Application of SVM to predict membrane protein types

As a continuous effort to develop automated methods for predicting membrane protein types that was initiated by Chou and Elrod (PROTEINS: Structure, Function, and Genetics, 1999, 34, 137-153), the support vector machine (SVM) is introduced. Results obtained through re-substitution, jackknife, and independent data set tests, respectively, have indicated that the SVM approach is quite a promising one, suggesting that the covariant discriminant algorithm (Chou and Elrod, Protein Eng. 12 (1999) 107) and SVM, if effectively complemented with each other, will become a powerful tool for predicting membrane protein types and the other protein attributes as well.     

Determination of the Role of Cold Acclimation-Induced Diverse Changes in Plant Cells from the Viewpoint of Avoidance of Freezing Injury

Received 4 January 1999/ Accepted in revised form 7 April 1999     

Inhibitory Effect of Sucrose on the Plasma Membrane H+ Pump Activity and Possible Involvement of the C-terminal Region in the Sucrose Effect

+ pump activity is unclear. In the plasma membrane vesicles from rye leaves high concentrations of sucrose had significant inhibitory effects on the plasma membrane H⁺ pump activity. Fusicoccin and trypsin treatment experiments suggest that the C-terminal region of the H⁺ pump may partially but significantly be involved in the sucrose effect. Received 20 August 1999/ Accepted in revised form 27 December 1999     

Effect of solution pH on protein transport through ultrafiltration membranes

Although a number of previous studies have demonstrated that solution pH can have a dramatic effect on protein transport through ultrafiltration membranes, the exact origin of this behavior has been unclear. Experimental data were obtained for the transport of a broad range of proteins with different surface charge and molecular weight. The effective hydrodynamic size of the proteins was evaluated using size‐exclusion chromatography. The membrane charge, both before and after exposure to a given protein, was evaluated using streaming potential measurements. In most cases, the electrostatic interactions were dominated by the distortion of the electrical double layer surrounding the protein, leading to a distinct maximum in protein transmission at the protein isoelectric point. Attractive electrostatic interactions did occur when the protein and membrane had a large opposite charge, causing a second maximum in transmission at a pH between the isoelectric points of the protein and membrane. The sieving data were in good agreement with theoretical calculations based on available models for the partitioning of charged solutes in cylindrical pores. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 27–37, 1999.     

Sphingomonads involved in the biodegradation of xenobiotic polymers

Sphingomonads involved in the microbial degradation of xenobiotic polymers are introduced. The metabolism of polyethylene glycol was the primary focus of the study. Several others, including polyvinyl alcohol, polyethylene and polyaspartate were also studied. It is suggested that these xenobiotic polymers are metabolized by intracellular enzymes located in the periplasmic space or bound to membranes, indicating that transport of these polymers through outer membranes is requisite for their metabolism. Involvement of specific membrane structures of sphingomonads such as unusual sphingolipids is suggested for membrane transport of xenobiotic compounds, especially hydrophobic materials. Received 01 May 1999/ Accepted in revised form 17 July 1999     

Chiral phase partitioning by enantioselective complexation: Characterisation by the semi‐empirical application of regular solution theory

The thermodynamics underlying enantioselective complexation and partitioning behaviour are poorly understood. This paper presents a model that decouples the effects of enantioselective complexation and subsequent diastereomer partitioning. Regular solution theory is applied in a semi‐empirical manner to describe the diastereomer partitioning process, which is reported to be governed by hydrophobic interactions. The model was shown to give a good fit to experimental partitioning for the enantioselective extraction of phenylalanine isomers by two chiral extractants; a modified amino acid [copper (II) N‐decyl‐(L)‐hydroxyproline] and a chiral crown ether [(S)‐bis(phenylnaphtho)‐20‐crown‐6]. A variety of aliphatic and aromatic solvents were tested. The predicted and observed experimental enantioselectivities were found to vary exponentially with the difference in the solubility parameters of the aqueous and organic phases and with those of the two diastereomeric complexes formed. This model provides the basis for a better understanding of enantioselective partitioning effects. Chirality 11:241–248, 1999. © 1999 Wiley‐Liss, Inc.     

