Antibiotic resistance in oral/respiratory bacteria

In the last 20 years, changes in world technology have occurred which have allowed for the rapid transport of people, food, and goods. Unfortunately, antibiotic residues and antibiotic-resistant bacteria have been transported as well. Over the past 20 years, the rise in antibiotic-resistant gene carriage in virtually every species of bacteria, not just oral/respiratory bacteria, has been documented. In this review, the main mechanisms of resistance to the important antibiotics used for treatment of disease caused by oral/respiratory bacteria--including beta-lactams, tetracycline, and metronidazole--are discussed in detail. Mechanisms of resistance for macrolides, lincosamides, streptogramins, trimethoprim, sulfonamides, aminoglycosides, and chloramphenicol are also discussed, along with the possible role that mercury resistance may play in the bacterial ecology.     

腐蚀微生物种类及腐蚀机理研究进展

目前微生物腐蚀（MIC）在工业环境中已成为普遍存在的严重问题,其是造成腐蚀损坏、设备故障和经济损失的主要原因之一.虽然部分经典的腐蚀理论能够解释一些微生物腐蚀的现象,但这些机理的片面性也逐渐暴露出来.随着对腐蚀菌种类的研究越来越多,人们对微生物腐蚀机理的认识也更加全面深入.本文重点介绍了易导致腐蚀的微生物种类及特征,如硫酸盐还原菌、硝酸盐还原菌和铁氧化菌等,并总结了微生物腐蚀机理中基于生物能学和生物电化学的最新研究进展,包括微生物胞外电子传递过程、代谢产物腐蚀和浓差电池作用等理论,为工业中厌氧及好氧条件下微生物腐蚀的诊断、预测及防治提供了理论指导.     

Characterization of a membrane calcium pathway induced by rotavirus infection in cultured cells

Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na+, K+, and Ca2+ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca2+ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca2+ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca2+, Ba2+, Sr2+, Mn2+, and Co2+. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La3+ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca2+ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca2+ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca2+ pools required for virus maturation and cell death.     

Interactions of surfactant protein D with pathogens, allergens and phagocytes

Surfactant protein D (SP-D) is considered to play an important role in innate immunity in the lungs by binding, via its multiple C-type lectin domains, to carbohydrate structures present on a range of viruses, bacteria, yeasts and fungi. The resulting agglutination of the target pathogens provides host defence which can be further enhanced by killing and clearance mechanisms mediated by phagocytic cells which carry receptors for SP-D. Recent findings suggest that SP-D, and the structurally related lung surfactant protein A (SP-A), may also modulate allergic reactions by binding certain glycosylated allergens. The finding of SP-D at a variety of other sites besides the lungs, such as the gastric mucosae, is suggestive that it may play a general protective role in several secretions.     

Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function alpha 4 beta 1 and alpha 5 beta 1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by "inside-out" mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn2+, Mg2+ and Ca2+ for alpha 4 beta 1 and alpha 5 beta 1 activation by "inside-out" mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-beta 1 integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of beta 1 integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, alpha 4 beta 1 and alpha 5 beta 1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca2+, Mg2+ or Mn2+ were able to restore adhesive functions of alpha 4 beta 1 and alpha 5 beta 1 when activated by 8A2, only Mg2+ and Mn2+ were able to support activation of alpha 4 beta 1 and alpha 5 beta 1 by cytokines. Furthermore, high concentrations of Ca2+ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn2+ and cytokines but not by 8A2. On the contrary, in the presence of both Ca2+ and Mg2+, Mn2+ had an additive effect on the activation of alpha 4 beta 1 and alpha 5 beta 1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca2+, Mg2+ and Mn2+ may modulate in vivo alpha 4 beta 1 and alpha 5 beta 1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

Nucleobase and nucleoside transport in mammalian cells

Membrane transport of nucleobases and nucleosides has been an actively pursued research field for the past 25 years. Not only are these substances of physiological interest; derivatives are in clinical use or under investigation for their pharmacological activity against viral and neoplastic disease. An understanding of the molecular pharmacology of these substances includes a detailed knowledge of how they reach their intracellular targets. Membrane transport systems which have so far been found in all cells examined play an important role in this process. Since the transporters are minor membrane components, little is known about them on a molecular basis. This review discusses methodological approaches used to measure initial rates of membrane transport and summarizes current knowledge of the various transport systems which have been characterized with these kinetic methods.     

