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At least 1,153 records · Page 64Linked to original sources
PubMed1996

Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis.

BACKGROUND: The present paper describes the immunocytochemical and morphometric characteristics of two major cell groups of the suprachiasmatic nucleus (SCN) in the human hypothalamus: the vasopressin (VP) and vasoactive intestinal polypeptide (VIP) neuronal subdivisions. The dimensions (volume and length) and the number of neurons expressing each peptide in the two subdivisions were obtained, as well as the mean diameter of the cell nuclei. All morphometric parameters were studied in relation to sex and age. METHODS: Brains of 42 human subjects (22 males and 20 females) ranging in age from 10 to 92 years were obtained at autopsy. The hypothalamic area containing the SCN was dissected from each brain, dehydrated, and embedded in paraffin. Serial sections of 6 microns were cut in a coronal plane and stained with thionin for general orientation. To determine the architectonic boundaries of the VP- and VIP-expressing cell populations every 25th section was immunocytochemically stained by means of antibodies against arginine VP or VIP using the peroxidase-antiperoxidase method. The VP- and VIP-expressing cell numbers in the SCN of each subject were estimated by unilaterally counting the number of nuclear profiles with the aid of a Zeiss microscope under x 500 magnification, using a deconvolution procedure and a correction for section thickness. RESULTS: The main portion of the VP positive neurons is located in the dorsomedial part of the SCN and is rostrocaudally longer in females than in males (1.76 +/- 0.12 mm and 1.40 +/- 0.10 mm, respectively). The volume of the VP subdivision is 0.244 +/- 0.017 mm3 and contains 6,890 +/- 520 VP-immunoreactive neurons, with a mean density of about 29,000 neurons/mm3. No significant sexual dimorphism or age-related alterations in the population of VP neurons is found. The VIP positive neurons are mainly located in the ventral and central part of the SCN and extend rostrocaudally in a similar way in females and males (1.07 +/- 0.08 mm and 1.02 +/- 0.11 mm, respectively). The volume of the VIP subdivision is 0.034 +/- 0.004 mm3 and contains 1,700 +/- 140 VIP-immunoreactive neurons, with a mean density of about 63,000 neurons/mm3. An age-dependent sexual dimorphism is observed in the number of VIP-expressing neurons in the SCN: young males have about twice as many VIP neurons as females of the same age, whereas in middle-aged subjects this sexual difference is reversed, and less robust, with females now having about 1.7 times as many VIP neurons as males. In old subjects the difference in VIP cell number between men and women disappears. CONCLUSIONS: The present study clearly shows that the population of VP neurons in the human SCN is considerably larger than the population of VIP neurons. Furthermore, the age-related sexual differences in the VIP cell number reinforces the idea that the SCN is not only involved in the timing of circadian rhythms but also in the temporal organization of reproductive functions.

Adolescent↗
PubMed1999

Demonstration of the capabilities of a parallel high performance liquid chromatography tandem mass spectrometry system for use in the analysis of drug discovery plasma samples.

There is a continuing need for increased throughput in the evaluation of new drug entities in terms of their pharmacokinetic (PK) parameters. This report describes an alternative procedure for increasing the throughput of plasma samples assayed in one overnight analysis: the use of parallel high performance liquid chromatography (HPLC) combined with tandem mass spectrometry (parallel LC/MS/MS). For this work, two HPLC systems were linked so that their combined effluent flowed into one tandem MS system. The parallel HPLC/APCI-MS/MS system consisted of two Waters 2690 Alliance systems (each one included an HPLC pump and an autosampler) and one Finnigan TSQ 7000 triple quadrupole mass spectrometer. Therefore, the simultaneous chromatographic separation of the plasma samples was carried out in parallel on two HPLC systems. The MS data system was able to deconvolute the data to calculate the results for the samples. Using this system, 20 compounds were tested in one overnight assay using the rapid rat PK screening model which includes a total of 10 standards plus samples and two solvent blanks per compound tested. This application provides an additional means of increasing throughput in the drug discovery PK assay arena; using this approach a two-fold increase in throughput can be achieved in the assay part of the drug discovery rat PK screening step.

Animals↗
PubMed1999

Strategies for locating disulfide bonds in a monoclonal antibody via mass spectrometry.

