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PubMed1987

Suppression of in vitro antibody production by dimethylnitrosamine in mixed cultures of mouse primary hepatocytes and mouse splenocytes.

The suppression of in vitro antibody responses by dimethylnitrosamine (DMN) was produced in a mouse hepatocyte and splenocyte co-culture system. Mouse hepatocytes were isolated from female B6C3F1 mice and cultured for 20-24 hr to allow the formation of a monolayer on collagen-coated plastic petri dishes. Spleen cells were isolated from the same hybrid and were co-cultured with the hepatocytes along with DMN. Cyclophosphamide (CP), an immunosuppressive agent requiring metabolic activation that was included as an initial positive control, produced a marked suppression of the in vitro antibody responses to LPS, DNP-Ficoll, and SRBCs in 4 hr in the co-culture system. Under comparable conditions DMN markedly suppressed the response to SRBCs, marginally suppressed the response to DNP-Ficoll, and did not suppress the polyclonal response to LPS. The suppression by DMN was related to the rocking speed during the 4-hr co-culture period and was optimally produced when the cultures were not rocked. Addition of serum into the medium (10% fetal calf serum) during the co-culture period did not change the effects of DMN on the antibody response. However, the addition of extracellular DNA (1 mg calf thymus DNA/ml) prevented the suppression of the antibody response by DMN. These results suggest that DNA represents the primary macromolecular target for the reactive intermediate of DMN, and indicate that a syngeneic co-culture system can be used to characterize the in vitro immunosuppression

Animals↗
PubMed1997

Quantitative evaluation of local drug delivery using the InfusaSleeve catheter.

BACKGROUND: Restenosis is the most common long-term complication after angioplasty. Local delivery of pharmacologic agents at the site of angioplasty holds promise as a means of achieving higher concentrations of drug in the arterial wall than can be obtained by systemic infusion. In this study, a novel local drug delivery catheter system, the InfusaSleeve catheter, was evaluated in a porcine coronary balloon injury model. The purpose of the study was to evaluate the efficacy of solute transfer to the arterial wall and the influence of varying supporting angioplasty balloon pressure. METHODS AND RESULTS: Ten pigs (total of 22 arterial segments) underwent overstretch balloon injury (artery/balloon ratio 1:1.29) with a standard angioplasty balloon. In 7 animals (16 arterial segments) horseradish peroxidase (HRP; 10 mg/ml) was administered locally after injury, by tracking the local infusion catheter as a sheath over the angioplasty balloon to the intended site of arterial drug delivery. Supporting angioplasty balloons were inflated to one of the three different pressures. In 3 pigs HRP (10 mg/ml) was administered intravenously. No significant arterial injury caused by the local delivery device was evident on histological examination (disruption of the internal lamina elastica, arterial media, or thrombosis). Radial concentrations of the HRP reaction product in the first 150 microns of the arterial wall were quantified against known standards by measurement of light transmission through tissue sections. Mean HRP concentrations were not significantly different from those obtained by intravenous infusion using a supporting pressure of 1 atm or a supporting pressure of 3 atm of the underlying angioplasty balloon. However, a supporting pressure of 6 atm resulted in a 6-fold greater mean HRP concentration in the arterial wall than that which could be achieved by systemic administration of an equal volume of tracer (P < 0.001). CONCLUSION: Thus solute can be delivered throughout the coronary media by the InfusaSleeve, with the magnitude of wall uptake related to support pressure. Local delivery at 6 atm support pressure produced substantially greater uptake than did systemic delivery.

Angioplasty, Balloon, Coronary↗
PubMed1989

SC5b-7, SC5b-8 and SC5b-9 complexes of complement: ultrastructure and localization of the S-protein (vitronectin) within the macromolecules.

Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all antisera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)n and (C5b-8)n, negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 less than 27 S) appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.

Avidin↗
PubMed1993

Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.

Animals↗
PubMed1995

Dielectric relaxation spectroscopy and some applications in the pharmaceutical sciences.