Coatomer (COPI)-coated vesicles: role in intracellular transport and protein sorting

Coatomer-coated vesicles have been proposed to play a role in many distinct steps of intracellular transport. Coatomer potentially plays a role in forward transport from the endoplasmic reticulum to the Golgi apparatus and through the Golgi apparatus. It may also function in retrograde transport and in the endocytic pathway. There are limitations to the various approaches used to study the role of coatomer, and looking at these helps us to better define the questions that remain to be answered.     

VO2+(IV) complexes with pyruvate carboxylase: activation of oxaloacetate decarboxylation and EPR properties of enzyme-VO2+ complexes

Chicken liver pyruvate carboxylase catalyzes a nonclassical ping-pong mechanism in which the carboxylation of biotin at subsite 1 of the active site is coupled to the biotin-dependent carboxylation of pyruvate at subsite 2. The functions of two divalent cation cofactors and at least one monovalent cation cofactor in catalysis are not well understood. The oxyvanadyl cation, VO2+ does not support phosphoryl transfer at the first subsite, and uncouples the decarboxylation of oxaloacetate at subsite 2 from the formation of ATP at subsite 1. Stimulation of this oxaloacetate decarboxylase activity in the presence of substrates and cofactors of the first subsite, including VO2+, VOADP-, Pi, and acetyl CoA, suggests that these cofactors and substrates induce the movement of carboxybiotin from the second subsite to the first subsite, where it is decarboxylated. VO2+ EPR has provided evidence for enzymic and nucleotide divalent cation binding sites within the first subsite. The EPR properties of enzyme bound VO2+ were altered by bicarbonate, suggesting that this substrate ligands directly to VO2+ at the enzymic metal site. Fluorescence quenching experiments suggest that a monovalent cation may interact with bicarbonate at the first subsite as well. The results of this study provide evidence that (i) the extrinsic metal ion cofactors interact with the substrates at the first subsite, and that (ii) divalent cations play a role in coupling catalysis at the two nonoverlapping subsites by inducing the decarboxylation of carboxybiotin at the first subsite.     

Interaction between thiol-modifying agents and P1075, a K(ATP) channel opener, in rat isolated aorta

In the past year progress in the study of cationic species has been made, particularly in our understanding of the factors which control the selective recognition of biologically important cations such as ammonium, alkali and alkaline earth metal ions, and of metal ions used in biomedicine such as lanthanides and iron(III). Based on this knowledge, several new hosts with improved transport, photophysical and biological properties have been designed.     

The Na+/K(+)-pump in rat peritoneal mast cells: some aspects of regulation of activity and cellular function

The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also have important clinical implications. The aim of the present work has been to document the existence of the Na+/K(+)-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na+/K(+)-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with 86Rb+ as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE directed stimulation of the cell was used as a parameter for cellular degranulation. Histamine was measured by spectrofluorometry. The finding of an ouabain-sensitive uptake mechanism in the mast cell documents the presence of a functional Na+/K(+)-pump in this cell. The pump activity is inhibited by lanthanides and by the divalent cations calcium, magnesium, barium and strontium. The pump has a large reserve capacity which probably is caused by a low intracellular concentration of sodium. This enables the pump to respond to changes in the intracellular sodium concentration. The inhibitory effect of di- and trivalent ions on the pump activity is probably a result of the inhibitory effect of these ions on the cellular sodium uptake. The digitalis glycosides, ouabain and digoxin, but not the more lipophilic drug digitoxigenin, increase both IgE-directed and non-IgE-directed histamine release from the mast cell in a calcium-free medium, while there is no effect of digitalis glycosides in a medium containing physiologically relevant concentrations of calcium. The effect of digitalis glycosides on the histamine release is dependent on the drug concentrations used and the time of preincubation. An increase in the intracellular concentration of sodium secondary to inhibition of the Na+/K(+)-pump is the effector mechanism likely to explain the effect of digitalis glycosides on the mast cell histamine release. Increases in intracellular sodium might affect the intracellular concentration of calcium via changes in Na+/Ca(2+)-exchange. IgE-directed and non-IgE-directed stimulation of the mast cell activates the Na+/K(+)-pump. In case of compound 48/80-induced histamine release, the pump is stimulated for at least 2 hr. It is proposed, that the poststimulatory activation of the Na+/K(+)-pump is due to increased cellular sodium uptake associated with the release process. This sodium uptake may occur via Na+/Ca(2+)-exchange, Na+/H(+)-exchange, Na+/K+/2Cl(-)-cotransport or a non-selective ion channel. Besides describing aspects of the function and regulation of the Na+/K(+)-pump in the rat peritoneal mast cells, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases.     

