Localization of aminopeptidase activity in freshly excised human skin: direct visualization by confocal laser scanning microscopy.

The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue.

Transport and metabolic pathway of thymocartin (TP4) in excised bovine nasal mucosa.

Thymocartin (TP4, Arg-Lys-Asp-Val) is the 32-35 fragment of the naturally occurring thymic factor (thymopoietin). Here studies on the nasal transport and metabolism of TP4 were performed. Freshly excised bovine nasal mucosa was taken as a model membrane.

Proteolysis of human calcitonin in excised bovine nasal mucosa: elucidation of the metabolic pathway by liquid secondary ionization mass spectrometry (LSIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI).

Purpose: Two calcitonins, i.e. human calcitonin (hCT) and, for comparison, salmon calcitonin (sCT), were chosen as peptide models to investigate nasal mucosal metabolism.

Broadband decoupled, 1H-localized 13C MRS of the human brain at 4 Tesla.

Broadband proton decoupling of the entire 13C spectrum was possible within power absorption guidelines and resulted in the detection of narrow (as low as 2-3 Hz), natural abundance signals from metabolites such as myo-inositol, glutamate, N-acetyl-aspartate, and glutamine from 72 cm3 volumes in the human brain. To overcome the chemical shift displacement error, three-dimensional localization on the 1H z magnetization was combined with polarization transfer. Efficiency of the heteronuclear localization method was demonstrated by the elimination of all scalp lipid resonances.

Induction of sustained and elevated immune responses to weakly immunogenic synthetic malarial peptides by encapsulation in biodegradable polymer microspheres.

Biodegradable microspheres (MS) based on poly(D,L-lactide) and poly(D,L-lactide-coglycolide) have the capacity to release encapsulated antigens over defined lengths of time depending on their composition and to elicit and sustain strong and long-lasting immune responses to protein antigens. In the present study, two synthetic multiple antigenic peptides (MAP), P30B2 and (NANP)6P2P30, were incorporated into MS of different compositions. P30B2 and (NANP)6P2P30 are composed of one or two universal T helper epitopes from tetanus toxin, 947-967 (P30) and 830-843 (P2), and of a B cell epitope derived from the repeat sequence of Plasmodium berghei or Plasmodium falciparum, respectively.

Relationships among lactate concentration, blood flow and histopathologic profiles in rat C6 glioma.

Increased capacity for glycolytic metabolism is a well-known characteristic of neoplastic cells. Because lactic acid is the end product of glycolysis, in vivo MRS measurements of tumor lactate concentration ([lac]t) may provide valuable information about tumor metabolism, which will aid the development of therapies and the clinical diagnosis and treatment of tumors. In the present study, several hemodynamic and histologic parameters were evaluated with respect to their influence on [lac]t.

Prevention of spontaneous autoimmune thyroiditis in the obese strain (OS) chickens by treatment with a monoclonal anti-CD4 antibody.

Untreated control OS chickens develop spontaneous autoimmune thyroiditis (SAT). In contrast, OS chickens treated with a monoclonal anti-CD4 antibody failed to develop SAT. The preventive effect of anti-CD4 antibody on SAT was associated with the marked depletion of CD4+ T-cells by anti-CD4 treatment.

Enzymatic cleavage of thymopoietin oligopeptides by pancreatic and intestinal brush-border enzymes.

The intestinal enzymatic degradation of the immunomodulating peptides thymotrinan (TP3), thymocartin (TP4), and thymopentin (TP5), three oligopeptides derived from the naturally occurring thymus hormone thymopoietin, was investigated to evaluate their potential for peroral drug delivery. In the presence of brush-border membrane vesicles, crude pancreas extract and everted rings from duodenum, jejunum, ileum, and colon, all peptides were shown to be degraded both by pancreatic enzymes and brush-border aminopeptidases. Degradation clearances (Cldeg) of TP3, TP4, and TP5 were calculated for a quantitative comparison of peptide stability.

Physical model relating diffusional transport and concurrent metabolism of peptides in metabolically active cell sheets.

The physical model developed describes the complex interplay of diffusion and saturable metabolism in metabolically active cell sheets under reflection kinetics in contact with a bulk solution containing a substrate. A prominent implication of this work is the enzymatic cleavage of drugs in the viable epidermis, which represents the major metabolic barrier of the skin. The mathematical solution is based on steady-state Fickian diffusion and nonlinear Michaelis-Menten kinetics.

Differential binding of two chicken beta-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells.

