Polymerization in biological systems. Introduction: the objectives.
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DNA↗
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Water structure in biological systems.
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Chemical Phenomena↗
Contractile processes in nonmuscular systems.
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Chemical Phenomena↗
Mechanisms of relaxation for protons in biological systems.
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Animals↗
Protein kinase A phosphorylation of the cardiac calcium release channel (ryanodine receptor) in normal and failing hearts. Role of phosphatases and response to isoproterenol.
The cardiac ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) comprises a macromolecular complex that includes a kinase and two phosphatases that are bound to the channel via targeting proteins. We previously found that the RyR2 is protein kinase A (PKA)-hyperphosphorylated in end-stage human heart failure. Because heart failure is a progressive disease that often evolves from hypertrophy, we analyzed the RyR2 macromolecular complex in several animal models of cardiomyopathy that lead to heart failure, including hypertrophy, and at different stages of disease progression. We now show that RyR2 is PKA-hyperphosphorylated in diverse models of heart failure and that the degree of RyR2 PKA phosphorylation correlates with the degree of cardiac dysfunction. Interestingly, we show that RyR2 PKA hyperphosphorylation can be lost during perfusion of isolated hearts due to the activity of the endogenous phosphatases in the RyR2 macromolecular complex. Moreover, infusion of isoproterenol resulted in PKA phosphorylation of RyR2 in rat, indicating that systemic catecholamines can activate phosphorylation of RyR2 in vivo. These studies extend our previous analyses of the RyR2 macromolecular complex, show that both the kinase and phosphatase activities in the macromolecular complex are regulated physiologically in vivo, and suggest that RyR2 PKA hyperphosphorylation is likely a general feature of heart failure.
Animals↗
Bile salt-enhanced rat jejunal absorption of a macromolecular tracer.
Bile salt deconjugation may occur in the jejunum under conditions of fecal colonic overgrowth of the small intestine. We studied the effects of conjugated and deconjugated bile salts on rat jejunal absorption of a macromolecular tracer, horseradish peroxidase, in an in vivo perfusion system. At a 0.5 mM perfusion concentration, only the deconjugated bile salts, cholate and deoxycholate, produced a significant increase in horseradish peroxidase absorption into serum. At a 0.5 mM concentration of the conjugated salt, taurocholate, horseradish peroxidase-absorption was indistinguishable from that seen in bile salt-free preparations. At a higher 5 mM concentration, both the conjugated and deconjugated salts increased jejunal HRP absorption into serum over that seen in bile salt-free preparations; this absorption wa most marked with the deconjugated salts. At a 0.5 mM level, the bile salts induced minimal sodium and glucose transport alterations but did not produce evidence of morphologic damage to villi or absorptive epithelial cell organelles. At a 5 mM level, the deconjugated salts induced glucose and sodium transport abnormalities and appeared to damage cellular organelles. Our observations suggest that an alteration in the tight junctional barrier to macromolecular absorption may play a role in some of the enhanced horseradish peroxidase absorption seen with deoxycholic acid in these experiments. The process of an increased absorption of intact macromolecules induced by products of bacterial metabolism may be of pathologic significance in the etiology of immunologically related intestinal disease or in toxigenic processes.
Animals↗
Effect of buffer on kinetics of proton equilibration with a protonable group.
The laser-induced proton pulse generates a massive, brief, proton pulse capable of perturbing biochemical equilibria. The time resolution of the monitoring system can follow the diffusion-controlled protonation of specific sites on macromolecular bodies [Gutman, M. (1984) Methods Biochem. Anal. 30, 1-103]. In order to apply this method in enzymology, one must first evaluate how the buffer capacity of biochemical systems (substrates and proteins) will affect the observed dynamics. Unlike equilibrium measurements, where buffer is an inert component, in kinetic studies buffer modulates the observed dynamics. In this paper we analyze the effect of buffer on the dynamics of protonation in a model system. We describe the experimental technique and introduce the mathematical formalism that determines the various rate constants involved in the reaction. The analysis of the experiments indicates that in buffered solution proton flux is carried by two mechanisms: (A) proton dissociation followed by free proton diffusion; (B) collisional proton transfer between small diffusing solutes. We demonstrate how to evaluate the contribution of each pathway to the overall proton flux.
