Accuracy of two filling systems for parenteral nutrient solutions.
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Automation↗
Biomedical subjects
P Arnaud
Publications and source records attributed to P Arnaud.
Laminar-airflow ceiling in a hospital pharmacy cleanroom.
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Child↗
Evaluation of a filling system for binary pediatric solutions.
The aim of this study was to assess the use of an automatic filling system (Siframix M31 and M32 system) to prepare pediatric parenteral nutrition. Volumetric accuracy was measured for each siframix system loads cells (< 5% for 5 ml with the Siframix M32 and < 5% for 9 ml with the Siframix M31) with sterile water for injection. The minimal 20 ml of flushing sterile water of the common tubulure of the Siframix M32 (p = 0.211 for 20 ml and p = 0.75 for 500 ml), the use of viscous solutions (70% dextrose) on the Siframix M31 (p = 0.28 for 20 ml and p = 0.12 for 500 ml) and the use of a special tubulure for using E.V.A. Luer-lock bags (p = 0.89 for 20 ml and p = 0.103 for 500 ml) do not modify the accuracy. Changing bags or bottles during the filling operation modify the accuracy (p = 0.004 for 20 ml and p = 0.009 for 500 ml). A flushing operation is necessary to lower the risk of electrolytic pollution for the filling of little bags. The filling speed for each module was also measured (the maximal filling speed was five liters per minute). The Siframix system allows one to prepare pediatric parenteral nutrition bags when volumes are above 4 ml and with adapted source solutions in terms of concentration and conditioning volumes.
Child↗
Human recombinant alpha 2-HS glycoprotein is produced in insect cells as a full length inhibitor of the insulin receptor tyrosine kinase.
Human Alpha 2-HS glycoprotein (AHSG), a glycoprotein synthesized by hepatocytes, was expressed in insect cells using the recombinant baculovirus system. The protein was purified from the cell supernatant, and appeared as a single band at about 52 kDa. Western blot using a specific antibody to the B-chain of AHSG indicated that the connecting peptide was present in the protein. When incubated with solubilized insulin receptors, recombinant AHSG inhibited the tyrosine kinase activity of the receptors in a dose-dependent fashion at concentrations in the range of those of the circulating protein. AHSG did not interfere with the binding of insulin to its receptor. These results indicate that human AHSG represents a natural inhibitor of the insulin receptor tyrosine kinase, is active as a single-chain protein and possesses a biological role similar to that of its homologue in rats, pp63, described by Auberger et al. (1).
Baculoviridae↗
Stability of nystatin in mouthrinses; effect of pH temperature, concentration and colloidal silver addition, studied using an in vitro antifungal activity.
Alkaline low concentration nystatin mouthrinses extemporanely prepared can be used to treat oropharyngeal candidiasis in immunodeficient patients. However, their expiration dates are not distinctly determined. The stability of nystatin, added (as Mycostatine) at a concentration of 14,400 U/ml in 10-4N hydrochloric acid, purified water and 1.4% injectable sodium hydrogen carbonate with or without 0.002% colloidal silver (an antiseptic agent added because of its known antifungal potency) was studied after storage in tinted glass bottles at 5 degrees C and 22 degrees C over 11 days, and compared with reconstituted 100,000 U/ml aqueous Mycostatine oral suspension. At 2, 4, 7, 9, and 11 days after preparation. Samples were tested for pH, microbial contamination, and assayed by an in vitro microbiological test. Neither significant variation of pH nor microbial contamination were in evidence. Nystatin 14400 U/ml maintained at least 90% of its initial concentration for 4 days in acid at both temperatures, for 7 days (5 degrees C) and 4 days (22 degrees C) in aqueous and alkaline environments, for 9 days (5 degrees C) and 7 days (22 degrees C) in 1.4% injectable sodium hydrogen carbonate containing colloidal silver which showed an antifungal potency. The 100,000 U/ml aqueous Mycostatine oral suspension was stable for 9 days and 4 days at 5 degrees C and 22 degrees C respectively. An ambulant patient can keep a low concentration alkaline antifungal mouthrinse at home for a week at 5 degrees C.
Antifungal Agents↗
Plant S1 SINEs as a model to study retroposition.