The location of the disulfide bonds in a recombinant monoclonal antibody was confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). A non-reduced Endoproteinase Lys-C (Endo Lys-C) digest of the antibody was analyzed directly by MALDI-TOFMS. The sample was then reduced on-plate by depositing dithiothreitol (DTT) on the sample spot and re-analyzed by MALDI-TOFMS. The disulfide bonds were assigned based on the disappearance of certain mass ions in the non-reduced digest and the appearance of product ions in the reduced digest. A rapid LC/ESI-MS protocol was also developed to determine the location of the disulfide bonds. The peptides generated from the Endo Lys-C digest of the antibody were partially separated on a high performance liquid chromatography (HPLC) column by utilizing a steep gradient and analyzed by ESI-MS. The masses of the partially resolved peptides were determined by deconvoluting the mass spectra.

Antibodies, Monoclonal↗
PubMed2000

Mass spectrometric determination of appearance energies for ions formed from CoF(4) and CoF(3) molecules.

Knudsen cell mass spectrometry was applied to the evaluation of the ionization efficiency curves for the ions originating from CoF(4) molecules. Cobalt tetrafluoride was obtained in the gas phase over the CoF(3)(s)-TbF(4)(s) system in the temperature range from 640 to 690 K. From the ionization efficiency curves the appearance energies of the ions formed from the CoF(4) molecules were determined by means of Vogt's deconvolution method. Clausius-Clapeyron plots for the ions from CoF(4) molecules were measured. Evaporation of pure CoF(3)(s) was carried out, and the appearance energies of the ions formed from CoF(3) molecules were determined. The ionization energies for CoF(4) and CoF(3) molecules were found to be (14.3 +/- 0.2) and (13.3 +/- 0.1) eV, respectively.

Cobalt↗
PubMed1997

CD conformational studies on synthetic peptides encompassing the processing domain of the ocytocin/neurophysin precursor.

Synthetic peptides of different size, reproducing the proteolytic processing site of proocytocin, were studied by CD under several experimental conditions in order to ascertain the ability of different solvents to stabilize secondary structural motifs, such as alpha-helix tracts and beta-turns. A combination of deconvolution methods and empirical calculations subtracting the contributions due to unordered structures from the spectra suggests that in solution (a) mainly two distinct families of ordered conformers containing structurally different beta-turns are present, (b) the relative stability of the different conformers depends from the nature of the solvent, and (c) in the case of the larger peptides, a population containing an alpha-helical conformation is also present. From the biological point of view the presence of at least two families of ordered conformers could be in line with current theories assuming that the catalytic effect of the receptor microenvironment may be determinant in shifting the equilibrium toward the active conformation.

Amino Acid Sequence↗
PubMed1997

Determination of secondary structure of normal fibrin from human peripheral blood.

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% alpha-helix (amide I and III components at 1653 cm-1, and 1312 and 1284 cm-1, respectively), 40% beta-sheets (amide I and III components at 1625 and 1231 cm-1, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm-1, and 1249 cm-1, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin beta-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (alpha-helix, beta-sheets, and turns) are accurate.

Algorithms↗
PubMed1997

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films.

A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100 degrees C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the alpha-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O-H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility.

Animals↗
PubMed1997

Solvent effects on the conformation and far UV CD spectra of gramicidin.

Solvent effects on the far-uv CD spectra of the polypeptide gramicidin have been studied systematically in a series of alcohols of increasing chain length, ranging from methanol to dodecanol. The effects observed are of two types: primary, involving a change in the equilibrium mixture of conformers present, and secondary, involving a shift in the spectral peak positions as a function of solvent polarizability. To quantitate the primary effect, the ratio of the individual conformers present was estimated by deconvolution of the spectra into their component species. For short chain length alcohols, both parallel and antiparallel double helices are found in considerable abundance. As the solvent chain length is increased and its polarity is decreased, the left-handed antiparallel double helical species is favored. For all alcohols with chain lengths of four or more carbon atoms, the ratio of the conformers present remains relatively constant. To quantitatively examine the secondary effect, the magnitudes of the spectral shifts on the dominant conformer (species 3) have been correlated with the dielectric constants and refractive indices of the solvents, thereby indicating what underlying physical properties are responsible for these shifts. This work thus demonstrates that for gramicidin, a flexible polypeptide, the solvent effects on the CD spectra can be resolved into two types: changes due to the mixture of conformers present and shifts in the spectral characteristics. Both effects need to be considered when interpreting CD spectra in terms of secondary structure for this and other polypeptides in nonaqueous solutions.

Alcohols↗
PubMed1999

New opioid peptides, peptidomimetics, and heterocyclic compounds from combinatorial libraries.

