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PubMed2005

Construction and validation of a population-based bone densitometry database.

Utilization of dual-energy X-ray absorptiometry (DXA) for the initial diagnostic assessment of osteoporosis and in monitoring treatment has risen dramatically in recent years. Population-based studies of the impact of DXA and osteoporosis remain challenging because of incomplete and fragmented test data that exist in most regions. Our aim was to create and assess completeness of a database of all clinical DXA services and test results for the province of Manitoba, Canada and to present descriptive data resulting from testing. A regionally based bone density program for the province of Manitoba, Canada was established in 1997. Subsequent DXA services were prospectively captured in a program database. This database was retrospectively populated with earlier DXA results dating back to 1990 (the year that the first DXA scanner was installed) by integrating multiple data sources. A random chart audit was performed to assess completeness and accuracy of this dataset. For comparison, testing rates determined from the DXA database were compared with physician administrative claims data. There was a high level of completeness of this database (>99%) and accurate personal identifier information sufficient for linkage with other health care administrative data (>99%). This contrasted with physician billing data that were found to be markedly incomplete. Descriptive data provide a profile of individuals receiving DXA and their test results. In conclusion, the Manitoba bone density database has great potential as a resource for clinical and health policy research because it is population based with a high level of completeness and accuracy.

Absorptiometry, Photon↗

PubMed2002

Binding of a C-terminal fragment (residues 369 to 435) of vitamin D-binding protein to actin.

The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.

Actins↗

PubMed1984

The effect of neurotensin on pituitary secretion of thyrotrophin and prolactin in vitro.

The effects of neurotensin on thyrotrophin (TSH) and prolactin (Prl) release were studied in two in vitro systems - anterior pituitary cells in culture and perifused anterior pituitary fragments. Neurotensin significantly reduced basal secretion of both TSH and Prl (P less than 0.001) from cultured pituitary cells, and abolished thyrotrophin releasing hormone (TRH)-stimulated TSH thyrotrophin releasing hormone (TRH)-stimulated TSH TSH and Prl release (P less than 0.02) from perifused pituitary fragments. These data indicate that neurotensin has a direct inhibitory effect on TSH and Prl secretion by the anterior pituitary.

Animals↗

PubMed2004

Artificial neural networks for prediction of antibacterial activity in series of imidazole derivatives.

Artificial neural networks (ANNs) have been applied for the quantitative structure-activity relationships (QSAR) studies of antibacterial activity against Escherichia coli, Serratia marcescens, Proteus vulgaris, Klebsiella pneumoniae and Pseudomonas aeruginosa of a large series of new imidazole derivatives. Antibacterial activity against individual bacteria, expressed as logarithm of reciprocal of the minimal inhibitory concentrations, log 1/MIC, has been related to a number of physicochemical and structural parameters of the imidazole derivatives investigated. Molecular descriptors of agents were obtained by quantum-chemical calculations combined with molecular modelling and from respective structure fragment reference data (e.g., log P). A high correlation resulted between the predicted from ANN model antibacterial activity, log 1/MIC(ANN), and that from biological experiments, log 1/MIC(exp), both for the data used in learning and in the testing sets of imidazoles. Correlation coefficient, R, depending on the type of bacteria and structural subset of analysed imidazole compounds, varies from 0.875 to 0.969. The applicability of ANNs has been demonstrated for the prediction of pharmacological potency of new imidazole derivatives based on their structural descriptors generated exclusively by calculation chemistry.

Anti-Bacterial Agents↗

PubMed2006

PRINS and C-PRINS: promising tools for the physical mapping of the lupin genome.

Two molecular cytogenetics methods, PRINS (primed in situ DNA labeling) and C-PRINS (cycling PRINS), were optimized for the physical mapping of several types of DNA sequences on the mitotic chromosomes of the narrow-leafed lupin (Lupinus angustifolius L.). The fragment of the FokI element from Vicia faba was localised by indirect PRINS reaction. Two other sequences, fragments of the coding sequences of L. luteus and of L. angustifolius, were localised by indirect C-PRINS. These techniques are faster and more sensitive than FISH, and they allowed the mapping of short DNA fragments. The data obtained shows that both types of PRINS are valuable tools for chromosome identification in lupin.

Chromosomes, Plant↗

PubMed1999

[Human dirofilariasis in Corsica: a new local case. Review of reported cases].