The S1 element is a plant SINE (Short INterspersed Element) that was first described and studied in Brassica napus and is widely distributed among Cruciferae, especially in species of the Brassiceae tribe. We propose that S1 amplification in Cruciferae could represent a good eukaryotic model to study retroposition. This is based on the fact that S1 elements share clear structural and evolutionary characteristics with mammalian SINEs but are present in a much lower copy number (500 loci by haploid genome for the S1 element in B. napus compared to 700,000 loci by haploid genome for the Alu element in human). This low copy number allows the characterization of a large portion of SINEs from a given plant species. This can lead to a more precise understanding of the evolutionary history of SINE amplification and can more easily allow an evaluation of the impact of retroposition on the evolution of that species. It can also lead more rapidly to the characterization of genomic elements active in transcription and retroposition so that the cellular control of these elements can be addressed. Finally, we show that the study of S1 insertion sites can reveal information on the RNA reverse transcription and integration step of the retroposition process.
Base Sequence↗
Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354.
We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.
Animals↗
The effects of phosphorylation on the functional regulation of an expressed recombinant human dopamine transporter.
Metabolic labeling experiments were performed using eukaryotic cells transfected with the human dopamine (DA) transporter cDNA. Autophosphorylation in the presence and absence of the transporter substrate DA, was analyzed. Dopamine transporter (DAT) was phosphorylated in the absence of DA and dephosphorylated in the presence of the substrate. The functional significance of this phenomenon was checked by incubating cells with phosphorylation promoting agents, all of which reduced substrate uptake and ligand binding significantly. It is shown that at least one site of phosphorylation on DAT is a serine residue. These experiments suggest that the state of phosphorylation of the DAT may play an important role in its biological function.
Animals↗
Electrophoretic and isoelectric focusing analysis of human recombinant alpha 2-HS glycoprotein produced in insect cells: analysis of the post-translational events.
Alpha 2-HS glycoprotein (AHSG) is a human serum glycoprotein synthesized by liver cells. It is a natural inhibitor of the insulin receptor tyrosine kinase activity. We produced this protein in insect cells by using a recombinant baculovirus expressing the whole coding sequence of the protein. By analyzing AHSG on isoelectric focusing and on sodium dodecyl sulfate (SDS) gels, followed by immunoblot, AHSG produced in insect cells was found to be phosphorylated and to possess the connecting peptide between the A and the B chains. The same features were found in the protein produced by Hep3B, a human liver cell line that synthesizes AHSG. By contrast, no phosphorylation could be detected in AHSG present in normal human plasma, and the connecting peptide was clipped. As the protein produced in insect cells is active on insulin receptors, in contrast to the plasma protein, our results suggest that the biological activity of the protein may be associated with its single chain form together with its phosphorylation.
Amino Acid Sequence↗
A transcriptional analysis of the S1Bn (Brassica napus) family of SINE retroposons.
S1Bn is a plant short interspersed element (SINE) whose amplification probably involves the reverse transcription of an RNA intermediate. In this report, we identified and characterized S1Bn transcripts from different Brassica napus tissues. Despite the presence of a consensus internal POL III promoter in a large number of genomic S1Bn elements, we observed that S1Bn transcripts are rare in B. napus cells. The use of two very sensitive methods (RT-PCR and RACE PCR) allowed the characterization of 102 independent S1Bn cDNA clones from three different tissues (shoot, root and callus). From this analysis, we conclude that the majority of S1Bn transcripts probably result from a small number of cotranscriptional events where an S1Bn element is transcribed due to its presence in a POL II transcriptional unit. Specific POL III RNA transcripts, initiating at the first 5' nucleotide of the DNA element, are also present in the tested tissues and possibly result from the transcriptional activity of as few as three genomic elements. Two of these transcripts could represent master transcripts responsible for the amplification of S1Bn subfamilies. We also observed that the population of specific POL III transcripts varies among the three tested tissues and that some transcripts appear completely tissue-specific.
Base Sequence↗
Antibodies to endometrial transferrin and alpha 2-Heremans Schmidt (HS) glycoprotein in patients with endometriosis.