Here we review the use of combinatorial libraries in opioid receptor assays. Following a brief description of the history of the combinatorial field, methods for the generation of synthetic libraries and the deconvolution of mixture-based libraries are presented. Case studies involving opioid assays used to demonstrate the viability of combinatorial libraries are described. The identification of new opioid peptides from combinatorial libraries is reviewed. The peptides found are composed of L-amino acids, D-amino acids, or L-, D-, and unnatural amino acids, and range from tetrapeptides to decapeptides. Likewise, new opioid compounds identified from peptidomimetic libraries, such as peptoids and alkylated dipeptides, and those identified from acyclic (e.g., polyamine, urea) and heterocyclic (e.g., bicyclic guanidine) libraries, are reviewed.

Combinatorial Chemistry Techniques↗
PubMed1996

Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells.

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.

Cell Division↗
PubMed1996

Improved oral absorption of L-365,260, a poorly soluble drug.

L-365,260, a specific and potent nonpeptide antagonist of the CCKB receptor, is under investigation for its potential utility in the treatment of anxiety and panic disorders. This drug has a very low aqueous solubility (< 2 micrograms mL-1). When L-365,260 was given orally as a suspension in 0.5% methylcellulose (5 mg kg-1), the absorption was rapid but incomplete. The peak concentrations occurred in about 30-40 min, and the bioavailability was 8.6% for the dog and 13.6% for the rat. The poor bioavailability could be attributed to poor absorption or extensive first-pass metabolism. By comparing the drug concentrations in the systemic circulation during portal and femoral (or cephalic) vein infusion, the hepatic first-pass metabolism was estimated to be 0.30 for the rat and 0.14 for the dog, suggesting that first-pass metabolism is not the main reason for the low bioavailability of the drug in rats and dogs. The limited bioavailability is, therefore, more likely due to its poor absorption as a result of its poor aqueous solubility and slow dissolution rate. However, while the absorption was substantially improved when the drug was given orally as a solution in PEG 600 solution, the bioavailability increased threefold to fourfold in rats and sixfold to sevenfold in dogs. Although the underlying mechanism for the improved absorption is unknown, PEG 600 may have exerted a cosolubilizing effect which enhances the dissolution rate of L-365,260 in the GI tract, resulting in better absorption. Kinetic analysis by a deconvolution technique revealed that PEG 600 increased both the extent and rate of absorption. These results are consistent with the notion that absorption of L-365,260 is rate limited by its dissolution rate. The data from these animal studies provides valuable information in selecting the formulation for clinical trials.

Absorption↗
PubMed1996

Theophylline controlled-release formulations: in vivo-in vitro correlations.

Four experimental controlled-release oral solid dosage formulations were developed and the in vitro dissolution characteristics of theophylline from these formulations were studied in USP apparatus I. Pharmacokinetic evaluation of these formulations was carried out in eight beagle dogs under fasting conditions. Theophylline in a 5% dextrose injection USP, oral solution, and Slo-Phyllin were used as controls to estimate the in vivo dissolution of these four formulations in the GI tract. The percentage cumulative amounts of drug absorbed and the percentage cumulative amounts of drug released into the GI tract from these four controlled-release formulations were obtained by numerical deconvolution methods. The in vivo and in vitro dissolution data demonstrated good correlation indicating that in vitro dissolution tests can be used to optimize the further design of controlled drug release oral solid dosage formulations for theophylline.

Animals↗
PubMed1998

A comparative study of ofloxacin and ciprofloxacin erythrocyte distribution.

The present work deals with the in vitro and in vivo distribution of ofloxacin and ciprofloxacin in erythrocytes. In vitro studies were carried out in standard solutions prepared using fresh blood for a concentration range between 100 and 0.25 micrograms mL-1. A 5 mg kg-1 bolus dose was administered to rabbits and erythrocyte and plasma kinetics were determined over 8 h. A linear model was used to establish the relationship between plasma and erythrocyte concentrations of both quinolones in vitro. The mean partition coefficient values obtained were 1.04 +/- 0.02 and 1.32 +/- 0.03 for ofloxacin and ciprofloxacin, respectively. A decrease in the ciprofloxacin partition coefficient was observed at higher concentrations. Values ranged between 2.54 +/- 0.40 and 1.38 +/- 0.15 as the concentrations increased. The partition coefficients obtained from the linear relationship between plasma and erythrocyte concentrations established from the in vivo data were 0.80 +/- 0.58 for ofloxacin and 0.61 +/- 0.30 for ciprofloxacin. In vivo plasma and erythrocyte data analysis was performed by a deconvolution method and the theoretical transfer curves in erythrocytes were estimated. The distribution of both quinolones to erythrocytes is very rapid, probably due to a high permeability of erythrocyte membranes to these drugs. This was also confirmed by the parallelism between plasma and erythrocyte kinetics.

Animals↗
PubMed1999

Single dose pharmacokinetics and bioavailability of nevirapine in healthy volunteers.

