Water in biological systems.
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Biochemical Phenomena↗
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Results for “MACROMOLECULAR SYSTEMS”
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The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.
Intestinal ischemia and reperfusion injury (I/R) is probably involved in the pathogenesis of intestinal barrier dysfunction, associated with the concomitant translocation of enteric bacteria and toxins and the potential development of multiple organ failure. The intestinal endothelial and epithelial layers play a major role preventing the entry of toxic substances from the gut, but the influence of protease-antiprotease systemic balance on these barrier functions and the relationship between epithelial DNA synthesis, apoptosis, and endothelial and epithelial barrier macromolecule permeability are not fully investigated. Endothelial and epithelial barrier macromolecular permeability, epithelial DNA synthesis, the endothelial and epithelial plasma membrane system, apoptosis and oncosis, plasma levels of proteinase inhibitors, and proenzymes were measured in rats subjected to 20 and 40 min intestinal ischemia and 1, 3, 6, or 12 h reperfusion. Endothelial permeability increased after both 20 and 40 min intestinal ischemia. Epithelial permeability significantly increased during 1-6 h reperfusion after 20 min ischemia and during 1-12 h reperfusion after 40 min ischemia. Epithelial DNA synthesis increased in animals with 20 min ischemia followed by 12 h reperfusion. Plasma levels of prekallikrein, C1-esterase inhibitor, and alpha1-macroglobulin were significantly lower following both 20 and 40 min ischemia from 3 h reperfusion and on. Apoptotic epithelial cells significantly increased in animals subjected to 20 min ischemia followed by 12 h reperfusion. The severity of reperfusion injury in the intestinal endothelial and epithelial barrier seems to correlate with the period of ischemia and the pathway of cell damage and death, together with proteinase-antiproteinase imbalance.
Albumins↗
The toxicity of phenothiazine.
Phenothiazine, the parent compound of a multitude of present-day drugs, has been employed on an extensive scale for its insecticidal, fungicidal, antibacterial and anthelmintic properties. Almost a catholicon, its widespread use in animals and man has led to the uncovering of many adverse reactions encompassing effects on blood elements, neuromuscular problems and photosensitization. The high lipophilicity of phenothiazine and the formation of two redox systems amongst its many metabolites can facilitate the occurrence of generalised macromolecular disruption. Information from the literature has been garnered and appraised in this review to enable an insight into the possible mode(s) of interaction of phenothiazine with living systems.
Animals↗
Altered cellular responsiveness during ageing.
The capacity of cells and organisms to respond to external stimuli and to maintain stability in order to survive decreases progressively during ageing. The mitogenic and stimulatory effects of growth factors, hormones and other agents are reduced significantly during cellular ageing. The sensitivity of ageing cells to toxic agents including antibiotics, phorbol esters, radiations and heat shock increases. This failure of homeostasis during cellular ageing does not appear to be due to any quantitative and qualitative defects in the receptor systems. Instead, metabolic defects in the pathways of macromolecular synthesis may be the basis of altered cellular responsiveness during ageing.
Aging↗
Chemical adaptor systems.
"Chemical adaptor systems" are molecules used to link different functionalities, based on unique reactivity that allows controlled fragmentation. Two different mechanistic reactivities were used to prepare chemical adaptor systems. The first is based on a spontaneous intra-cyclization reaction to form a stable ring molecule. Cleavage of the trigger generates a free nucleophile, for example, an amine group, which undergoes intra-cyclization to release the target molecule from the handle part (e.g., a targeting antibody or a solid support for synthesis). The second applied reactivity is an elimination reaction, which is usually based on a quinone-methide-type rearrangement. Similarly, cleavage of the trigger generates a free phenol functionality, which can undergo a self-elimination reaction through a quinone-methide rearrangement to release the target molecule. The adaptor molecules have been applied in the field of drug delivery to release a drug from a targeting device and in the field of solid-phase synthesis to release a synthetic molecule from the solid support. A chemical adaptor molecule has also been used as a building unit to construct dendrimers with a triggered fragmentation.
Antineoplastic Agents↗
The delivery of proteins and peptides to the lung.
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Administration, Inhalation↗
Equilibrium analysis of high affinity interactions using BIACORE.
