Biomedical subjects
Publications and source records attributed to A Perrin.
Returning home after stoma surgery can be fraught with difficulties. Identifying potential problems and dealing with them promptly is an important part of the stoma care nurse's role. This article is the third in a series of three guides on stoma care.
Low bulk concentrations of thyroid stimulating hormone (TSH) were detected by scanning force microscopy (SFM) using gold-labeled conjugates. Anti-TSH antibodies were covalently bound onto amino-modified silicon oxide wafers. Surface modification was examined by contact-angle measurements, ellipsometry, X-ray photoelectron spectroscopy, and SFM. Antibodies were found to form a monolayer of prone molecules with an average surface density of 5000 IgG/mum2. TSH molecules were then allowed to bind to immobilized antibodies. The immunological reaction was quantified by SFM using gold-labeled species. Two scanned force microscopic immunoassays (SFMIA) were compared: first, a competitive test which used gold-labeled TSH molecules mixed with free TSH antigens was performed . Afterward, a sandwich assay was carried out, using gold-labeled anti-TSH antibodies. This latter method was found to be far more sensitive than competitive SFMIA. Gold conjugates were also found to be of great use to quantify antigens in large volumes by a sandwich test: a sensitivity threshold as low as 0.015 ng of TSH/ml (0.075 UI/ml or 6 x 10(-13) M) was estimated.
A reanalysis of the nu3, nu4, and nu6 interacting bands of D2CO has been carried out in the region 850-1250 cm-1 using high-resolution Fourier transform spectra recorded at Giessen. As compared to the previous study of these bands (1987, K. Nakagawa, R. H. Schwendeman, and J. W. C. Johns, J. Mol. Spectrosc. 122, 462-476) higher J and Ka transitions were assigned for the three bands, leading to a better determination of the upper state constants. The v3 = 1, v4 = 1, and v6 = 1 experimental energy levels were introduced in a least-squares fit calculation together with the microwave measurements available in the literature in order to obtain the upper state parameters (band centers, rotational and coupling constants). In this calculation, which allowed us to reproduce both the infrared and the microwave measurements to within their experimental accuracies, the A-, B-, and C-type Coriolis interactions involving the rotational levels belonging to the v4 = 1 and v6 = 1, v3 = 1 and v4 = 1, and v3 = 1 and v6 = 1 interacting states respectively were explicitly taken into account. Finally, from the intensities, a new determination of the relative values of the q3, q4, and q6 first derivatives of the D2CO dipole moment was performed. Copyright 1998 Academic Press. Copyright 1998Academic Press
The two triad systems of hydrogen sulfide (2nu2, nu1, and nu3 near 4 µm and 3nu2, nu1 + nu2, and nu2 + nu3 near 2.7 µm) were analyzed using 14 spectra recorded at 0.0056 and 0.011 cm-1 resolution with the McMath Fourier transform spectrometer located at Kitt Peak National Observatory. Experimental upper state levels of H232S, H234S, and H233S were obtained from assigned positions (as high as J = 20 and Ka = 15 for the main isotope). These were fitted to the A-reduced Watson Hamiltonian to determine precise sets of rotational constants through J10 and up to nine Fermi and Coriolis coupling parameters. Intensities of the two H232S triads were modeled with rms values of 2.5%, using the transformed transition moment expansion with 19 terms for 568 intensities of the first triad and 11 terms for the 526 intensities of the second triad. The second derivatives of the dipole moment with respect to normal coordinates were estimated in Debye to be: 22µx = -0.004873 (90); 12µx = 0.01372 (30); and 23µz = 0.01578 (30). This confirmed that for hydrogen sulfide some of the second derivatives are larger than the first derivatives. The calculated line intensities were summed yielding integrated band strengths (in cm-2/atm at 296 K) as follows: 0.3315 for 2nu2, 0.4522 for nu1, 0.1201 for nu3, 0.0303 for 3nu2, 1.820 for nu1 + nu2, and 2.869 for nu2 + nu3. In addition, the hot band transitions were identified in both regions. Finally, a composite database of hydrogen sulfide line parameters was predicted for the 5- to 2.5-µm region. Copyright 1998 Academic Press.
Using both high-resolution (R = 0.003 cm-1) and medium-resolution (R = 0.12 cm-1) Fourier transform spectroscopy, it has been possible to measure accurately a large set of individual line intensities for the nu1 and nu3 bands of SO2 in the 950-1400 cm-1 spectral region. These intensities were introduced into a least-squares fit calculation allowing one to obtain the expansion of the transition moment operator of the nu1 and nu3 bands. For these intensity calculations, the theoretical model takes into account the vibration-rotation interactions linking the upper levels involved in the nu1, 2nu2, and nu3 interacting SO2 bands. Finally, a synthetic spectrum of the 8-µm SO2 bands has been generated using the dipole moment expansion determined in this work and the molecular parameters and the Hamiltonian matrix given in a previous analysis. Copyright 1998 Academic Press.