Plant lectins can be potent modulators of vertebrate immune functions. Biochemical characterization of lectins from animal tissues enables the determination of whether these endogenous activities display a comparable immunological potency. Focusing on chicken beta-galactoside-binding lectins, the monomeric intestinal (CL-14) and the dimeric liver lectin (CL-16) were purified and the lack of cross-contamination was ascertained.

Fast anatomical imaging of the heart and assessment of myocardial perfusion with arrhythmia insensitive magnetization preparation.

A new contrast preparation based on modified driven equilibrium Fourier transfer is introduced and evaluated for generation of T1-weighted images for assessment of the myocardial perfusion with contrast agent first-pass kinetics. The new preparation scheme produces T1 contrast with insensitivity to arrhythmias in prospectively triggered sequential imaging thereby eliminating one of the major sources of problems in potential patient studies with previously employed contrast preparations schemes.

Effect of left ventricular hypertrophy secondary to chronic pressure overload on transmural myocardial 2-deoxyglucose uptake. A 31P NMR spectroscopic study.

Background: This study tested the hypothesis that 31P nuclear magnetic resonance (NMR)-detectable 2-deoxyglucose (2DG) uptake is increased in chronically pressure-overloaded hypertrophied left ventricular myocardium.

Methods And Results: 31P NMR spectroscopy was used to determine the transmural distribution of high-energy phosphate levels and 2-deoxyglucose-6-phosphate (2DGP) accumulation during intracoronary infusion of 2DG (15 mumol.kg body wt-1.

Myocardial tagging with B1 insensitive adiabatic DANTE inversion sequences.

A new technique, based on adiabatic delays alternating with mutations for tailored excitation (DANTE) inversion sequences, is presented for generating uniform contrast tags across the myocardial wall even in the presence of B1 inhomogeneities. The utility of this pulse was demonstrated using a surface coil for both transmission and signal reception in phantom and animal heart tagging studies. The experimental data demonstrated uniform grid contrast over a sixfold variation of B1 magnitude, sharp tagging profiles, and the ability to follow the cardiac wall motion through the deformation of the fine rectangular tagging grid at different phases throughout the cardiac cycle.

Preferential TCR V beta 1 gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis of the obese strain of chickens.

We studied T cell receptor variable beta (TCR V beta) gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis (SAT) of obese strain (OS) chickens. Chicken alpha beta T cells may express either V beta 1 or V beta 2 genes, the products of which can be recognized by TCR2 and TCR3 monoclonal antibodies, respectively. Selective depletion of V beta 1+ or V beta 2+ T cells in OS chickens was accomplished by repeated injections of TCR2 or TCR3 antibodies into embryonic and 1-3-week-old chickens.

Membrane modulated dissolution of oral drug delivery systems.

A kinetic model analogous to two chemical reactions occurring in series describes the dissolution behavior of tablets and pellets coated with either microporous or semipermeable membranes. Microporous membranes permit free water influx, but moderated core dissolution and dialytically controlled solution efflux. Semipermeable membranes allow osmotically limited water influx, moderated core dissolution, but relatively unimpeded solution efflux through a release orifice.

Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin.

The human calcitonin (hCT) peptide hormone has a marked tendency to aggregate in aqueous solutions and to form long, thin fibrillar aggregates resulting in viscous and turbid dispersions. In this study, the in vitro aggregation products of hCT were systematically investigated using conventional transmission electron microscopy (CTEM) and in-lens field emission scanning electron microscopy. The mass per length of unstained/air-dried specimens was determined by scanning transmission electron microscopy.

Transmural bioenergetic responses of normal myocardium to high workstates.

The response of myocardial high-energy and inorganic phosphates (HEP and Pi, respectively) and associated changes in myocardial blood flow, lactate uptake, and O2 consumption (MVo2) rates were examined in an open-chest canine model during progressively increasing workloads achieved by catecholamine infusion. HEP and Pi levels (measured with transmurally localized 31P-nuclear magnetic resonance spectroscopy) were unaffected by moderate increases in the level of energy expenditure but were significantly altered by high workloads, especially in the subepicardium. The MVo2 and HEP data from three different protocols that utilized pharmacological augmentation of blood flow demonstrated that the maximal rate of myocardial energy production during inotropic stimulation was dictated by perfusion limitation.

A single administration of tetanus toxoid in biodegradable microspheres elicits T cell and antibody responses similar or superior to those obtained with aluminum hydroxide.

