiation. Through physical and functional assays, we have found an activating transcription factor/cAMP response element binding protein binding site (or cAMP response element) that is crucial for OCA-B promoter activity. Furthermore, we demonstrate that IL-4 and anti-CD40 induce both the OCA-B promoter and octamer-dependent promoters, thus implicating OCA-B in B cell signaling events in the nucleus.

Activating Transcription Factor 4↗

PubMed2005

Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase.

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2'-5' phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Deltadbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure-activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching.

Alanine↗

PubMed1990

Functional analysis of clonally expanded CD8, TCR gamma delta T cells in a patient with chronic T-gamma lymphoproliferative disease.

Leukemic cells from a patient with T-gamma lymphocytosis were found to have the surface phenotype, CD3+, CD4-, CD8+, Leu19+, TCR delta 1+, WT31-. The clonal nature of the TCR gamma delta T cell proliferation was documented by flow cytometry and Southern blot analysis. Morphologically, they were large to medium-sized mature lymphocytes with cytoplasmic granules. Functionally, the cells revealed strong cytotoxic activities against NK-sensitive target cells, but had neither killer T cell activity nor suppressive activity on PWM-driven immunoglobulin synthesis by B cells. Interestingly, both suppressive and cytotoxic T cell activities were recovered with the depletion of CD8+ T cells. These studies may suggest some functions of the CD8+ population of human TCR gamma delta T cells in a normal immune system.

Aged↗

PubMed1991

Cost function analysis of Medicare policy: are reimbursement limits for rural home health agencies sufficient?

This paper estimates a hybrid total cost function to determine to what extent an urban/rural differential exists in home health agency expenditures in Wisconsin in 1987-1988. We find that costs are over $16 higher per visit in urban Milwaukee than in rural and small MSA counties, providing no evidence that Medicare reimbursement limits should be raised to reflect 'ruralness'. However, the cost of providing skilled nursing care exceeds both urban and rural reimbursement limits. Because rural agencies depend more on Medicare clients and provide disproportionately more skilled nursing visits, this might represent the source of any financial difficulty.

Health Care Costs↗

PubMed2006

Structure-function analysis of Frizzleds.

Frizzleds, cell surface receptors that mediate the actions of Wnt ligands on early development, are heptahelical (based upon hydropathy analysis) and couple to heterotrimeric G proteins. The primary structure of all ten mammalian Frizzleds display many landmarks observed in virtually all G protein-coupled receptors, including an exofacial N-terminus that is N-glycosylated, the presence of seven hydrophobic transmembrane segments predicted to form alpha-helixes, and three intracellular loops as well as a cytoplasmic, C-terminal tail that harbor suspected sites for protein phosphorylation. Prediction of the G proteins to which Frizzleds mediate signaling based upon a bioinformatic analysis of the primary sequence of the intracellular domains are in good agreement with functional screens in Drosophila, zebrafish, and mouse models of development, e.g., predicting Frizzled-1 to interact with members of the Gi/Go protein family. Likewise various Wnt signaling pathways are sensitive to treatment with pertussis toxin and knock-down of specific G protein alpha-subunits. Homology among the sequences encoding the cytoplasmic domains of human Frizzleds is high and the various Frizzleds can be segregated into subsets predicted to share some common downstream signaling elements. Among different species, homologies can reveal conservation of signaling to cognate G protein partners. Additionally, cytoplasmic domains of the prototypic beta2-adrenergic receptor can be substituted with those from either Frizzled-1 or Frizzled-2 to create chimeric receptors that are activated by beta-adrenergic agonists, yet signal with high fidelity to the Wnt/beta-catenin and Wnt/Ca2+, cyclic GMP pathways, respectively, regulating key aspects of early development. The nature of Frizzled-based signaling complexes, their temporal assembly, and spatial distribution via scaffold protein remains to be elucidated, as does whether or not these Wnt receptors display agonist-induced desensitization, internalization, and re-cycling to the cell membrane.

Amino Acid Sequence↗