BIACORE biosensors are useful for measuring reaction kinetics and calculating affinity constants for macromolecular interactions. However, one drawback with the flow system used in these instruments is that the standard injection procedures limit the amount of time available to collect association-phase data. This is especially problematic during equilibrium analysis of high affinity interactions. Using protein-DNA interactions as a model system, we demonstrate a simple method for overcoming this limitation. By placing the analyte directly into the running buffer we were able to deliver a continuous supply of protein to the sensor surfaces for greater than 12 h at a time. Complete equilibrium binding profiles were generated by changing the concentration of analyte and allowing the surface reactions to reequilibrate. Analyte concentrations were also decreased to demonstrate that the binding reactions were fully reversible. This method of analysis is a simple and convenient way of directly measuring equilibrium dissociation constants for very high affinity interactions.
Base Sequence↗
Two different enzymes are primarily responsible for retinoic acid synthesis in rabbit liver cytosol.
Retinoic acid biosynthesis in rabbit liver was catalyzed by cytosolic NAD(+)-dependent dehydrogenase and oxygen-dependent oxidase, with an activity ratio of 59% and 41% in the presence of 2 mM dithiothreitol under aerobic conditions. The two enzymes could be well separated by fractionation involving ammonium sulfate precipitation. Purification of the enzymes indicated that the oxygen-dependent enzyme was a flavoenzyme, retinal oxidase (EC 1.2.3.11), composed of two 135 kDa subunits; and the NAD(+)-dependent enzyme was a basic pI retinal dehydrogenase composed of four 55-kDa subunits. A high concentration (1-2 mM) of DTT was required to stabilize the activity of retinal dehydrogenase during the purification procedures and storage, but inhibited the activity of retinal oxidase by 13-38%. The physiological roles of the two retinoic acid synthases in liver cytosol were discussed.
Aerobiosis↗
The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain.
Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.
Anticodon↗
Freeze-fracture observations on the visceral yolk sac placenta of rats, mice and hamsters. With special reference to endodermal cell tight junctions.
Freeze-fracture replicas of visceral yolk sac from rats, mice and hamsters in late stages of gestation were studied by electron microscopy. Special attention was directed toward determining the types of junctional specializations that exist between the columnar endoderm cells of this placental membrane. In all three species, well-developed, zonular tight (occluding) junctions were found on the contiguous lateral surfaces of the endoderm cells. The tight junctional network, located in an immediate subluminal position, was from 0.2--0.5 micrometers in depth and consisted at any point of 2--5, interconnecting, approximately 9 eta wide, strands (P-face) or shallow furrows (E-face). Patch-like aggregations of irregular intramembrane particles, characteristic of desmosomes (maculae adherentes), also were observed at scattered sites below the tight junctions. However, no evidence of gap (communicating) junctions was encountered. The endoderm cells of the rodent visceral yolk sac have been shown to play a central role in the selective transport of macromolecular substances from the maternal to the fetal system. Tight junctions may be vital to this endodermal cell function by preventing random paracellular fluxes of macromolecules.
Animals↗
Life at low temperatures: is disorder the driving force?
The thermodynamic characterization of various biological systems from psychrophiles points to a larger entropic contribution when compared to the corresponding mesophilic or (hyper) thermophilic counterparts, either at the level of the macromolecules (thermodynamic and kinetic stabilities) or of their function (ligand binding, catalytic activity). It is suggested here that in an environment characterized by a low heat content (enthalpy) and at temperatures that strongly slowdown molecular motions, the cold-adapted biological systems rely on a larger disorder to maintain macromolecular dynamics and function. Such pre-eminent involvement of entropy is observed in the experimental results and, from a macroscopic point of view, is also reflected for instance by the steric hindrances introduced by cis-unsaturated and branched lipids to maintain membrane fluidity, by the loose conformation of psychrophilic proteins or by the local destabilization of tRNA by dihydrouridine in psychrophilic bacteria.
Adaptation, Physiological↗
Performance studies of particle acceleration for transdermal drug delivery.
