The cystic fibrosis transmembrane conductance regulator (CFTR) and its stability

The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the disease cystic fibrosis (CF). It is a membrane protein belonging to the ABC transporter family functioning as a chloride/anion channel in epithelial cells around the body. There are over 1500 mutations that have been characterised as CF-causing; the most common of these, accounting for ~70 % of CF cases, is the deletion of a phenylalanine at position 508. This leads to instability of the nascent protein and the modified structure is recognised and then degraded by the ER quality control mechanism. However, even pharmacologically ‘rescued’ F508del CFTR displays instability at the cell’s surface, losing its channel function rapidly and it is rapidly removed from the plasma membrane for lysosomal degradation. This review will, therefore, explore the link between stability and structure/function relationships of membrane proteins and CFTR in particular and how approaches to study CFTR structure depend on its stability. We will also review the application of a fluorescence labelling method for the assessment of the thermostability and the tertiary structure of CFTR.     

Oxytoxins, bioactive molecules produced by the marine opisthobranch mollusc Oxynoe olivacea from a diet-derived precursor

The ethereal extract of the mucous secretion from the opisthobranch mollusc Oxynoe olivacea was examined and found to contain two novel ichthyotoxic compounds, named oxytoxin 1 and 2 (1,2). The structures of 1 and 2 are closely related to the metabolites previously isolated from the alga Caulerpa prolifera. The activity of the most stable compound was studied in order to investigate the possibility of a further biological role for these metabolites, which represent an uncommon example of bioactive molecules produced in vivo from a dietary precursor.     

Lipopolysaccharide-binding molecules: transporters, blockers and sensors

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, can be beneficial to the host by activating the innate immune system, or harmful, by inducing inflammation, disseminated intravascular coagulation, multiple organ failure, shock and often death. On the bacteria, and in host biological fluids and cells, LPS is never free but constantly attached to cognate-binding proteins. Understanding how LPS is transported and further recognized by sensors able to deliver a signal, or by inactivating molecules able to neutralize its biological effects, is an important goal. This review describes the large panel of peptides and proteins reported to associate with LPS, and provides information on their origin, their structure and the location of amino acid residues involved in their interaction with LPS. A better understanding of the mode of recognition of LPS by cognate proteins prompted many laboratories to design on a rational basis synthetic molecules which can be used to detect low amounts of endotoxin, or to act as efficient blockers of in vitro and in vivo responses to LPS.Received 15 January 2004; received after revision 20 February 2004; accepted 25 February 2004     

Proteases and protein degradation in Escherichia coli.

In E. coli, protein degradation plays important roles in regulating the levels of specific proteins and in eliminating damaged or abnormal proteins. E. coli possess a very large number of proteolytic enzymes distributed in the cytoplasm, the inner membrane, and the periplasm, but, with few exceptions, the physiological functions of these proteases are not known. More than 90% of the protein degradation occurring in the cytoplasm is energy-dependent, but the activities of most E. coli proteases in vitro are not energy-dependent. Two ATP-dependent proteases, Lon and Clp, are responsible for 70-80% of the energy-dependent degradation of proteins in vivo. In vitro studies with Lon and Clp indicate that both proteases directly interact with substrates for degradation. ATP functions as an allosteric effector promoting an active conformation of the proteases, and ATP hydrolysis is required for rapid catalytic turnover of peptide bond cleavage in proteins. Lon and Clp show virtually no homology at the amino acid level, and thus it appears that at least two families of ATP-dependent proteases have evolved independently.     

Oxytoxins,bioactive molecules produced by the marine opisthobranch molluscOxynoe olivacea from a diet-derived precursor

Summary The ethereal extract of the mucous secretion from the opisthobranch molluscOxynoe olivacea was examined and found to contain two novel ichthyotoxic compounds, named oxytoxin 1 and 2 (1, 2). The structures of1 and2 are closely related to the metabolites previously isolated from the algaCaulerpa prolifera. The activity of the most stable compound was studied in order to investigate the possibility, of a further biological role for these metabolites, which represent an uncommon example of bioactive molecules produced in vivo from a dietary precursor.     