The role of the divalent cation in the structure of the I domain from the CD11a/CD18 integrin

BACKGROUND: The integrin family of cell-surface receptors mediates a wide variety of cell-cell and cell-extracellular matrix interactions. Integrin-ligand interactions are invariably dependent on the presence of divalent cations, and a subset of integrins contain a approximately 200 amino acid inserted (I) domain that is important for ligand binding activity and contains a single divalent cation binding site. Many integrins are believed to respond to stimuli by undergoing a conformational change that increases their affinity for ligand, and there is a clear difference between two crystal structures of the CD11b I domain with different divalent cations (magnesium and manganese) bound. In addition to the different bound cation, a 'ligand mimetic' crystal lattice interaction in the CD11b I domain structure with bound magnesium has led to the interpretation that the different CD11b I domain structures represent different affinity states of I domains. The influence of the bound cation on I domain structure and function remains incompletely understood, however. The crystal structure of the CD11a I domain bound to manganese is known. We therefore set out to determine whether this structure changes when the metal ion is altered or removed. RESULTS: We report here the crystal structures of the CD11a I domain determined in the absence of bound metal ion and with bound magnesium ion. No major structural rearrangements are observed in the metal-binding site of the CD11a I domain in the absence or presence of bound manganese ion. The structures of the CD11a I domain with magnesium or manganese bound are extremely similar. CONCLUSIONS: The conformation of the CD11a I domain is not altered by changes in metal ion binding. The cation-dependence of ligand binding thus indicates that the metal ion is either involved in direct interaction with ligand or required to promote a favorable quaternary arrangement of the integrin.     

Regulation of multicatalytic enzyme activity by insulin and the insulin-degrading enzyme

The insulin-degrading enzyme (IDE) plays an important role in the cellular metabolism of insulin. Recent studies have also suggested a regulatory role for this protein in controlling the activity of cytoplasmic protein complexes, including the proteasome [multicatalytic proteinase (MCP)] and the glucocorticoid and androgen receptors. Binding of IDE to these complexes increases their activity, whereas the addition of substrates for IDE inhibits activity. This provides a potential mechanism of action for internalized insulin and other IDE substrates in the control of protein turnover. To examine further the interactions, partially purified IDE-MCP complex was treated with EDTA or EGTA, and activity was measured in the absence and presence of various divalent cations (Ca2+, Mn2+, Co2+, and Zn2+) and insulin. EDTA treatment reduced MCP activity and eliminated the effect of insulin on the complex. Divalent cations partially or completely restored MCP activity, but did not restore the effect of insulin. EGTA treatment had a lesser effect on MCP activity, but abolished insulin inhibition of activity. Divalent cations restored the insulin effect. Inhibitors of IDE also blocked the insulin effect on MCP activity, as did treatment with SDS. These findings suggest that conformational changes in the complex may play a role in the insulin control of MCP activity.     

New insights on P2X purinoceptors

Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported. A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor. Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.     

Functions of neutrophil motility of human blood: locomotion and chemotaxis in simplified systems

Polymorphonuclear leukocytes (PMN, granulocytes) employ their plasma membranes and subjacent microfilament-rich peripheral cytoplasm for such motile functions as adherence and spreading, random locomotion, chemotaxis (directed locomotion), and phagocytosis. All of these functions are preserved in certain anucleate, granule-poor, cytoplasmic fragments (cytoplasts) derived from PMN. Thus, the sensing, transduncing, and effector capacities involved in these functions remain integrated without control from nuclei or from the other cellular organelles left behind when the cytoplast forms. More recently, we have begun to examine in intact PMN the role of divalent cations, which have been thought to be essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In slide preparations under direct microscopic observation, EDTA (10 mM; to chelate divalent cations) did not impair either random locomotion or chemotaxis, nor did specific antibodies to beta-2 (CD 18) integrins or to other PMN integrins. Motile behavior appeared to benefit from the close approximation of slide and coverslip ("chimneying"). Thus, in "close quarters", PMN can generate the force for locomotion even when adhesion molecules are lacking or disabled. We relate these findings to the reported independence from integrins of PMN in certain experimental and diseases states.     