The analysis of the nu6, nu7, nu8, and nu9 bands of H15N16O3 located at 646.9641, 578.4719, 743.6166, and 458.2917 cm-1, respectively, has been carried out in the 400-800 cm-1 region using high-resolution Fourier transform spectra recorded at Ottawa. Using the ground state energy levels calculated from the v = 0 rotational constants of H15N16O3 [A. P. Cox, M. C. Ellis, C. J. Attfield, and A. C. Ferris, J. Mol. Struct. 320, 91-106 (1994)], it was possible to assign the A-type nu6 and nu7 bands and the C-type nu8 and nu9 bands of H15N16O3 up to high J and Ka rotational quantum numbers. The v6 = 1, v7 = 1, v8 = 1, and v9 = 1 experimental energy levels were then introduced in a least-squares fit calculation and precise upper state Hamiltonian constants (band centers and rotational constants) were determined allowing one to reproduce the infrared data to within the experimental uncertainty. Copyright 1998 Academic Press.
Ouabain or a closely related isomer, and 'ouabain-like compound' (OLC), has been identified in plasma, by Hamlyn et al., using several physico-chemical and biological methods. Using a radioimmunoassay, the same authors later characterized an identical compound in adrenal cortex tissue and culture medium from adrenocortical cells. Nevertheless, other groups, using different immunosera, were not able to detect OLC in adrenal cortex and adrenocortical cells medium. In this report, we confirm the presence of OLC in bovine adrenal cortex and in fasciculata cells culture medium. The compound that we obtained has the same chromatographic properties as ouabain on HPLC using two types of elution systems. It presents the same mass spectrum and is able to bind to erythrocytes membranes Na(+)-K(+)-ATPase. In primary cultures of adrenocortical cells, its biosynthesis is increased after addition of pregnenolone or progesterone suggesting that these compounds may represent intermediate substrates in the biosynthetic pathway. Rhamnose readily enters the adrenocortical cell and increases slightly the biosynthesis of OLC. The present studies confirm that bovine adrenocortical cells in primary culture release an OLC with no differences with authentic ouabain using, HPLC, mass spectrometry and radioreceptor assay and suggest that OLC may be a product related to the adrenocortical steroidogenic pathway.
OBJECTIVE: To investigate the stability of 50 microg/ml tropisetron in 0.9% sodium chloride or 5% dextrose injections on storage in either 100 ml polyvinylchloride (Tuliflex) or polyolefin (Clear-flex) infusion bags, at +4 degrees C and -20 degrees C. METHOD: A stability-indicating high-performance liquid chromatography (HPLC) assay was used to measure residual drug at day 0 (D0), D8, D15, D30, D60 and D90 for each bag. Samples were tested for pH at D0, D60 and D90. Frozen samples were thawed in a microwave oven according to a validated procedure before analysis. RESULTS: In 0.9% sodium chloride and 5% dextrose, no degradation products were observed in any of the chromatograms. No admixtures, at any time, contained less than 98.2% of the initial concentration. Only minor changes (-0.12 unit) in pH occurred over the storage period. No colour or other visual changes were seen in any sample. CONCLUSION: Tropisetron (50 microg/ml) in either 0.9% sodium chloride or 5% dextrose is chemically stable in polyvinylchloride or polyolefin bags for at least 3 months when stored in a refrigerator or in a frozen state.
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In steroidogenic tissues, cytochrome P-450(17) alpha catalyzes both steroid 17 alpha-hydroxylation and 17,20-lyase reactions. The ratio of the two activities, hydroxylase over lyase (H/L) depends upon the tissue of origin; this ratio is low in the testis whereas it is high in the adrenal cortex. To examine the factors responsible for this specific regulation, two approaches were followed: (i) the purified enzyme was incorporated into liposomes made of microsomal lipids of testis or adrenal cortex; and (ii) the effects of disorganization of the microsomal membrane on the activities were observed. The results show that the cytochrome 17,20-lyase activity is stimulated by the presence of lipids from testicular origin. In the adrenal microsomes, this activity appears to be dependent upon the local membrane organization. Specific component(s) associated with the neutral fraction of the microsome lipid extract may be responsible for the repression of lyase activity in the adrenal.
Tn5Map, a Tn5 derivative containing the 18 bp I-SceI site, was delivered from a RP4-mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-SceI sites in their genomes. Digestion of the DNA from Tn5Map-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion. These digests also gave direct and independent proof for the single circular chromosome of E. coli, and for the presence of two circular chromosomes in B. melitensis and of a circular and a linear chromosome in A. tumefaciens C58 (which also contains two large circular plasmids). This rapid and versatile technique is potentially applicable to the study of the genomic organization in all Gram-negative bacteria which support Tn5 transposition. Moreover, linearization of circular replicons could be the first step for a rapid method of physical mapping.
The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.
13C-NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron-transport system responsible for steroid hydroxylation. [13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin. The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.
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Double-strand breaks in genomic DNA stimulate recombination. Until now it was not possible to induce in vivo site-directed double-strand breaks in a mammalian chromosomal target. In this article we describe the use of I-Sce I meganuclease, a very rare cutter yeast endonuclease, to induce site-directed double-strand breaks mediated recombination. The results demonstrate the potential of the I-Sce I system for chromosome manipulation in mammalian cells.
Group I intron-encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site-specific sequences. We have studied the mechanism of interaction of the I-SceI endonuclease with different DNA substrates derived from its natural site in the intron-less gene or from intron-exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron-containing gene, the enzyme recognizes the downstream intron-exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.
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