The use of biodegradable polymer microspheres as a single dose vaccine delivery system was investigated by using tetanus toxoid (TT). In order to compare the immunogenicity of TT-microspheres (TT-MS) with aluminum hydroxide (alum)-based TT, BALB/c mice were immunized with TT in different formulations including individual or mixtures of MS and TT-alum. All TT-MS formulations elicited high proliferative and antibody responses comparable to those obtained with TT-alum formulation.

Dermal metabolism of topically applied drugs: pathways and models reconsidered.

The study of skin metabolism is of prime importance not only in the field of transdermal drug delivery but also for the safe and efficient local skin treatment with topically applied substances. Since it has become clear that even peptides may be delivered across the permeation barrier of the stratum corneum, e.g.

Regional myocardial blood volume and flow: first-pass MR imaging with polylysine-Gd-DTPA.

The authors investigated the utility of an intravascular magnetic resonance (MR) contrast agent, poly-L-lysine-gadolinium diethylenetriaminepentaacetic acid (DTPA), for differentiating acutely ischemic from normally perfused myocardium with first-pass MR imaging. Hypoperfused regions, identified with microspheres, on the first-pass images displayed significantly decreased signal intensities compared with normally perfused myocardium (P < .0007).

A new localization method using an adiabatic pulse, BIR-4.

A new method is described for accomplishing localized spectroscopy with an adiabatic pulse, BIR-4. The method has advantages similar to previously described combinations of outer-volume suppression (OVS) and ISIS, with the additional advantages that localization is achieved with only three radiofrequency pulses and the localization remains accurate even in the presence of intense signals with short relaxation times. This new localization pulse sequence is referred to as integrated OVS-ISIS.

Effects of dobutamine on myocardial blood flow, contractile function, and bioenergetic responses distal to coronary stenosis: implications with regard to dobutamine stress testing.

To determine the effects of dobutamine stimulation on myocardium distal to a coronary stenosis, transmural spatially localized phosphorus 31 nuclear magnetic resonance measurements of myocardial high-energy phosphate compounds (adenosine triphosphate and phosphocreatine), inorganic phosphate, and blood flow and systolic wall thickening were made in 8 open-chested dogs. Data were collected under (1) control conditions, (2) after the application of a moderate coronary stenosis, (3) during infusion of dobutamine with continuing stenosis, and (4) after the release of the stenosis with continuing dobutamine. Stenosis was associated with concordant reductions of subendocardial blood flow, wall thickening, and high-energy phosphate, and mild elevation of inorganic phosphate; subepicardial measurements were essentially unchanged.

An in vivo 31P magnetic resonance spectroscopy study of uridine excess in rats fed orotic acid.

Spatially localized 31P NMR spectroscopy was used to assay in vivo the liver of intact rats fed orotic acid (OA) in a diet which produces hepatic steatosis. Twenty-three sets of multiple volume spectra were obtained from twenty-one 265- to 315-g female rats after 0-9 days of feeding either a 1% OA/64% sucrose diet (12 rats) or a 65% sucrose control diet (9 rats). The intensity of the in vivo diphosphodiester resonance ascribed to UDP-hexos(amin)es increased and the phosphomonoester resonance decreased in intensity prior to fatty infiltration.

Transmural distribution of 2-deoxyglucose uptake in normal and post-ischemic canine myocardium.

This investigation was performed to determine (i) whether 31P spatially localized 31P NMR spectroscopy could be utilized to determine the transmural distribution of 2-deoxyglucose (2DG) uptake in the in vivo canine heart and (ii) whether transmural 2DG uptake would be affected by a preceding ischemic insult. 2DG was infused and the accumulation of 2-deoxyglucose-6-phosphate (2DGP) was monitored (by means of spatially localized 31P NMR) in control hearts, in pharmacologically hyperperfused hearts, and in hearts subjected to four (5 min) occlusions of the left anterior descending coronary artery. Myocardial blood flow was measured with radioactive microspheres.

Quality improvement of spray-dried, protein-loaded D,L-PLA microspheres by appropriate polymer solvent selection.

The aim was to study the effect of the type of polymer solvent on characteristics of microspheres produced by spray drying. The water-soluble model protein, bovine serum albumin (BSA) was microencapsulated into biodegradable poly(D,L-lactic acid) using the following 10 different polymer solvents: acetaldehyde dimethyl acetal, acetone, dichloromethane, dioxane, ethyl acetate, ethyl vinyl ether, nitromethane, tetrahydrofuran, 1,1,1-trichloroethane, and 1,1,2-trichloroethylene. These solvents having similar toxicity levels differ greatly in their physico-chemical characteristics such as boiling point, vapour pressure, miscibility and interfacial tension with an aqueous phase, and solubility parameter.