A case of human dirofilariasis with Dirofilaria (Nochtiella) repens, located in a subcutaneous nodule of the arch of the foot is reported in a 64-year-old man living in the surroundings of Porto Vecchio in Corsica. The histological examination of this nodule has revealed a gravid female nematode, dead some weeks or months before its surgical excision. It is the second case of human dirofilariasis observed in a patient living on the island. The authors are surprised by the number of patients reported in medical literature (10 altogether) as having contracted the infection probably during a short stay in Corsica. This paper seeks to collect the fragmented available data on the existence in Corsica of nematodes in dogs and its possible vectors.

Animals↗

PubMed1992

Cloning and structural analysis of the human c-kit gene.

The recent identification of the mouse White spotting and Steel loci as genes encoding the c-kit receptor and its ligand, respectively, has shed light on the importance of this ligand and receptor in embryogenesis, melanogenesis and hematopoiesis. In order to determine if the c-kit proto-oncogene is involved in human disease, we isolated seven overlapping lambda recombinants, using a fetal brain cDNA, and characterized the normal human gene (KIT). The longest mapped transcript is 5230 bp, is alternatively spliced and includes 21 exons that span more than 70 kb of DNA. From the exon-intron structure, we have localized an alternative splice site to the 3' end of exon 9. The overall c-kit gene structure closely resembles that found in the CSF-1R gene (c-fms). This similarity includes a large first intron, the same number of exons containing translated sequence and very similar exon-intron boundaries. Using pulsed-field gel electrophoresis, we have linked KIT to the platelet-derived growth factor receptor A gene, with both residing on a 700-kb BssHI fragment. These data will allow investigation into the control of KIT expression and the potential to identify mutations or altered expression of this gene in human disease.

Base Sequence↗

PubMed1977

Mucopolysaccharide accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV.

Increased concentrations of total sulfated mucopolysaccharides (MPS), threefold, and hyaluronic acid (HA), 10-fold, were found in ML IV fibroblast extracts when compared to normal controls. Such accumulations altered the distribution of MPS:HA comprised 70% of total MPS in ML IV but only 30% in control cells. Intracellular sulfated MPS was observed accumulating almost linearly in ML IV fibroblasts. "Pulse-chase" experiments indicate that both HA and the sulfated MPS remain in the ML IV cells for long periods of time; in control cells, they are rapidly removed as low molecular weight, dialyzable fragments. These data suggest that the MPS accumulation in ML IV fibroblasts, is the consequence of a catabolic block, probably involving the lysosome.

Child, Preschool↗

PubMed1992

Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.

Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.

Albumins↗

PubMed2004

Baicalein induces apoptosis through ROS-mediated mitochondrial dysfunction pathway in HL-60 cells.

Baicalein is one component of the dried root of Scutellaria Baicalensis Georgi. (Huang Qin) which is widely used in the traditional Chinese herbal medicine. In this study, we report that baicalein was able to induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by poly-(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6 h. Mechanistic analysis demonstrated that baicalein induced the cleavage of Bid protein, cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8 and -9. Moreover, baicalein caused elevation of intracellular hydrogen peroxide level. Catalase could effectively block baicalein-induced DNA fragmentation. These data indicate that baicalein may trigger an apoptotic death program through reactive oxygen species (ROS)-mediated mitochondrial dysfunction pathway. The findings enhance our understanding of anticancer function of baicalein in herbal medicine.

Apoptosis↗

PubMed2005

Study: CPOE facilitates 22 types of medication errors.

Fragmentation of data and human/machine interface are two key sources of potential errors. Make sure your system accounts for the total flow of products, services, and people. Avoid complacency; newer systems mean newer potential sources of errors.

Humans↗

PubMed1991

Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr1b.

P-glycoprotein is an energy-dependent drug efflux pump with broad specificity for hydrophobic antitumor agents such as vinblastine, doxorubicin, and taxol. We have previously shown that [3H]azidopine and [125I] iodoaryl azidoprazosin, which are photoaffinity probes for the alpha 1-subunit of the L-type calcium channel and alpha 1-adrenergic receptor, respectively, specifically interact with P-glycoprotein, partially reverse multidrug resistance, and bind to a 6-kDa common domain in the 140-kDa P-glycoprotein molecule (Greenberger, L., Yang, C.-P. H., Gindin, E., and Horwitz, S. B. (1990) J. Biol. Chem. 265, 4394-4401). An immunological approach was used to identify the position of photoaffinity drug-binding domains in P-glycoprotein. Analysis was done with a series of site-specific rabbit polyclonal antibodies to peptides that mimic domains in the mouse mdr1b gene product. The antibodies were made against amino acid residues 269-284, 356-373, 665-682, 740-750, 907-924, and 1203-1222. Upon trypsin digestion, cleavage products of 95 and 55 kDa were obtained, which after further digestion migrated at 60 and 40 kDa, respectively. The 40-kDa fragment was recognized by the antibodies to residues 1203-1222 and 919-1276, while the 55-kDa fragment was recognized by these antibodies plus antibodies to residues 740-750 and 907-924. In contrast, the 95- and 60-kDa trypsin fragments were recognized only by the antibody to residues 269-284. The 55- and 40-kDa fragments, as well as the 95- and 60-kDa fragments, were major photolabeled species after digestion of P-glycoprotein. The previously identified 6-kDa photo-labeled P-glycoprotein fragment was within the 40-kDa trypsin fragment. These data suggest that there are two photoaffinity drug-binding domains in P-glycoprotein encoded by mouse mdr1b. The C-terminal site most likely resides within or in close proximity to putative transmembrane domains 11-12.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Multicolor in situ hybridization and linkage analysis order Charcot-Marie-Tooth type I (CMTIA) gene-region markers.