Sphingolipids in mammalian cell signalling

Sphingolipids and their metabolites, ceramide, sphingosine and sphingosine-1-phosphate, are involved in a variety of cellular processes including differentiation, cellular senescence, apoptosis and proliferation. Ceramide is the main second messenger, and is produced by sphingomyelinase-induced hydrolysis of sphingomyelin and by de novo synthesis. Many stimuli, e.g. growth factors, cytokines, G protein-coupled receptor agonists and stress (UV irradiation) increase cellular ceramide levels. Sphingomyelin in the plasma membrane is located primarily in the outer (extracellular) leaflet of the bilayer, whilst sphingomyelinases are found at the inner (cytosolic) face and within lysosomes/endosomes. Such cellular compartmentalisation restricts the site of ceramide production and subsequent interaction with target proteins. Glycosphingolipids and sphingomyelin together with cholesterol are major components of specialised membrane microdomains known as lipid rafts, which are involved in receptor aggregation and immune responses. Many signalling molecules, for example Src family tyrosine kinases and glycosylinositolphosphate-anchored proteins, are associated with rafts, and disruption of these domains affects cellular responses such as apoptosis. Sphingosine and sphingosine-1-phosphate derived from ceramide are also signalling molecules. In particular, sphingosine-1-phosphate is involved in proliferation, differentiation and apoptosis. Sphingosine-1-phosphate can act both extracellularly through endothelial-differentiating gene (EDG) family G protein-coupled receptors and intracellularly through direct interactions with target proteins. The importance of sphingolipid signalling in cardiovascular development has been reinforced by recent reports implicating EDG receptors in the regulation of embryonic cardiac and vascular morphogenesis. Received 16 May 2001; received after revision 29 June 2001; accepted 3 July 2001     

The functioning and interrelationships of blood capillaries and lymphatics

Summary the structure and function of blood capillaries, as related to permeability, depends on tight, close and (in injured vessels) open junctional regions, small vesicles, vacuoles (in injured vessels) and fenestrae. The basement membrane presents a hindrance to the larger macromolecules, at high flow rates, but not to small molecules. The connective tissue channels are probably the paths by which macromolecules, and most of the small ones, pass from the arterial-limbs to the venous ones, and to the lymphatics. In some regions these channels are grouped in special systems: the prelymphatics. The initial lymphatics take up material via open junctions, which close during tissue-compression. The collecting lymphatics retain the lymph because they do not have open junctions.In the close junctional regions the motive force for water flow is the result of Starling's forces; diffusion is very important for other small molecules. The small vesicles transport macromolecules slowly by Brownian motion, as may the vacuoles, but possibly these latter are moved actively.There is much evidence that colloids can develop high effective osmotic pressures even across pores much larger than their molecules, and that proteins can be dragged up a concentration gradient by the resultant fluid flow. On the basis of this, hypotheses have been developed about the functioning of venous-limb fenestrae and the initial lymphatics, for which there is much theoretical, in vitro, and in vivo evidence. Thus, in fenestrae and regions there is held to be a large local circulation through the tissues, of which a quantitatively small, but qualitatively vital, part goes to the lymphatics. Material is considered usually to enter these latter because of the relative concentration of the lymph.It is becoming increasingly evident that in the study of the microvasculature, as with other systems, there is much to be gained by quantifying fine structural observations and by combining and contrasting this data, via physical laws, with that obtained by other methods where the characteristics of whole organs and regions are studied. Thus one can obtain interrelated information, which is not possible by either method alone, and which gives us a vital, comprehensive, perspective of the ways in which whole systems function, and how different systems interact. In this paper I shall show how this approach has yielded much that is new about the functioning of different kinds of blood capillaries, of the tissue channels, of the whole lymphatic system, and of the ways they affect each other.Most of the author's work reported here was financed by the Australian Research Grants Committee.     

Lipid transfer in plants

Summary Plant cells contain cytosolic proteins, called lipid transfer proteins (LTP), which are able to facilitate in vitro intermembrane transfer of phospholipids. Proteins of this kind from three plants, purified to homogeneity, have several properties in common: molecular mass around 9 kDa, high isoelectric point, lack of specificity for phospholipids, and binding ability for fatty acids. The comparison of their amino acid sequences revealed striking homologies and conserved domains which are probably involved in their function as LTPs. These proteins could play a major role in membrane biogenesis by conveying phospholipids from their site of biosynthesis to membranes unable to form these lipids. Immunochemical methods were used to establish an in vivo correlation between membrane biogenesis and the level of LTP or the amount of LTP synthesized in vitro from mRNAs. The recent isolation of a full-length cDNA allows novel approaches to studying the participation of LTPs in the biogenesis of plant cell membranes.     

Protein folding in membranes

Separation of cells and organelles by bilayer membranes is a fundamental principle of life. Cellular membranes contain a baffling variety of proteins, which fulfil vital functions as receptors and signal transducers, channels and transporters, motors and anchors. The vast majority of membrane-bound proteins contain bundles of α-helical transmembrane domains. Understanding how these proteins adopt their native, biologically active structures in the complex milieu of a membrane is therefore a major challenge in today’s life sciences. Here, we review recent progress in the folding, unfolding and refolding of α-helical membrane proteins and compare the molecular interactions that stabilise proteins in lipid bilayers. We also provide a critical discussion of a detergent denaturation assay that is increasingly used to determine membrane-protein stability but is not devoid of conceptual difficulties.     