With a few exceptions, dielectric relaxation spectroscopy (DRS) has been largely neglected by pharmaceutical scientists, despite the potential for this technique as a noninvasive and rapid method for the structural characterization and quality control of pharmaceutical materials. DRS determines both the magnitude and time dependency of electrical polarization (i.e. the separation of localized charge distributions) by either measuring the ability of the material to pass alternating current (frequency domain DRS) or by investigating the current that flows on application of a step voltage (time domain DRS). DRS is thus (i) sensitive to molecular mobility and structure, (ii) non-invasive, and (iii) employs only mild stresses (a weak electromagnetic field) in order to measure the sample properties. The technique covers a broad-band frequency window (from 10(-5) to 10(11) Hz) and therefore enables the investigation of a diverse range of processes, from slow and hindered macromolecular vibrations and restricted charge transfer processes (such as proton conductivity in nearly dry systems) to the relatively fast reorientations of small molecules or side chain groups. The dielectric response provides information on (i) structural characteristics of polymers, gels, proteins, and emulsions, (ii) the interfacial properties of molecular films, (iii) membrane properties, (iv) water content and states of water (and the effects of water as a plasticizer), and (v) lyophilization of biomolecules. This review article details the basis of dielectric theory and the principles of measuring dielectric properties (including a comprehensive account of measurement artifacts), and gives some applications of DRS to the pharmaceutical sciences.

Chemistry, Pharmaceutical↗
PubMed2003

Prediction of the unknown: inspiring experience with the CAPRI experiment.

We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our submitted models included acceptable, medium accuracy predictions of the structures of the complexes, and for a fifth target we identified the location of the binding site of one of the molecules. We used a weighted-geometric docking algorithm in which contacts involving specified parts of the surfaces of either one or both molecules were up-weighted or down-weighted. The weights were based on available structural and biochemical data or on sequence analyses. The weighted-geometric docking proved very useful for five targets, improving the complementarity scores and the ranks of the nearly correct solutions, as well as their statistical significance. In addition, the weighted-geometric docking promoted formation of clusters of similar solutions, which include more accurate predictions.

Algorithms↗
PubMed1994

The quaternary structures of untransformed steroid hormone receptors: an open issue.

Oligomeric forms of steroid hormone receptors have been observed since the original detection of these proteins in cytosols from target tissues, and the capacity of receptors to arrange in supramolecular entities was found to be related to their functional untransformed states of low affinity for nuclear acceptor sites. In this paper we discuss the models of quaternary structures of untransformed receptors and steroid-receptor complexes proposed in the last two decades, including homo-oligomers as well as hetero-oligomers containing non-steroid binding proteins and/or RNA. The multiplicity of forms detected in cell-free systems, their stabilization by unphysiological experimental conditions, and their sensitivity to the ionic species employed to prepare and analyze steroid receptors, are critically evaluated, and it is concluded that the extrapolation of models to account for quaternary structure of receptors in intact cells is unwarranted. We have also discussed the experimental strategies which have been developed to circumvent the possible artefactual changes in the arrangements of oligomeric receptors following cell rupture, in order to probe the existence of these forms in vivo, and to characterize their composition and structure. The data obtained by these studies support the concept that oligomeric untransformed steroid receptors exist in intact cells, where they can be present in multiple supramolecular arrangements whose quaternary structures remain to be established.

Animals↗
PubMed1992

Present and emerging applications of polymeric biomaterials.

An important trend in biomaterials research and development is the synthesis of polymers, which combine capabilities of biologic recognition ('biomimetic') with special physicochemical properties of the synthetic polymer system. For example, an antibody may be conjugated to the backbone of a polymer which precipitates upon small changes in pH, temperature, or ionic strength. Crosslinked gels may also be synthesized from such polymers, and a biomolecule such as an enzyme may be chemically or physically entrapped in these gels. Such gels will shrink and swell in response to small changes in environmental stimuli. Another approach is to 'engineer', perhaps via computer-aided molecular design, new artificial biomimetic systems by exact placement of functional groups on rigid polymer backbones, crosslinked structures, or macromolecular assemblies. In this way, biocatalytic functioning or biorecognition similar to enzymes and antibodies can be achieved without the inherent instability often encountered with the native biomolecules or assemblies. In addition to these synthetic approaches, new and exciting analytical tools, such as the scanning tunnelling microscope and the atomic force microscope, are permitting visualization of individual and small clusters of proteins and other biomolecules on surfaces. Cell attachments and spreading may also be visualized at various depths within the cell using the confocal laser microscope. Such analytical techniques can lead to important new knowledge about biologic interactions with biomaterials, and, therefore, to development of even more biocompatible implants and devices. This paper overviews the field of polymeric biomaterials and highlights the important emerging trends in synthesis and analysis of these materials.

Biocompatible Materials↗
PubMed1985

Electron microprobe analysis of intracellular electrolytes in resting and isoproterenol-stimulated exocrine glands of frog skin.

