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Properties of a hemoglobin from the chromatrope of the nematode Mermis nigrescens.

The chromatrope pigment of Mermis nigrescens (Phylum: Aschelminthes, Class: Nematoda) was previously thought to have a role in photoreception. In this work it is shown to be an oxyhemoglobin whose absorption spectrum in vivo and in extracts resembles that of Ascaris oxyhemoglobins in having a weak alpha-band absorptivity. The extracted hemoglobin binds O2 and CO reversibly and with an affinity higher than that of the same concentration of horse hemoglobin. The Soret band of the deoxy derivative is unusually low and broad. Absorptivities and lambdamax for absorption bands of the oxy, deoxy and CO derivatives are tabulated for comparison with other hemoglobins. Microchemical procedures were developed which revealed that the chromatrope contains an average of 5-10(-12) mol of non-dialysable protoheme. Thus the hemoglobin concentration in the approx. 0.5 nl chromatrope volume is on the order of 10 mM (heme). The O2 binding ability and high in vivo concentration of this hemoglobin make possible a role in O2 storage or facilitation of O2 diffusion.

Animals↗

Effects of ethinyl estradiol on substrate uptake and efflux by isolated rat hepatocytes.

Ethinyl estradiol is a well documented, predictable cholestatic agent. The direct effects of this drug on hepatocellular uptake and efflux have been investigated. Uptake of taurocholate and ouabain, which are both actively transported, was inhibited, while uptake of cadmium, which occurs by a combination of facilitated and simple diffusion, was unaffected. The Vmax for taurocholate uptake was not altered by the presence of ethinyl estradiol, whereas the Km was increased, suggesting a reduced affinity of receptor for taurocholate. Efflux of taurocholate from pre-loaded cells remained unchanged in the presence of ethinyl estradiol. The data are not entirely consistent with an interference of Na+,K+-ATP ase by ethinyl estradiol being an important site of action. The observations may help to explain the mechanism of biliary dysfunction induced by ethinyl estradiol.

Animals↗

The effect of denervation on the synchronization of contraction of the rat vas deferens.

Denervation of the rat vas deferens results in both quantitative and qualtitative changes in the contraction of the isolated smooth muscle. The maximum contraction induced by norepinephrine, acetylcholine or serotonin is enhanced by denervation. The response of the tissue to 2 X 10(-4) M BaCl2 initiates a contraction which is more synchronized than the response of the control tissue. By use of 14C-sorbitol and Co-EDTA for estimating extracellular space and from fluorescence histochemical observations, it appears that facilitation of drug diffusion through the extracellular space plays an unimportant role in the altered response to drugs. In experiments which utilized a double-compartment tissue bath it was found that denervation enhanced the propagation of the contraction in the longitudinal direction. From measurements of the longitudinal tissue impedance it was determined that the resistance between smooth muscle cells was significantly decreased by denervation. These results suggest that denervation improves the propagation of excitation in this smooth muscle.

Animals↗

Substitution of arginine for lysine 134 alters electrostatic parameters of the active site in shark Cu,Zn superoxide dismutase.

The complete amino acid sequence was determined for the Cu,Zn superoxide dismutase from the shark Prionace glauca. The active site region shows the substitution of an Arg for Lys at position 134, which is important for electrostatic facilitation of the diffusion of O2- to the catalytically active copper. This change may be related to observed alterations of electrostatic parameters of the enzyme (pK of the pH dependence of the enzyme activity, rate of inactivation by H2O2), although it preserves a high efficiency of dismutation at neutral pH.

Amino Acid Sequence↗

The role of capsaicin-sensitive sensory neurons in healing of HCl-induced gastric mucosal lesions in rats.

BACKGROUND/AIMS: Capsaicin-sensitive sensory neurons are important in protecting the stomach against damage. This study investigated the role of these sensory neurons in the healing of gastric lesions. METHODS: Rats fasted for 18 hours were given 1 mL of 0.6N HCl orally for induction of gastric lesions; they were then fed normally from 1 hour later. On various days after HCl treatment, the area of lesion, acid secretion, mucosal DNA synthesis, mucosal H+ permeability, and blood flow responses were measured. RESULTS: Ablation of sensory neurons by capsaicin pretreatment did not affect the development of gastric lesions in response to HCl but significantly delayed the healing of these lesions. The damaged mucosa secreted less acid and had minimal changes in DNA synthesis but showed a marked increase in H+ permeability, resulting in luminal acid loss accompanied by an increase of mucosal blood flow. Sensory deafferentation did not have any influence on such changes in the damaged stomach, except the mucosal hyperemic response to acid. CONCLUSIONS: Capsaicin-sensitive sensory neurons may contribute to the healing of gastric lesions by mediating the mucosal hyperemic responses associated with acid back-diffusion and by facilitating acid disposal in the mucosa.