Probing the stability of S-layer-supported planar lipid membranes

Isolated protein subunits of the crystalline bacterial cell surface layer (S-layer) of Bacillus coagulans E38-66 have been recrystallized on one side of planar black lipid membranes (BLMs) and their influence on the electrical properties, rupture kinetics and mechanical stability of the BLM was investigated. The effect on the boundary potential, the capacitance or the conductance of the membrane was negligible whereas the mechanical properties were considerably changed. The mechanical stability was characterized by applying voltage pulses or ramps to induce irreversible rupture. The amplitude of the voltage pulse leading to rupture allows conclusions on the ability of membranes to resist external forces. Surprisingly, these amplitudes were significantly lower for composite S-layer/lipid membranes compared to undecorated BLMs. In contrast, the delay time between the voltage pulse and the appearance of the initial defect was found to be drastically longer for the S-layer-supported lipid bilayer. Furthermore, the kinetics of the rupture process was recorded. Undecorated membranes show a fast linear increase of the pore conductance in time, indicating an inertia-limited defect growth. The attachment of an S-layer causes a slow exponential increase in the conductance during rupture, indicating a viscosity-determined widening of the pore. In addition, the mechanical properties on a longer time scale were investigated by applying a hydrostatic pressure across the BLMs. This causes the BLM to bulge, as monitored by an increase in capacitance. Compared to undecorated BLMs, a significantly higher pressure gradient has to be applied on the S-layer face of the composite BLMs to observe any change in capacitance. Received: 4 May 1999 / Revised version: 1 July 1999 / Accepted: 1 July 1999     

Lipid domains in the exoplasmic and cytoplasmic leaflet of the human erythrocyte membrane: a spin label approach

The existence of different lipid domains in the monolayers of the human erythrocyte membrane was investigated at 4 °C by employing spin-labelled phospholipid analogues. Spectra of analogues located exclusively either in the exoplasmic or in the cytoplasmic leaflet of erythrocyte membranes were recorded. Spectra were simulated by variation of order parameter describing the average amplitude of motion of the long molecular axis of the nitrogen 2pπ orbital of the spin label and of the respective correlation times. For both leaflets at least three components were required to fit the experimental spectra, differing mainly in the order parameter. While the parameters of each component are not very different between both membrane halves, the relative contribution of each component to the spectrum is different between the exoplasmic and cytoplasmic leaflet. The order parameter of the most fluid component, presumably resembling the lipid bulk phase, is smaller in the cytoplasmic leaflet in comparison to the exoplasmic one. The lateral coexistence of different lipid domains in the human red blood cell membrane is concluded. The molecular nature of those domains is discussed. Received: 6 November 1998 / Revised version: 25 January 1999 / Accepted: 29 January 1999     

A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori. Received: 10 May 1999 / Accepted: 7 July 1999     

Chemical structure and function of glycosphingolipids of Sphingomonas spp and their distribution among members of the α-4 subclass of Proteobacteria

Sphingomonas spp are phylogenetically placed in the α-4 subclass of Proteobacteria. They have glycosphingolipids (GSL) in their membranes instead of lipopolysaccharide (LPS) as in other Gram-negative bacteria. S. paucimobilis, the type species of the genus, has GSL-1, which contains only glucuronic acid (GlcA) as a sugar moiety, and GSL-4A, which contains a tetrasaccharide including GlcA. GSL-1 and GSL-4A form the outer membrane of S. paucimobilis with outer membrane proteins and phospholipids. In the outer membrane, GSLs are assumed to locate and function as does the LPS of other Gram-negative bacteria. Sphingomonas spp closely related to the type species contain both GSL-1 and the oligosaccharide-type GSL such as GSL-4A, but other Sphingomonas spp and other genera in the α-4 subclass of Proteobacteria contain only GSL-1. Structural variations of fatty acids and dihydrosphingosines in the GSL-1 are presented. Received 19 April 1999/ Accepted in revised form 18 June 1999     

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5‐(octa‐decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity. Mol. Reprod. Dev. 52:207–215, 1999. © 1999 Wiley‐Liss, Inc.     

Requirement of sperm‐oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5 ± 0.6 and 4.3 ± 0.6 for ICSI; 3.0 ± 0.3 and 3.8 ± 0.4 for activated and 0.2 ± 0.1 and 0.6 ± 0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore‐puromycin‐activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes. Mol. Reprod. Dev. 52:183–188, 1999. © 1999 Wiley‐Liss, Inc.     