Arylsulfonates↗
Post-traumatic pulmonary insufficiency.
Pulmonary insufficiency is occasionally a life-threatening sequel to severe nonthoracic trauma. A similar disturbance of pulmonary function may be a feature of hepatic cirrhosis, fulminant hepatic failure, liver homotransplantation and major hepatic resection for blunt injury. In addition to the respiratory changes, other associations common to both post-traumatic pulmonary insufficiency and liver disease are disturbance of consciousness and susceptibility to infection by organisms normally found in the gastrointestinal tract. If failure of some aspect of liver function is responsible for the development of pulmonary insufficiency, it is likely that it is the hepatic reticulo-endothelial cells rather than the parenchymal cells which are concerned, because hepatic parenchymal cell failure is not a feature of post-traumatic pulmonary insufficiency. It is suggested that the hepatic reticuloendothelial system normally protects the lungs by acting as a prepulmonary filter removing and inactivating noxious macromolecular and particulate matter in the blood, such matter arising mainly from the intestine and the blood coagulation and fibrinolytic systems. Experimental evidence indicates that hypovolemia increases the load of circulating phagocytosable material and depresses activity of the hepatic reticuloendothelial system, while trapping of the pulmonary reticuloendothelial system is considerably enhanced. This noxious material, normally dealt with by the hepatic reticuloendothelial system, may accumulate during and after hypovolemia in active form in the lungs as a result of the action of three mechanisms: direct pulmonary reticuloendothelial phagocytosis from the blood, transfer in hepatic reticuloendothelial cells from liver to lungs and uptake from the blood by polymorphonuclear leukocytes which become sequestered in the lungs. The ensuing pulmonary injury caused by this material may then be manifested by progressive pulmonary insufficiency and the impairment of activity of the hepatic reticuloendothelial system by the presence of gram-negative infection. The reticuloendothelial system has been largely neglected in man, and it is considered that evaluation of reticuloendothelial activity in injured patients may lead to the definition of a syndrome of failure of the reticuloendothelial system of which pulmonary insufficiency may be an integral part.
Blood Coagulation↗
What are antibiotics? Archaic functions for modern activities.
Secondary metabolites are proposed to have played important roles in the evolution of the reactions of living forms on earth, in effecting and modulating reactions during biochemical evolution by chemical and structural interaction with 'receptor' sites in primitive macromolecular templates. For example, in the evolution of the translation system, as the polymerizing reactions became more complex and proteins became involved, the low molecular-weight effectors were functionally replaced by polypeptides, but retained their ability to interact with receptor sites in nucleic acids and proteins. Many of these low molecular-weight effectors now play a different role, that of antagonists, by interacting with the original receptor sites in macromolecular structures; this explains their contemporary activity as antibiotics.
Animals↗
Scaffolding proteins and their role in viral assembly.
Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.
Adenoviridae↗
Physicochemical and functional characterization of the C1r subunit of the first complement component.
C1r was isolated from serum by an improved method and found to be a glycoprotein with a sedimentation coefficient of 7.0S. Under conditions of physiologic ionic strength and pH, C1r consist of two apparently identical noncovalently linked 95,000 dalton polypeptide chains. Antisera to C1r detected a protein of gamma-mobility on electrophoresis of serum in agarose in the presence of calcium, and a Beta-mobility protein when the electrophoretic separation was carried out in EDTA. On sucrose gradient ultracentrifugation of normal human serum in the presence of calcium, C1r antigenicity was found in the 19 S region of the gradient. On the other hand, when the gradient contained EDTA, C1r antigenicity was found in the 7 S region. No reaction of anti-C1r with C1r-deficient sera was observed. C1r had a high affinity for active C1s or proenzyme C1s in the presence of calcium and was able to activate C1s and to form C1 in conjunction with C1q and C1s. Activation of C1s by C1r was inhibited by calcium, C1 inactivator, polyanethol sulfonate, and DFP. Activation of C1s by C1r occurred only after a preliminary incubation of C1r for a brief time at 37 degrees C before addition of C1s. The ability of C1r to form C1 in conjunction with C1q and C1s was, however, progressively lost on incubation at 37 degrees C. Trypsin, although potentiating the activity of crude C1r, did not modify the activity of purified C1r. Its action was on a trypsin-sensitive inhibitor separated from C1r in the final step of the isolation procedure. The binding of 125I-C1r to sensitized sheep erythrocytes required the presence of C1q and calcium but not C1s, whereas the binding of 125I-C1s required C1q, C1r and calcium. Thus, C1r functions as not only the activator of C1s, but also serves as the physical link between C1q and C1s in macromolecular C1.