We have proposed a transdermal biolistic method to accelerate a powder formulation of drugs to penetrate human skin for the treatment of a range of diseases. One of the key issues for designing and evaluating transdermal biolistic system is ensuing that the powder drugs are delivered into the skin with a controllable velocity range and spatial distribution. The aerodynamics of supersonic nozzles and performance of the delivery system were initially studied, mainly analytically and experimentally. In this paper, computational fluid dynamics is utilized to characterize two existing prototype devices, in order to further investigate the transient gas and particle dynamics in their supersonic nozzles. To validate the implemented numerical approach, calculated pressure histories, two-dimensional flow structures and particle velocity distributions are made and compared with the reported experimental measurements. The key features of gas dynamics, gas-particle interaction and performance of the prototype transdermal biolistics are discussed and interpreted.
Administration, Cutaneous↗
Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.
Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.
Aniline Compounds↗
Selective chemical modification of plasma membrane ectoenzymes.
As part of an investigation of the organization of cell surface macromolecular assemblies, we have treated intact central nervous system cells with chemical probes which react convalently with proteins and aminophospholipids. Selective alterations of the enzymatic activities of ecto-ATPases, ecto-5'-nucleotidases and cholinesterases were obtained under appropriate reaction conditions. The cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene, was a potent inactivator of ecto-ATPase of C6 glioblastoma, IMR-32 neuroblastoma and of a primary rat astroblast cell line (RB). Ecto-5'-nucleotidase and acetylcholinesterase were less sensitive to difluorodinitrobenzene. 1-Fluoro-2,4-dinitrobenzene at concentrations which inactivated ecto-ATPase had little effect on ecto-5'-nucleotidase. Conversely, 2,4,6-trinitrobenzenesulfonic acid was a potent inactivator of ecto-5'-nucleotidase but had no effect on ecto-ATPase. The difluorodinitrobenzene inactivation of ecto-ATPase and of ecto-5'-nucleotidase as well as the fluorodinitrobenzene inactivation of ecto-ATPase could be prevented by the presence of the appropriate substrates in the reaction medium. In the presence of protecting nucleotide substrates, a decrease in reactivity with proteins and lipids was observed when the isotopic probe fluorodinitro[3H]-benzene was used.
5'-Nucleotidase↗
Purification and properties of cytochrome p-450 (11beta- and 18-hydroxylase) from bovine adrenocortical mitochondria.
We describe an improved procedure for the preparation of a cytochrome P-450 from bovine adrenocortical mitochondria which catalyzes 11beta- and 18-hydroxylation of steroids. The preparation is based upon chromatography on DEAE cellulose which separates the enzyme from the side-chain cleavage P-450, which can also be prepared in highly purified form from the same tissue extracts. The enzyme behaves as a single compound in glycerol density gradients. The enzyme aggregates at protein concentrations greater than 1 mg/ml to a series of forms of various molecular weights. On Sepharose 4B the enzyme shows a molecular weight of 185 000, while on glycerol density gradients a molecular weight of 1 - 10(6) is observed. The subunit molecular weight determined by electrophoresis on polyacrylamide gels with sodium dodecyl sulfate is 47 500 and the protein appears as a single band. The ratio of 11beta-/18-hydroxylase activities does not change significantly during purification and is constant through the protein peak on glycerol density gradients. Since there appears to be only one subunit species, it seems likely that the two hydroxylase activities are catalyzed by one protein.
Adrenal Cortex↗
Synthesis of high molecular weight thyroglobulin peptides by thyroid polysome in vitro.
Bovine thyroid polysomes were isolated under conditions which had yielded large polysomes in other systems. Between 25 and 40% of the protein synthesized by these polysomes could be precipitated by thyroglobulin antibody. When these immunoprecipitates were separated by dodecyl sulfate-polyacrylamide gel electrophoresis (4% running gel), over 50% of the radioactivity was located in the regions of polypeptides greater than 100,000 daltons. Between 11 and 13% of the total radioactivity was found as a single peak co-migrating with the main band of bovine thyroglobulin (Mr = 330,000). Peaks of radioactivity were also found in regions of molecular weights between 130,000 and 200,000. When the immunoprecipitates were separated in a 10% running gel, about 50% of the radioactivity was located in the top 8 mm of the gel. Most of the remaining radioactivity was distributed in regions corresponding to molecular weights greater than 68,000. No peak of radioactivity was seen corresponding to peptides of 15,000 daltons.
Animals↗
Effect of double stranded RNA on protein synthesis in an in vitro wheat germ embryo system.
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Animals↗
Binding of Triton X-100 to purified cytochrome P-450scc and enhancement of the cholesterol side chain cleavage activity.
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Adrenal Cortex↗