Animals↗

One-step purification of rat heart-type fatty acid-binding protein expressed in Escherichia coli.

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance intracellular transport of lipids by facilitating their cytoplasmic diffusion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective method was developed to purify recombinant rat H-FABP from cell lysates in a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H-FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30-40 mg/l culture. Characterization showed that recombinant rat H-FABP was indistinguishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focusing was observed, presumably due to differences in N-terminal processing of the proteins. In conclusion, a method is presented for efficient high-yield production of recombinant rat H-FABP.

Amino Acid Sequence↗

pH regulation and swimbladder function in fish.

Gas gland cells of the European eel (Anguilla anguilla) are specialized for the production and secretion of acidic metabolites. Although typically exposed to high oxygen partial pressures, they convert glucose mainly into lactate, but also produce CO2 in the pentose phosphate shunt. Only a very small fraction of glucose is oxidized via aerobic metabolism. Although the buffer capacity of gas gland cells appears to be high, even at low extracellular pH values intracellular pH is always kept about 0.2-0.3 pH-units more acidic. Thus, under all physiological conditions proton concentration within gas gland cells is higher than in the extracellular fluid, facilitating proton extrusion. Diffusion of CO2, Na+/H+-exchange, sodium-dependent anion exchange and a V-ATPase represent the pathways available for proton secretion. While under resting conditions the sodium-dependent pathways and diffusion of CO2 appear to be the dominating mechanisms for acid secretion, at low intracellular pH the contribution of Na+/H+-exchange and of V-ATPase appear to increase, while sodium-dependent anion exchange becomes less important. The mechanisms regulating the activity of these acid-secreting pathways and of the metabolism responsible for the production of protons are largely unknown.

Acids↗

Nutritional adaptations to parasitism within the platyhelminthes.

Some of the most significant alterations to the basic turbellarian plan are evident in the adaptations that relate to the acquisition of food by parasitic flatworms, reflecting the most potent of selection pressures in initiating and maintaining the host-parasite association. Nutritionally, ectoparasitic monogeneans show most correspondence with the predatory turbellarians, with certain monopisthocotylean members feeding by means of a protrusible pharynx and extracorporeal digestion, as skin-browsers of fish, with extensive intracellular digestion involving lysosomal enzymes in a well-differentiated gut. The more sheltered vascularised gill chamber of fish provides many polyopisthocotylean monogeneans with a totally renewable and more comprehensive diet in the form of blood, but haematophagy has necessitated a number of digestive adaptations, not least in resolving the problem of intracellular accumulations of haematin pigment. Haematophagy is the predominant feeding strategy of digeneans, but in contrast to monogeneans digestion of blood is largely extracellular; in schistosomes digestion is rapid, involving a battery of cathepsin-like cysteine proteinases and aminopeptidases. The external surfaces of all parasitic flatworms depart from turbellarian character and are composed of a multifunctional syncytial tegument, which is permeable to a variety of small organic solutes, some crossing by passive diffusion, others via facilitated or active mediated transport. The relative roles of the tegument and gut in trematode nutrition are difficult to assess, but can be related to the nature of the microhabitat within the host. Cestodes are highly adapted intestinal parasites bereft of any vestige of gut, and their tegument has become elaborated into a sophisticated and highly efficient digestive-absorptive layer, rivalling the vertebrate mucosa in its ability to gain kinetic advantage in the selective uptake of nutrient at the host-parasite interface. The patterns of energy metabolism in adult flatworm parasites are generally anaerobic and based on glycogen, with abbreviated metabolic pathways and the loss of biosynthetic capacities.

Animal Nutritional Physiological Phenomena↗

Diagnostic radiographic findings and symptom-free teeth.

The determination of whether a symptom-free tooth has pulpal and periapical disease relies heavily on radiographic findings and pulp tests. A case of a symptom-free upper left first molar is presented. The radiographic evidence of pathological changes associated with the endo-antral syndrome coupled with diffuse pulpal calcifications facilitated the diagnosis of chronic pulpal periapical disease. Non-surgical root canal therapy resulted in periapical healing as shown on a follow-up radiograph.

Adult↗

Nutrient transport across the placenta.

The placenta forms a selective barrier that functions to transport nutrients that are of critical use to the fetus. Nutrient transport across the placenta is regulated by many different active transporters found on the surface of both maternal and fetal facing membranes of the placenta. The presence of these transporters in the placenta has been implicated in the facilitation of nutrient diffusion and proper fetal growth. In this review, recent developments concerning nutrient transporters that regulate glucose, amino acid, fatty acid, and nucleoside transplacental movement are discussed.

Journal Article↗

Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity.