Animals↗
Interpretation of the kinetics of consecutive enzyme-catalyzed reactions. Studies on the arginase-urease system.
Physiocochemical properties of beef liver arginase are reported, particular attention being given to its state of aggregation in the concentration range encountered in enzymic assays. It is shown that a species of molecular weight 114,000 is the operational kinetic unit. Evidence is also provided that arginase does not associate heterogeneously with urease, and therefore, in the absence of macromolecular interactions, the arginase-urease couple provides a suitable experimental system to test the applicability of theory previously developed to guide the interpretation of coupled assay results. Application of the theory led to values of the Michaelis constant and maximal velocity describing the first reaction in the sequence, catalyzed by arginase, which agreed within experimental error with the corresponding values obtained by studying the arginase-catalyzed reaction alone. Comment is also made on the product inhibition of arginase by ornithine, which must be considered in the comparison of experimental results describing the time course of a coupled assay with theoretical solutions obtained by numerical integration.
Animals↗
Membrane localization of the S1 subunit of pertussis toxin in Bordetella pertussis and implications for pertussis toxin secretion.
Pertussis toxin is secreted from Bordetella pertussis with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type B. pertussis, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of B. pertussis that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of B. pertussis. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of Escherichia coli (J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of B. pertussis. Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.
Biological Transport↗
The role of endothelin-1 in ischemia-reperfusion induced acute inflammation of the bladder in rats.
PURPOSE: Endothelin (ET)-1 is causatively involved in ischemia-reperfusion induced acute inflammatory reactions and microcirculatory disturbances in many organs. We investigated the role of endothelin-1 in the microcirculatory consequences of ischemia-reperfusion of the bladder using intravital fluorescence videomicroscopy. MATERIALS AND METHODS: Male Sprague-Dawley rats were used in the experiments. The animals were randomly assigned to a sham operated group or to 1 of 2 ischemia-reperfusion groups that underwent 60 minutes of ischemia followed by 30 minutes of bladder reperfusion. In 1 ischemia-reperfusion group the animals were pretreated with BQ 610, a specific ET-A receptor blocker. The bladder was placed on an especially designed stage for intravital fluorescence videomicroscopy measurements. Venular red blood cell velocity, functional capillary density, venular and arteriolar diameter, venular and arteriolar macromolecular leakage, and leukocyte-endothelial cell interactions in postcapillary venules were determined using a computer assisted analyzing system. RESULTS: Functional capillary density, red blood cell velocity, venular and arteriolar diameter were significantly decreased and macromolecular leakage was significantly enhanced after bladder ischemia-reperfusion. The number of rolling and adherent leukocytes was significantly increased in postcapillary venules. Pretreatment with BQ 610 was effective for attenuating the effects of ischemia-reperfusion induced inflammation but could not completely prevent microcirculatory failure. CONCLUSIONS: Ischemia-reperfusion induced cystitis leads to significant impairment of the microcirculation and ET-1 is suggested to have an important role in this process. Pretreatment with an ET-A receptor antagonist reduces ischemia-reperfusion related microvascular disturbances in the bladder.
Acute Disease↗
The transport of albumin: a critique of the vesicular system in transendothelial transport.