Calbindin D-28k and monoamine oxidase A immunoreactive neurons in the nucleus basalis of Meynert in senile dementia of the Alzheimer type and Parkinson''s disease

The lungs must be kept "dry" for efficient gas exchange. The mechanisms that contribute to clear alveoli from fetal lung fluid at birth are still present during adult life and allow recovery from alveolar flooding. It has recently been shown with the use of different approaches in vitro, as well as in vivo, that alveolar epithelium performs solute-coupled fluid transport. Fluid absorption from alveoli occurs chiefly as a result of active transepithelial Na+ transport. The mechanisms of Na+ transport have been partly elucidated; Na+ enters alveolar cells through apical Na+ channels and Na(+)-coupled solute transporters and is pumped out at the basolateral membrane by a Na(+)-K(+)-adenosinetriphosphatase (ATPase). Transepithelial Na+ transport and fluid absorption are stimulated by beta-adrenergic agonists, with adenosine 3',5'-cyclic monophosphate being the likely intracellular second messenger. K+ is probably secreted into alveoli because its concentration in the epithelial lining fluid is larger than expected for passive distribution. K+ channels have been described that, in conjunction with Na(+)-K(+)-ATP-ase, might provide pathways for active transport. Active proton secretion or bicarbonate absorption have been reported, which may explain the low pH of the alveolar epithelial lining fluid. It is probable that active solute transports are the main determinants of epithelial lining fluid depth and composition. A challenge for the future is to understand how this homeostasis is achieved.     

A possible role for multidrug resistance-associated protein in the secretion of basic fibroblast growth factor by osteogenic sarcoma cell line (MG-63)

Basic fibroblast growth factor (bFGF) lacks signal sequence and therefore the mechanism of its secretion is unknown. Multidrug resistance-associated protein (MRP) has been shown to transport a variety of molecules. Therefore, in this study we examined the role of MRP in the secretion of bFGF by osteogenic sarcoma MG-63 cells which spontaneously secrete bFGF. We show that MG-63 cells express MRP both at the protein and at the mRNA level. Furthermore, probenecid (a putative inhibitor of transport activity of MRP), in a concentration-dependent manner, inhibited secretion of bFGF from MG-63 cells with concomitant increase in intracellular contents of bFGF. These results suggest that MRP may have a possible role in the secretion of bFGF.     

A 1.3-A resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation

The crystal structure of an HIV-1 trans-activation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-A resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the influence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T in vivo.     

The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.     

Interactions of surfactant protein A with pathogens

The lung is an organ with a large inner surface that is continuously in contact with the environment. Infection of this organ is prevented by several mechanisms. A recently described defence system is collectin-mediated innate immunity of the lung. Collectins are multimeric proteins characterized by carbohydrate recognition domains bound to collagen stalks. Surfactant protein (SP)-A and SP-D are collectins that are present in the epithelial lining fluid of the lung. SP-A interacts with viruses, bacteria and fungi. Furthermore, SP-A binds to various other inhaled glycoconjugates. SP-A receptors on phagocytic cells have been described that are important to ensure rapid pathogen clearance. This innate defence system of the lung may be particularly important during infections in young children when the acquired immune system has not yet become fully established. Also in later life SP-A could be very important to prevent the lungs from infections by pathogens not previously encountered. In addition, SP-A may limit the inflammatory response in the lungs, thus preventing damage to the delicate lung epithelia. Recently, evidence was presented that SP-A may modulate the allergic response to various glycosylated inhaled antigens. The presence of SP-A (and SP-D) in other organs indicates that these collectins may have a general role in mucosal immunity. In this review the interactions of SP-A with a variety of pathogens and its implications are discussed.