The results of two randomized, single-dose, crossover bioavailability studies are presented which describe the pharmacokinetics and oral bioavailability of nevirapine, a novel nonnucleoside antiretroviral drug. In the first study 12 healthy male volunteers received nevirapine 15 mg via short-term i.v. infusion or orally as a 50 mg tablet or reference solution (50 mg/200 mL). Following the i.v. dose, nevirapine had a low systemic clearance (Mean +/- S.D., Cl = 1.4 +/- 0.3 L/h) and a prolonged elimination phase (t(1/2beta) = 52.8 +/- 14.8 h; MRT = 81.4 +/- 22.4 h). Nevirapine absolute bioavailability was 93 +/- 9% and 91 +/- 8% for the tablet and oral solution, respectively. In the second study, 24 healthy male volunteers were administered nevirapine as a 200 mg production-line tablet or oral reference solution (200 mg/200 mL). There was no significant difference in bioavailability between the tablet and reference solution. Overall, comparison of the pharmacokinetic parameters between the 50 and 200 mg doses indicates that nevirapine is well absorbed at clinically relevant doses. The absorption profiles using deconvolution revealed no evidence of differential enzyme induction between the two doses or routes of administration following a single dose.

Administration, Oral↗
PubMed2000

Facilitated tip-positioning and applications of non-electrode tips in scanning electrochemical microscopy using a shear force based constant-distance mode.

In scanning electrochemical microscopy (SECM) a microelectrode is usually scanned over a sample without following topographic changes (constant-height mode). Therefore, deconvolution of effects from distance variations arising from non-flat sample surface and electrochemical surface properties is in general not possible. Using a shear force-based constant distance mode, information about the morphology of a sample and its localized electrochemical activity can be obtained simultaneously. The setup of the SECM with integrated constant-distance mode and its application to non-flat or tilted surfaces, as well as samples with three-dimensional surface structures are presented and discussed. The facilitated use of non-amperometric tips in SECM like enzyme-filled glass capillaries is demonstrated.

Electrochemistry↗
PubMed2000

Application of Combinatorial Procedures in the Search for Serine-Protease-Like Activity with Focus on the Acyl Transfer Step.

Recursive deconvolution of a 729-membered peptide library has identified three active sequences, in which both Ser and His are present in one of the two tripeptidic chains generated on a steroidal scaffold (see structural formula), for the cleavage of an activated p-nitrophenyl ester. This combinatorial approach aims at searching for serine-protease-like activity.

Journal Article↗
PubMed1999

Use of the mean transit time of an intravascular contrast agent as an exchange-insensitive index of myocardial perfusion.

A simple two-compartment model was used to study the effects of water exchange on the signal produced by an inversion recovery prepared rapid gradient-echo sequence during the first passage of a low dose of an intravascular contrast agent. Water exchange at intermediate rates of exchange (1-10 Hz) between the vascular and extravascular spaces caused the form of the signal changes during the first pass to be dependent on both the fractional sizes of the vascular and extravascular compartments and on the exchange rate. Unless the effects of exchange are minimized by using a very short inversion time, parameters such as the peak height and area under the curve will be affected by regional and/or pathological variations in the exchange rate and the size of the vascular fraction. The mean transit time (MTT) is, however, less affected by water exchange. Experimental first-pass data produced by intravascular low-dose injections of iron oxide particles were studied in five pigs at 0.5 T. The MTT as derived from the first-pass curves, without deconvolution with the arterial input function, was well correlated with the myocardial blood flow (MBF) as measured using radioactive microspheres (r = 0.70, n = 52, P < 0.01). Other first-pass parameters such as the peak height or area under the curve exhibited either a poorer, or no, correlation with the MBF. The data suggest that the MTT of the first pass of an intravascular contrast agent may be a robust, quantitative method for assessing myocardial blood flow in patients.

Animals↗
PubMed1999

Cerebral blood flow measurement by dynamic contrast MRI using singular value decomposition with an adaptive threshold.

Singular value decomposition (SVD) is a promising deconvolution technique for use in dynamic contrast agent magnetic resonance perfusion imaging. Computer simulations, however, show that the selection of the threshold for SVD affects the accuracy of the cerebral blood flow measurements and may distort the shape of the vascular residue function. In this report, a pixel-by-pixel thresholding method is proposed based on the signal-to-noise ratio of the concentration time curve at maximum concentration (SNRC). Monte Carlo simulations were used to determine the optimal threshold for different SNRC. This technique was used to analyze data from six healthy volunteers, resulting in a mean gray to white matter cerebral blood flow ratio of 2.67 +/- 0.07. This value is in excellent agreement with values published in the literature.

Blood Flow Velocity↗