Although albumin circulating in the plasma can exchange with the contents of the extensive vesicular system of microvascular endothelium, the mechanism and quantitative significance of vesicular transport remains unclear. It has recently been shown that transport of electron opaque tracers can occur via the vesicular system, but the detailed ultrastructure is inconsistent with the transcytotic shuttling of single vesicles. Although most physiologic measurements show a major degree of convective coupling in the microvascular permeation of perfused tissues, recent reports suggest that convective coupling is low when albumin escapes from the intact circulation. Because albumin clearance in these studies is consistent with values calculated from ferritin-labeling of vesicles in perfused microvessels, the possibility remains that macromolecular transendothelial transport occurs through transient communications within the vesicular system.
Albumins↗
Stochastic dynamics of macromolecular-assembly networks.
The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The inherent complexity of assembling macromolecular structures makes current computational methods strongly limited for understanding how the physical interactions between cellular components give rise to systemic properties of cells. Here, we present a stochastic approach to study the dynamics of networks formed by macromolecular complexes in terms of the molecular interactions of their components. Exploiting key thermodynamic concepts, this approach makes it possible to both estimate reaction rates and incorporate the resulting assembly dynamics into the stochastic kinetics of cellular networks. As prototype systems, we consider the lac operon and phage lambda induction switches, which rely on the formation of DNA loops by proteins and on the integration of these protein-DNA complexes into intracellular networks. This cross-scale approach offers an effective starting point to move forward from network diagrams, such as those of protein-protein and DNA-protein interaction networks, to the actual dynamics of cellular processes.
Computational Biology↗
The permeability of the basal lamina at the neuromuscular junction. An ultrastructural study of rat skeletal muscle using particulate tracers.
Various macromolecular substances, such as toxins and antibodies, may interfere with neuromuscular transmission. The neuromuscular junction is also known to be a site for the uptake of macromolecular substances into the axon for subsequent transport to the central nervous system. The synaptic cleft of the neuromuscular junction is separated from the rest of the extracellular compartment of muscle by the basal lamina, the permeability properties of which are not known. The penetration of macromolecular substances of varying size into the synaptic cleft of the neuromuscular junction has been studied in rats. Four different tracers: Imferon (an iron-dextran measuring 11 X 7 X 7 nm), ferritin (a spherical iron-protein 12 nm in diameter), Imposil (an iron-dextran measuring 21 X 12 X 12 nm) and colloidal gold-protein (20-25 nm in diameter) were injected into the palmaris longus muscle. Fifteen and 120 min after injection, the distribution of these particulate tracers was studied by electron microscopy. Imferon and ferritin penetrated rapidly through the basal lamina along the muscle fibres and also into the synaptic cleft of the neuromuscular junction. The larger Imposil and colloidal gold particles were restricted from penetrating the basal lamina even after 2 h, and these particles were traced only occasionally within the synaptic cleft. The results indicate that the basal lamina of muscle acts as a diffusion barrier to large macromolecules, preventing them from entering the synaptic cleft.
Animals↗
Transglutaminase-independent binding of gliadin to intestinal brush border membrane and GM1 ganglioside.
Anti-ganglioside antibodies have been described in celiac disease or gluten sensitivity, in conjunction with the presence of central and peripheral nervous system deficits. The observed antibody reactivity to gangliosides is postulated to be related to the anti-gliadin immune response, either through antigenic mimicry, or by formation of gliadin-ganglioside complexes and haptenization. We examined the possibility of the presence of ganglioside-like epitopes in gliadin, as well as the potential for complex formation between gliadin and GM1 ganglioside. Low levels of glycosylation were present in gliadin, but ganglioside-like carbohydrate epitopes were not detected. However, gliadin was found to bind to GM1 ganglioside and to the GM1-rich intestinal brush border membrane. The described complex formation and possible haptenization of GM1 by gliadin may be responsible for driving the anti-ganglioside antibody response in some patients with gluten sensitivity. Furthermore, binding of gliadin to GM1 on the intestinal epithelium might have a role in the anti-gliadin immune response and contribute to the intestinal inflammatory reaction in celiac disease.
Animals↗