PROBLEM: Identifying the endometrial antigens inciting autoimmunity is important in setting up an antibody assay for a non-invasive diagnosis and clinical monitoring of endometriosis. METHODS: Two-dimensional gel electrophoresis of endometrial extracts, Western blot analysis, passive hemagglutination and enzyme-linked immunosorbent assay (ELISA), amino acid sequencing and molecular studies were done on chosen antigens. Forty-six women with endometriosis, 4 women with uterine leiomyomata, 4 with pelvic adhesions, 3 with repeat Cesarean sections (conditions that coexist with or predispose to endometriosis) and 46 controls participated. RESULTS: Antigens with molecular weights (MW) of 64 kDa [isoelectric point (pI) of 3.5-4.0] and 72 kDa (pI of 4.5) bound to IgG in all patients with endometriosis, but not the controls. Amino acid sequencing of the proteins revealed that they had homology to alpha 2-Heremans Schmidt (HS) glycoprotein (MW: 64 kDa) and transferrin (MW: 72 kDa). Endometriosis patients had significant antibody levels to these two proteins (predictive value of 80-90%). The analysis of patients' endometrial RNA detected the message for alpha 2-HS glycoprotein and transferrin. Albumin (pI 5.5) and collagen (pI 3.5) failed to elicit antibody responses. CONCLUSIONS: Patients with endometriosis have significant antibodies to endometrial transferrin and alpha 2-HS-glycoprotein. We can effectively use an antibody assay using these antigens for diagnosing endometriosis.
Adult↗
Restriction polymorphisms of the ceruloplasmin gene on chromosome 3.
Using a probe isolated from a human liver cDNA library, polymorphisms were observed in the human ceruloplasmin gene with the enzymes PstI and MspI. The PstI polymorphism was frequent (allele frequencies, 0.46 and 0.54) whereas the polymorphisms found with MspI were rare.
Ceruloplasmin↗
Binding of GC (VDBP) to membranes of human B lymphocytes following stripping of extant protein.
The presence of Gc (vitamin D binding protein) has been consistently demonstrated on the membrane of B lymphocytes. This protein appears to be spatially associated with surface immunoglobulins. The origin of this surface protein has not yet been determined and the purpose of the present paper was to investigate if Gc may bind to human lymphocytes after immunoglobulin (Ig) capping. For this purpose the presence of Gc on B lymphocytes was examined by three different approaches. First, when cells were examined by immunofluorescence and quantified by flow cytometry, membrane Ig capping was followed by a dramatic decrease in positivity for Gc when compared to native cells. In addition, incubation of capped cells with purified Gc was followed by a significant increase in fluorescence, indicating that this protein had been able to bind again. Second, analysis of solubilized lymphocytes by Western blotting showed that native lymphocytes and capped cells incubated with purified Gc contained a large quantity of a 56kDa protein which was immunoreactive with anti Gc antibodies. This protein band was much weaker on blots from capped cells not treated with Gc. Third, radiobinding assays indicated that, following capping, cells were able to bind Gc in saturable fashion. These results suggest that membrane Gc could play a role in the entry of vitamin D metabolites into lymphocytes.
B-Lymphocytes↗
Sequencing of the ovine interleukin-8-encoding cDNA using the polymerase chain reaction.
The nucleotide (nt) sequence encoding the ovine homologue of interleukin-8 (IL-8) was determined. The mRNA is 1494-nt long with an ORF of 101 codons. The long 3' non-coding element contains several ATTTA repeats implicated in the swift turnover of other chemokine mRNAs. The encoded protein of 11 kDa before processing, and 9 kDa as mature protein, contains the Cys-Xaa-Cys motif common to alpha-chemokines, and has conserved amino acids (aa) at positions identified as receptor contact sites for IL-8. Identities with other published IL-8 aa sequences are: dog, 91%; pig, 87%; rabbit, 84%; human, 78%; guinea pig, 69%. A 49% aa identity is also found with a chicken embryo fibroblast protein.
Amino Acid Sequence↗
Splitting tablets in half.
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Dosage Forms↗
Rapid purification of native SecA from Escherichia coli: development of a new affinity chromatography procedure.
The SecA protein occupies a pivotal position in the public protein export pathway in Escherichia coli. The multifunctional SecA protein recognizes cytoplasmic factors associated with export including the presecretory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of SecA to bind ATP was the basis for the development of a novel, rapid purification scheme involving a single chromatographic step. Affinity chromatography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts of E. coli binds strongly to this dye-ligand matrix, and active protein was purified to greater than 90% homogeneity. The protein isolated by this procedure retained the previously described ATPase and RNA-binding activities of SecA. This approach should permit the rapid purification of SecA homologs from a variety microorganisms.