Alterations of Intracellular pH in Response to Low Temperature Stresses

+ -ATPase is one of the primary cellular events directly resulting from cold exposure. We demonstrate here that cold-induced inactivation of the proton translocating enzyme is closely linked to the rapid acidification of the cytoplasm and the concomitant alkalization of the vacuoles, suggesting an important role of the enzyme in maintaining homeostasis of the cellular pH in a cold environment. The stability of the vacuolar H⁺-ATPase to cold both in vivo and in vitro is distinctly different between species sensitive and insensitive to cold. These findings provide further insight into the way in which the vacuolar H⁺-ATPase is involved in cold adaptation of plants. In addition, the temperature reduction and the concentration of the cytoplasm as a consequence of freeze-induced dehydration may also result in changes in the cellular pH. In fact, we demonstrate here that the cytoplasm is markedly acidified upon freezing; in particular, in cells of less hardy plants. Freeze-induced acidification is presumably due to changes in the physico-chemical properties of the cytoplasm and the changes in the permeability of the vacuolar membrane both of which result from severe dehydration. The physiological significance of freeze-induced acidification of the cytoplasm is discussed. Received 26 March 1999/ Accepted in revised form 30 March 1999     

Demonstration of efficient trichloroethylene biodegradation in a hollow‐fiber membrane bioreactor

Rapid cometabolism of trichloroethylene (TCE) by pure cultures of Methylosinus trichosporium OB3b PP358 was demonstrated in a two‐stage hollow‐fiber membrane bioreactor over the course of 3 weeks. PP358 was grown in a continuous‐flow chemostat and circulated through the shell of a hollow‐fiber membrane module (HFMM), while TCE contaminated water (160 to 1450 μg/L) was pumped through the fiber lumen (fiber interior). In parallel‐flow HFMM biological experiments, 82% to 89% of the influent TCE was removed from the lumen (5.1‐min residence time) with 99% of the transferred TCE undergoing biodegradation. Biological experiments in a larger capacity baffled radial‐flow HFMM resulted in 66% to 99% TCE transferred and 93% to 96% TCE biodegradation at lumen residence times of between 1.5 and 3.7 min. Biodegradation was maintained throughout the experiments at pseudo‐first‐order biodegradation rate constants of 0.41 to 2.8 L/mg TSS/day. Best‐fit computer modeling of the baffled radial‐flow biological process estimated mass transfer coefficients as large as 2.7 × 10⁻² cm/min. The computer model was also shown to simulate the experimental results quite well. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 681–692, 1999.     

The study of the conformation and interaction of two tachykinin peptides in membrane mimicking systems by NMR spectroscopy and pulsed field gradient diffusion

Pulsed‐field gradient diffusion has been used to study the binding of two tachykinin peptides, [Tyr⁸]‐substance P (SP) and [Tyr⁰]‐neurokinin A (NKA) to two membrane‐mimicking micelles, dodecylphosphocholine, and sodium dodecylsulfate. The structure of these peptides bound to the micelles have also been studied by using two‐dimensional nmr and restrained simulated annealing calculations. No major difference in the structures of each peptide in the two micellar media was found. The difference between the micelle‐bound structure of [Tyr⁸]SP and that of SP was also minor. The longer helical conformation on the C‐terminus for [Tyr⁰]NKA was observed, compared with that for NKA. The relationship between the difference in the biological potencies of [Tyr⁸]SP and SP and the differences in their structure, especially the interaction of the side chains of the two aromatic residues, and the difference in their binding affinities to membrane was discussed. In addition, differences between the result of restrained molecular dynamics simulations of [Tyr⁸]SP in the presence of an explicit micelle and the present results were observed and discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 555–568, 1999     