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O(2) supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a hexacoordinated heme. O(2) and CO bind to the heme iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold adapted to host the reversible bis-histidyl heme complex and an elongated protein matrix cavity, held to facilitate O(2) diffusion to the heme. The neuroglobin structure suggests that the classical globin fold is endowed with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.

Amino Acid Sequence↗

Preparation and properties of O-dansyltyrosine gramicidin C.

Gramicidins A, B, and C are a family of poly-peptide antibiotics which facilitate the passive diffusion of alkali cations and protons through lipid bilayer membranes. It is clear that gramicidin forms a multimeric transmembrane channel and it has been suggested that the channel is an io-conducting dimer in equilibrium on the membrane with non-conducting monomer. We describe the preparation and purification of a derivative of gramicidin C in which the phenolic hydroxyl of the tyrosine at position 11 has been esterified to 8-dimethylaminonaphthalene-1-sulfonate (dansyl). This derivative fluoresces strongly in the visible with an emission maximun in dioxane of 530 nm, an emission lifetime of 16 ns, and a quantum yield of 0.8. Veatch et al. ((1975),J. Mol. Biol. 99, 75) have shown this 0-dansyltyrosine gamicidin C to be a fully active analogue of gramicidin A in artificial lipid bilayer membranes. We here utilize this derivative to further characterize the state of aggregation and rotational mobility of the four interconvertible conformational species formed by gramicidin in nonpolar organic solvents (Veatch et al. (1974), Biochemsitry 13, 5249; Veatch and Blout (1974), Biochemistry 13, 5257). Fluorescence energy transfer from the tryptophans of gramicidin A to the 0-dansyltyrosine of this derivatives supports the conclusion that all of these gramicidin isolated species are aggregates. Decay of fluorescence polarization anisotropy measurements yield a rotational correlation time of 1 ns for the 0-dansyltyrosine chromophore in ethanol in good agreement with the more detailed information previously obtained by 13C-nuclear magnetic resonance for the monomer in dimethyl sulfoxide (Fossel et al. (1974), Biochemistry 13, 5264). However, it is likely that the chromophore has much more rotational mobility than the rest of the gramicidin molecule in the aggregated comformational states.

Amino Acids↗

Localization of D-2 dopamine receptors to intrinsic striatal neurones by quantitative autoradiography.

Recent work with positron emission and single photon emission computed tomography has demonstrated the feasibility of studying striatal dopamine receptors in the living human brain. For the proper interpretation of these studies in normal and diseased states, the cellular localization of these receptors must be definitively established. It has been claimed, on the basis of receptor binding studies with tissue homogenates in rats, that 30-50% of striatal D-2 dopamine receptors are located on axons or terminals of the corticostriatal pathway. This finding has been incorporated into major reviews and classifications of dopamine receptors. The recent development of quantitative autoradiographic methods for diffusible ligands has facilitated the study of neurotransmitter receptors in cytoarchitechtonically intact tissue. Because this technique provides the necessary anatomic resolution that is lacking in homogenate binding studies, we have used it to re-examine the localization of striatal dopamine receptors. Here we present evidence that D-2 receptors are located exclusively on kainic acid-sensitive intrinsic neuronal elements in the striatum. We report that discrete cortical ablation does not alter 3H-spiperone binding to rat striatum and thus our results do not support the existence of D-2 dopamine receptors on the terminals of the corticostriatal pathway.

Animals↗

Mechanisms of disease: psychomotor retardation and high T3 levels caused by mutations in monocarboxylate transporter 8.

The actions and the metabolism of thyroid hormone are intracellular events that require the transport of iodothyronines across the plasma membrane. It is increasingly clear that this process does not occur by simple diffusion, but is facilitated by transport proteins. Only recently have iodothyronine transporters been identified at the molecular level, of which organic anion transporting polypeptide 1C1 and monocarboxylate transporter 8 (MCT8) deserve special mention, because of their high activity and specificity for iodothyronines. Organic anion transporting polypeptide 1C1 is almost exclusively expressed in brain capillaries, and may be crucial for the transport of the prohormone T4 across the blood-brain barrier. MCT8 is also expressed in the brain--in particular, in neurons--but also in other tissues. MCT8 seems to be especially important for the uptake of active hormone T3 into neurons, which is essential for optimal brain development. T3 is produced from T4 by type 2 deiodinase in neighboring astrocytes. Neurons express type 3 deiodinase, the enzyme that terminates T3 activity. The SLC16A2 (formerly MCT8) gene is located on chromosome Xq13.2 and has recently been associated with a syndrome combining severe, X-linked, psychomotor retardation and high serum T3 levels. In over 20 families, where affected males have developed this syndrome, several mutations in MCT8 have been identified. The disease mechanism is thought to involve a defect in the neuronal entry of T3 and, therefore, in the action and metabolism of T3 in these cells. This defect results in impaired neurological development and a decrease in T3 clearance.