Proteases and protein degradation inEscherichia coli

InE. coli, protein degradation plays important roles in regulating the levels of specific proteins and in eliminating damaged or abnormal proteins.E. coli possess a very large number of proteolytic enzymes distributed in the cytoplasm, the inner membrane, and the periplasm, but, with few exceptions, the physiological functions of these proteases are not known. More than 90% of the protein degradation occurring in the cytoplasm is energy-dependent, but the activities of mostE. coli proteases in vitro are not energy-dependent. Two ATP-dependent proteases, Lon and Clp, are responsible for 70–80% of the energy-dependent degradation of proteins in vivo. In vitro studies with Lon and Clp indicate that both proteases directly interact with substrates for degradation. ATP functions as an allosteric effector promoting an active conformation of the proteases, and ATP hydrolysis is required for rapid catalytic turnover of peptide bond cleavage in proteins. Lon and Clp show virtually no homology at the amino acid level, and thus it appears that at least two families of ATP-dependent proteases have evolved independently.     

Introduction: lipids as regulators of cell function

It is becoming increasingly clear that lipids are key regulators of cellular function and that these effects are quite diverse. First, the lipid environment in the cellular membrane bilayer is important in maintaining the normal function of receptors, enzymes, transporters and so on that are localized in the membrane. Phosphoinositides are important regulators of signalling molecules. Lipid metabolites formed by a number of enzymes including the cyclooxygenases, lipoxygenases and P450s also mediate important cellular functions. Fatty acids and lipid metabolites can also activate the nuclear peroxisome proliferator-activated receptors. Finally, a wide variety of lipid molecules are generated nonenzymatically by free-radical mechanisms that also exert potent biological effects in a wide variety of organs. Presented are a series of eight reviews that broadly cover all of these topics in some detail.     

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

Deoxyribozymes (DNA enzymes; DNAzymes) are catalytic DNA sequences. Using the technique of in vitro selection, individual deoxyribozymes have been identified that catalyze RNA cleavage, RNA ligation, and a growing range of other chemical reactions. DNA enzymes have been used in vitro for applications such as biochemical RNA manipulation and analytical assays for metal ions, small organic compounds, oligonucleotides, and proteins. Deoxyribozymes have also been utilized as in vivo therapeutic agents to destroy specific mRNA targets. Although many conceptual and practical challenges remain to be addressed, deoxyribozymes have substantial promise to contribute meaningfully for applications both in vitro and in vivo.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells

Summary The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Electrophysiology of mammalian hypothalamic and interpeduncular neurons in vitro.

Electrical activities of the hypothalamic and interpeduncular neurons were studied in vitro in brain slices prepared from the guinea-pig brain stem. Neurons preserved resting membrane potentials comparable to those of neurons in vivo, responded to stimulation of the afferent fibres, and retained stable spontaneous firings for more than several hours.     

Electrophysiology of mammalian hypothalamic and interpeduncular neurons in vitro

Summary Electrical activities of the hypothalamic and interpeduncular neurons were studied in vitro in brain slices prepared from the guinea-pig brain stem. Neurons preserved resting membrane potentials comparable to those of neurons in vivo, responded to stimulation of the afferent fibres, and retained stable spontaneous firings for more than several hours.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells.

The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

车辆转弯制动ABS模糊控制及稳定性仿真分析

在Matlab／Simlink下建立了八自由度车辆系统的仿真模型。采用模糊控制方法,设计了基于滑移率的ABS控制算法。针对车辆转弯制动工况,对15m／s、20m／s、25m／s三种车速进行了仿真分析。仿真结果表明所建立的ABS控制器能有效提高车辆的制动性能以及中低车速下的稳定性,但定性分析表明车辆在高速工况下出现不稳定现象。为此利用稳定性判定式,对车辆转弯制动ABS控制下的稳定性做了定量判定。判定结果表明,高速时车辆仅有ABS控制并不能达到稳定性控制的要求。     

Effects of estradiol-7 alpha-butyric acid on hypothalamus cells]

When Rat uterus was incubated at 37 degrees with estradiol-7 alpha-butyric acid (OII-7 alpha-bu), no interference was observed with the intracellular estradiol receptors. In addition, OII-7 alpha-bu did not display estrogenic effect such as in vivo inhibition of LH secretion in Rat and in vitro increased activity of the enzyme ornithine-decarboxylase in the chick oviduct. Contrary to these negative findings, we have observed preoptic and septal cells in the guinea pig where micro-iontophoresis of OII-7 alpha-bu triggers changes of the electric activity within a second. We submit therefore, that this latter response is due to an interaction between the acid estrogen and the neuron membrane.     

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.