S‐perindopril assay using a potentiometric,enantioselective membrane electrode

A potentiometric, enantioselective membrane electrode based on graphite paste (graphite powder and paraffin oil) has been constructed. The graphite paste is impregnated with a 10⁻³ mol/L 2‐hydroxy‐3‐trimethylammoniopropyl‐β‐cyclodextrin (as chloride salt) solution. The potentiometric, enantioselective membrane electrode can be used reliably for enantiopurity tests of S‐perindopril using a chronopotentiometric (zero current) technique, in the 10⁻⁵–10⁻² mol/L concentration range (detection limit 5 × 10⁻⁶ mol/L), with an average recovery of 99.58% (RSD = 0.33%). The enantioselectivity was determined over R‐perindopril and d ‐proline. The response characteristics of the enantioselective, potentiometric membrane electrode were also determined for R‐perindopril. It was shown that l ‐proline is the main interfering compound. The surface of the electrode can be regenerated simply by polishing, obtaining a fresh surface ready to be used in a new assay. Chirality 11:631–634, 1999. © 1999 Wiley‐Liss, Inc.     

Analysis of the role of detergent mixtures on the crystallization of the reaction center of Photosystem II

We have recently reported the crystallization of the reaction center of Photosystem II in the presence of detergent mixtures [Adir N (1999) Acta Crystallogr D Biol Crystallogr D55: 891–894]. We have used high performance liquid chromatography, dynamic light scattering, native gel electrophoresis and thermoluminescence measurements to characterize the interaction between these detergent mixtures and RC II, to try and understand their role in the crystallization process. Size exclusion HPLC and dynamic light scattering confirmed that the isolated RC II used for crystallization was exclusively monomeric. Dynamic light scattering measurements show that the detergent mixtures formed single micelles within a limited range of hydrodynamic radii. Both size exclusion HPLC and dynamic light scattering were used to follow the interaction between the detergent mixtures and monomeric RC II. These techniques revealed a decrease in the detergent mixture treated RC II particle size (with respect with the untreated RC II), and that RC II from solubilized crystals contained particles of the same size. Native gel electrophoresis showed that this change in apparent size is not due to the disintegration of the internal structure of the RC II complex. Thermoluminescence measurements of solubilized RC II crystals showed charge recombination from the S_2,3Q_A ⁻ state, indicating that RC II remains functionally viable following detergent mixture treatment and crystallization. The role of the detergent mixtures in the crystallization of RC II is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interaction of peptides and proteins with bacterial surface glycolipids: a comparison of glycosphingolipids and lipopolysaccharides

The bacterial cell wall of Gram-negative bacteria consists, in addition to the cytoplasmic membrane, of another permeability barrier, the outer membrane. The lipid distribution between both sides of this membrane is strictly asymmetric. The outer leaflet is made up of glycolipids, usually lipopolysaccharides. In Sphingomonas spp glycosphingolipids were found to substitute for lipopolysaccharides. In this review, it is shown by an electrophysiological approach that glycosphingolipid can replace lipopolysaccharide with respect to its function as antigenic surface structure as well as to its contribution to the diffusion barrier properties of the outer membrane. This review is focused on: (i) the function of porins, as examples of transmembrane proteins, in the different glycolipid environments; (ii) the interaction of polymyxin B with the outer membrane, as an example of polycationic antibacterial peptides; and (iii) the activation of the human complement system by lipopolysaccharides and glycosphingolipids. Received 14 April 1999/ Accepted in revised form 19 June 1999     

Ultrastructural changes in hamster spermatogenic cell nuclei after incorporation into homologous oocytes by electrofusion

Isolated hamster spermatogenic cells at various stages of spermatogenesis were examined by thin‐sectioning techniques after electrofusion with activated homologous oocytes. The nuclei and attached organelles of the cells remained almost unchanged within the ooplasm three to five hours after the fusion pulse, by which time the oocytes had developed to the pronuclear stage. Only the spherical nuclei of primary spermatocytes and early spermatids in the Golgi and cap phases underwent modifications in their fine structures. They gained the morphological characteristics of well‐developed mammalian pronuclei; e.g., electron‐dense round nucleolus‐like bodies and blebbing of the nuclear envelope. In contrast, the elongated nuclei of later spermatids in the acrosome and maturation phases retained their original features, except that their acrosomes were deformed. Thus, ooplasm‐mediated transformation within activated oocytes at the pronuclear stage occurred only in nuclei containing dispersed chromatin and having nuclear pores in the envelope. Mol. Reprod. Dev. 52:66–73, 1999. © 1999 Wiley‐Liss, Inc.