Amino Acid Sequence↗

The urban geography of SARS: paradoxes and dilemmas in Toronto's health care.

BACKGROUND: Severe acute respiratory syndrome (SARS) has impacted heavily on both Canada's society and its health care system. Quite unexpectedly, hospitals and health care workers became facilitators of disease diffusion. This experience has demonstrated the need for a comprehensive disease control strategy. AIMS: The aims are threefold. First, to examine the subtle and changing spatial characteristics of SARS during the short but critical period of its rapid spread in the Greater Toronto Area. Second, to make salient three paradoxes, and their associated dilemmas, faced by nurses and other health care professionals challenged with caring for the sick and containing the rapid spread of the disease. Third, to propose some ways of approaching these dilemmas, as well as some broader preventative and mitigating strategies. METHODS: The crosscutting concepts of 'clinical uncertainty' and 'flow of human contacts' are used to explain disease transmission characteristics and urban spatial diffusion and to guide the particular strategies developed. Evidence for the paper comes from public health records, governmental and non-governmental health statements and the initial epidemiological research on SARS. Direct insights are also gained from recent first-hand experiences of Toronto's health care system during the crisis. DISCUSSION: The concepts of clinical uncertainty and the flow of human contacts provide in-depth insights that complement the findings of large-scale epidemiological studies, and help operationalize their general calls for enhanced control measures. The comprehensive disease control strategy proposed includes the creation of a hospital infrastructure specific to the containment of biological threats; an advisory coalition of disease control specialists; the development of a biological threat-and-containment simulation laboratory and three specific programmes in patient safety, risk assessment and community mobilization. CONCLUSION: It is argued that containment alone, while a necessary and urgent priority, cannot be seen as an end in itself and might better be understood as one possible trajectory within a comprehensive problem-solving strategy. The experiences in Toronto may offer insights to other cities and countries that currently lack such strategies and hence may be vulnerable to similar outbreaks of dangerous infectious diseases.

Canada↗

Crystal structure of the yeast cytochrome bc1 complex with its bound substrate cytochrome c.

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins. Specific and transient complexes need to be formed between the redox partners to ensure fast turnover. In respiration, the mobile electron carrier cytochrome c shuttles electrons from the cytochrome bc1 complex to cytochrome c oxidase. Despite extensive studies of this fundamental step of energy metabolism, the structures of the respective electron transfer complexes were not known. Here we present the crystal structure of the complex between cytochrome c and the cytochrome bc1 complex from Saccharomyces cerevisiae. The complex was crystallized with the help of an antibody fragment, and its structure was determined at 2.97-A resolution. Cytochrome c is bound to subunit cytochrome c1 of the enzyme. The tight and specific interactions critical for electron transfer are mediated mainly by nonpolar forces. The close spatial arrangement of the c-type hemes unexpectedly suggests a direct and rapid heme-to-heme electron transfer at a calculated rate of up to 8.3 x 10(6) s(-1). Remarkably, cytochrome c binds to only one recognition site of the homodimeric multisubunit complex. Interestingly, the occupancy of quinone in the Qi site is higher in the monomer with bound cytochrome c, suggesting a coordinated binding and reduction of both electron-accepting substrates. Obviously, cytochrome c reduction by the cytochrome bc1 complex can be regulated in response to respiratory conditions.

Benzoquinones↗

Transfection and overexpression of the calcium binding protein calbindin-D28k results in a stimulatory effect on insulin synthesis in a rat beta cell line (RIN 1046-38).

Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.

Animals↗

Disruption of myoglobin in mice induces multiple compensatory mechanisms.

Myoglobin may serve a variety of functions in muscular oxygen supply, such as O(2) storage, facilitated O(2) diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo(-/-)) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo(-/-) mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo(-/-) mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during beta-adrenergic stimulation with dobutamine. Myo(-/-) hearts showed no alteration in contractile parameters either under basal conditions or after maximal beta-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine ((31)P-NMR), and myocardial O(2) consumption were not altered. However, coronary flow [6.4 +/- 1.3 ml.min(-1).g(-1) [wild-type (WT)] vs. 8.5 +/- 2.4 ml.min(-1).g(-1) [myo(-/-)] [and coronary reserve [17.1 +/- 2.1 (WT) vs. 20.8 +/- 1.1 (myo(-/-) ml. min(-1).g(-1) were significantly elevated in myo(-/-) hearts. Histological examination revealed that capillary density also was increased in myo(-/-) hearts [3,111 +/- 400 mm(-2) (WT) vs. 4,140 +/- 140 mm(-2) (Myo(-/-)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO(2) gradient and reduce the diffusion path length for O(2) between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

Adenosine↗