In the intact, in vitro frog skin, isoproterenol (ISO) stimulates an amiloride-insensitive increase in short-circuit current (SCC) that can be localized to the exocrine glands and is associated with secretion of chloride. To determine which cells in the glands respond to stimulation we measured the intracellular electrolyte concentrations of the various cell types of the mucous and seromucous glands of the skin using freeze-dried cryosections and electron microprobe analysis. In the resting state, the various cell types of the glands have intracellular electrolyte concentrations similar to the epithelial cells of the skin. Exposure to amiloride (10(-4) M) has little effect on the concentration of Na and Cl in the cells of the glands. The effect of isoproterenol has two distinct phases. Analysis of glands in tissues frozen at the peak of the SCC response (13 min after addition of isoproterenol) shows that the only significant change is an increase in Na and Ca in a group of cells at the ductal pole of the acini of both gland types. These are termed "gland" cells. The duct cells and cells that secrete macromolecules did not show any significant changes at this timepoint. In the gland cells, after a one-hour exposure to isoproterenol the Na concentration is at prestimulation levels while Cl drops. There is also a smaller drop in Cl in the duct and skin epithelial cells. Ouabain, which can completely block the isoproterenol SCC response, has little short-term effect on Na and Cl in the control gland but accentuates the gain of Na and drop in Cl in the isoproterenol-treated condition. Bumetanide and, to a lesser extent, furosemide, also blocks the isoproterenol SCC response and causes a further drop in Cl. The results provide indirect evidence that a major portion of the ionic component of the gland secretion is produced by a distinct group of cells separate from those producing the macromolecular component and that the mechanism of secretion involves a Na: Cl coupled transport system linked to the activity of the basolateral Na pump.

Amiloride↗
PubMedDate not supplied

1994 Whitaker Lecture: polymers for drug delivery and tissue engineering.

This paper reviews three areas of the author's research. The first area concerns the development of technologies to release macromolecules continuously from solid polymers. By embedding solid protein (or other macromolecule) powders at the correct concentration in hydrophobic polymers, prolonged release for over 100 days can be achieved. The second area involves the synthesis of new biodegradable polymers specifically designed for drug delivery. A novel family of polymers, polyanhydrides, now being explored in a number of medical applications is examined. The use of these polymers to deliver chemotherapeutic agents locally may provide a new approach to treat brain cancer. The final research topic is in the area of tissue engineering. By placing mammalian cells on biodegradable polymer scaffolds, a variety of tissues have been created in animal models. Cartilage is discussed as a model tissue.

Animals↗
PubMed2006

The macromolecular peptide-loading complex in MHC class I-dependent antigen presentation.

A challenging task for the adaptive immune system of vertebrates is to identify and eliminate intracellular antigens. Therefore a highly specialized antigen presentation machinery has evolved to display fragments of newly synthesized proteins to effector cells of the immune system at the cell surface. After proteasomal degradation of unwanted proteins or defective ribosome products, resulting peptides are translocated into the endoplasmic reticulum by the transporter associated with antigen processing and loaded onto major histocompatibility complex (MHC) class I molecules. Peptide-MHC I complexes are transported via the secretory pathway to the cell surface where they are then inspected by cytotoxic T lymphocytes, which can trigger an immune response. This review summarizes the current view of the intracellular machinery of antigen processing and of viral immune escape mechanisms to circumvent destruction by the host.

ATP-Binding Cassette Transporters↗
PubMed1992

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.

Animals↗
PubMed1992

Stimulation of cholecystokinin (CCK) release from superfused rat hypothalamo-neurohypophyseal complexes by interleukin-1 (IL-1).

This in vitro study examined the effects of interleukin-1 (IL-1) and interferon-gamma (Ifn-gamma) on the release of cholecysto-kinin (CCK) from superfused hypothalamo-neurohypophyseal complexes (HNC) of rats. An increase of CCK from HNC was elicited in a dose-dependent manner by recombinant human IL-1 alpha and -1 beta in concentrations of 0.1-10 nM. In contrast, the release of CCK from HNC was not affected by recombinant human Ifn-gamma at any dose tested (0.1, 1 and 10 nM). The increased release of CCK elicited by IL-1 was calcium-dependent, as was that induced by potassium (60 mM), but it was biphasic and had a different time course and a lower magnitude than those induced by potassium and veratridine. These results suggest that IL-1 activates pituitary-adrenal axis by stimulating CCK neurons in the hypothalamus and/or neurohypophysis to release CCK, since CCK has been implicated in the regulation of adrenocorticotropin release.

Animals↗