This study demonstrates a clear and current role for multicolor in situ hybridization in expediting positional cloning studies of unknown disease genes. Nine polymorphic DNA cosmids have been mapped to eight ordered locations spanning the Charcot-Marie-Tooth type 1 (CMT1A) disease gene region in distal band 17p11.2, by multicolor in situ hybridization. When used with linkage analysis, these methods have generated a fine physical map and have firmly assigned the CMT1A gene to distal band 17p11.2. Linkage analysis with four CMT1A pedigrees mapped the CMT1A gene with respect to two flanking markers (8B10-5 cM[LOD 5.2]-CMT1A-3.5 cM[LOD 5.3]-10E4). Additional loci were physically mapped and ordered by in situ hybridization and analysis of phase-known recombinants in CMT1A pedigrees. The order determined by multicolor in situ hybridization was 17cen-LEW301-8B10-5H5/6A9-VAW409- 5G7-6G1-4A11-VAW412-10E4-pter. Two ordered probes, 4A11 and 6G1, reside on the same 440-kb partial SfiI restriction fragment. These data demonstrate the ability of in situ hybridization to resolve loci within 0.5 Mb on early-metaphase chromosomes. Multicolor in situ hybridization also excluded the possibility of pericentric inversions in two unrelated patients with CMT1 and neurofibromatosis type 1. When used with pulsed-field gel electrophoresis, multicolor in situ hybridization can establish physical location, order, and distance in closely spaced chromosome loci.

Charcot-Marie-Tooth Disease↗

PubMed1991

Mechanism of cell death following thymidylate synthase inhibition: 2'-deoxyuridine-5'-triphosphate accumulation, DNA damage, and growth inhibition following exposure to CB3717 and dipyridamole.

The thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717) inhibits the growth of human lung carcinoma A549 cells. The cytotoxicity of CB3717 is potentiated by the nucleoside transport inhibitor dipyridamole (DP), which not only inhibits the uptake and therefore salvage of thymidine but also inhibits the efflux of deoxyuridine, thereby enhancing the intracellular accumulation of deoxyuridine nucleotides. Measurement of intracellular deoxyuridine triphosphate (dUTP) pools, by sensitive radioimmunoassay, demonstrated a large increase in response to CB3717, in a dose- and time-related manner, and this accumulation was enhanced by coincubation with DP. In untreated cells and those treated with DP alone, dUTP was close to or below the limit of detection of the assay. In cells treated for 24 h with 3 microM CB3717 (concentration producing 50% growth inhibition) the intracellular dUTP was 46.1 +/- 9.6 (SEM) pmol/10(6) cells and after 24 h exposure to 30 microM CB3717, 337.5 +/- 37.9 pmol dUTP/10(6) cells was detected. There was significant enhancement by DP of the accumulation of dUTP in cells treated with CB3717; coincubation of cells with 1 microM DP + 3 microM CB3717 for 24 h resulted in intracellular dUTP levels of 174.7 +/- 57.7 pmol/10(6) cells. Accumulation of DNA strand breaks, measured by alkaline elution, also increased in response to CB3717 concentration and exposure period. Newly synthesized (nascent) DNA was more sensitive to damage by CB3717 than was mature DNA. As with the accumulation of dUTP, coincubation with DP also enhanced the accumulation of strand breaks, whereas DP alone had little or no effect on DNA fragmentation. When data for cells treated with CB3717 alone and CB3717 in combination with DP were combined, there was a significant correlation of intracellular dUTP levels with the level of DNA strand breaks. This strongly suggests that growth inhibition following thymidylate synthase inhibition is mediated through an increase in intracellular dUTP, leading to uracil misincorporation into DNA, its subsequent excision, and resultant strand breakage.