Adenosine Triphosphatases↗
Neurointermediate pituitary lobe cells synthesize and release interleukin-6 in vitro: effects of lipopolysaccharide and interleukin-1 beta.
The cytokine interleukin-6 (IL-6) is produced by a variety of cells, including macrophages, T-cells, and B-cells. Recent studies have confirmed a neuroendocrine role for IL-6 in the regulation of anterior pituitary (AP) hormone release. Because the neurointermediate pituitary lobe (NIL) may modulate AP hormone release, we investigated the production of IL-6 by NIL cells in vitro. NIL tissue removed from pituitary glands of male Long-Evans rats was enzymatically and mechanically dispersed, and the cells were subsequently cultured in 96-well tissue culture plates for 4-6 days in 10% serum-containing RPMI-1640. Test incubations were performed in serum-free RPMI-1640, and IL-6 concentrations were determined using the 7TD1 cell bioassay. Preliminary studies revealed a cell-dependent release of IL-6: increasing the number of NIL cells per well from 6.25 to 50 x 10(3) revealed detectable basal release of IL-6 between 25-50 x 10(3) cells/well. The endotoxin lipopolysaccharide (LPS; 100 ng/ml) and IL-1 beta (100 ng/ml) stimulated IL-6 release at 25 and 50 x 10(3) cells/well. Subsequent studies used a cell density of 50 x 10(3) cells/well and demonstrated time-dependent 3- to 6-fold inductions of IL-6 release by 100 ng/ml IL-1 beta and LPS. Concentration-response studies revealed maximal stimulation of IL-6 release by 1 ng/ml and a minimally effective concentration of 1 pg/ml for both IL-1 beta and LPS. Treatment of NIL cells with 1-10 mM (Bu)2cAMP increased IL-6 release by 7- to 14-fold. Endotoxin and IL-1 beta also enhanced the accumulation of IL-6 messenger RNA in these cells. Vasopressin and oxytocin (1 microM) inhibited LPS and IL-1 beta stimulation of IL-6 release from NIL cells, but did not inhibit IL-6 release from AP cells. Immunofluorescent dual labeling of NIL cells for flow cytometry revealed that greater than 95% of the cells did not stain for CD11b/c (common epitope found on monocytes, granulocytes, and macrophages) or CD45 (leukocyte common antigen). These results demonstrate for the first time the synthesis and release of IL-6 from cultured NIL cells. Agents that enhance IL-6 release [LPS, IL-1 beta, and (Bu)2cAMP] from other cell types also increase IL-6 release from NIL cells. Vasopressin and oxytocin inhibition of IL-6 release suggests a role for these neuropeptides in feedback inhibition in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The intestinal permeability test applied to the diagnosis of food allergy in paediatrics.
Intestinal permeability was evaluated in 17 healthy young children (control group), aged 4.3 +/- 1.9 years (mean +/- SD), weight 15.4 +/- 5.5 kg and in 11 infants with food allergy (allergic group, aged 2.3 +/- 1.6 years, weight 9.5 +/- 1.5 kg). In the two groups, the urinary excretion of two non-metabolized sugars (mannitol, lactulose) was measured by gas-chromatography after oral administration. Measurement of urinary content of these sugars 5 hours after administration in the control group showed a clearance of 20.2 +/- 8.6% for mannitol and 0.44 +/- 0.18% for lactulose. The lactulose/mannitol ratio in the same group was equal to 0.023 +/- 0.008. When compared to the control group, the 11 patients of the allergic group presented a normal mannitol urinary excretion (16.5 +/- 13.4%, p = NS, Student's t-test) and an increase in the lactulose excretion (1.36 +/- 0.92%, p < 0.001). Moreover, the allergic group showed a lactulose/mannitol ratio that was significantly different (0.105 +/- 0.071, p < 0.001). The intestinal permeability test for the diagnosis of food allergies seems to be a sensitive and non-invasive test that is well suited to the paediatric practice.
Cell Membrane Permeability↗