Cell Division↗

PubMed1988

[The main difference between the repertoire of receptors of effector T-killers and their secondary precursors (memory cells)].

Receptor repertoire analysis of in vivo induced secondary cytotoxic T lymphocyte precursors (pCTL-2) was performed, using the technique of their specific adherence to macrophage monolayers with subsequent elution of the adherent pCTL-2 and their activation by heat-killed donor stimulator cells. The capacity of anti-H-2Kb pCTL-2 receptors to contact H-2Kbm has been revealed only in a minor pCTL-2 component whose progenitors were able to lyse mutant bm1 target cells (TC). Unlike poor cross-reactivity of CTL descended from anti-Kb, pCTL-2 which were eluted from the donor monolayer, CTL-progenitors of anti-Kb pCTL-2 eluted from bm 1 or B10. A (4R) third-party monolayers lyse in equal quantities the donor TC and those third-party TC from which they have been eluted, but fail to lyse other TC. Enrichment of pCTL-2 eluted from bm 1 or B10. A (4R) monolayers is 6- or 12-fold lower, respectively, than after their elution from the donor monolayer. The findings indicate that anti-class I MHC pCTL-2 are separated into fractions, with their receptors being strongly specific (with high affinity) to the particular fragment of the same complex epitope without cross-reactivity to other fragments. These data differentiate pCTL-2 receptors from effector CTL ones which are homogenous in specificity to a whole (single) complex epitope with variable degree of complementarity. A cardinal distinction of receptor repertoire between CTL, pCTL-2 and suppressor T cells specific to the same class I MHC molecule and alteration of the active site during pCTL-2 differentiation have been suggested.

Animals↗

PubMed1989

Diversity of surface protein antigen A gene in the chromosomal DNA of Streptococcus sobrinus.

A recombinant plasmid, pYA724, containing an 8.45-kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of Streptococcus sobrinus B13N. Restriction endonuclease BamHI-digested pYA724 DNA was 32P radio-labeled by nick-translation, and a Southern blot DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with a BamHI fragment of B13N chromosomal DNA, and the size of this fragment was larger than 8.45 kb. The chromosomal DNA was digested with BamHI-Pst I, BamHI-Hind III, and BamHI-Sal I, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of DNA fragments of B13N chromosomal DNA; however, the sizes were not identical to those of internal fragments expected from the pYA724 restriction map. SpaA gene clone derivative plasmids, pYA745 and pYA756, were digested with EcoRI and Hinc II, respectively, and electrophoresed together with the digested B13N chromosomal DNA on the same gel; then Southern blot analysis was carried out. Fragments with sizes corresponding to the EcoRI or HincII internal fragments of pYA745 and pYA756 were not observed among the B13N fragments. These data indicate that serotype d S. sobrinus B13N has DNA homologous with the spaA gene from serotype g strain 6715, although within the same biotype strain has a diversity of arrangement within the chromosome.

Antigens, Bacterial↗

PubMed1988

Characterization of the class III collagen receptor, a phosphorylated, transmembrane glycoprotein expressed in nucleated human cells.

We previously identified a 90-kDa cell surface glycoprotein, termed the class III collagen receptor (CRIII), that bound to collagen in affinity chromatography experiments (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). Here, we utilize monoclonal antibodies to define three domains of the CRIII, hydrophobic transmembrane, phosphorylated cytoplasmic, and glycosylated extracellular. The domain designations are based on the following characteristics. (i) Differential extraction, phase partitioning with Triton X-114, and incorporation into liposomes all indicate that the CRIII is an intrinsic membrane receptor with a hydrophobic domain. After incorporation into liposomes the CRIII binds collagen. (ii) Immunofluorescence microscopy reveals that most nucleated cells express the CRIII and that after extraction with Triton X-100, the Triton-insoluble CRIII distributes in a fibrillar pattern at the cell periphery and in closed loops that partially co-distributed with vimentin. The CRIII contains phosphoserine residues which are located on a cytoplasmic domain that may interact with the cytoskeleton. (iii) The CRIII contains 25% carbohydrate in 8-10 asparagine-linked carbohydrate chains of 2800 daltons each bound to a 65-kDa core peptide in the extracellular domain. Peptide mapping with trypsin defined a glycosylated 27-kDa extracellular fragment and a phosphorylated and glycosylated 35-kDa transmembrane fragment. These data suggest a model for the CRIII that links the cytoskeleton with the extracellular matrix.

Alkaline Phosphatase↗

PubMed1988

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis.

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.

Affinity Labels↗