The lipopeptide antibiotic A21978C has a specific interaction with DMPC only in the presence of calcium ions

The A21978C group are lipopeptide antibiotics which kill Gram-positive bacteria only in the presence of calcium ions. The calcium requirement of the antibacterial activity of A21978C correlates well with an in vitro calcium-dependent insertion into phospholipid vesicles. In this paper the interaction of A21978C with phosphatidylcholine is investigated in mixed monomolecular films. The spontaneity of the antibiotic-lipid mixing was determined by calculating the free energy change. On a Ca2+ containing subphase there is a specific interaction between the components at all antibiotic-lipid ratios. This is not true on K+ subphases, where specific interactions never occur. On Mg2+ subphases specific interactions occur only in monolayers containing very little lipid. By analysing the fluorescence of the kynurenine residue we have followed the effects of two factors on the penetration of the antibiotic into lipid bilayer vesicles. Firstly, the phospholipid gel to liquid crystalline phase transition which in the absence of calcium leads to an exclusion of the antibiotic from the bilayer. This trend is completely reversed in the presence of Ca2+. Secondly, the role of this lipopeptide's lipid tail was clarified by use of a series of versions of increasing fatty acyl chain length. The results indicate that the interaction promoted by calcium is not simply a hydrophobic attraction between fatty acyl chains but is more likely to be a specific interaction between polar headgroups.     

Euglena sp. as a suitable source of lipids for potential use as biofuel and sustainable wastewater treatment

Prolific algal growth in sewage ponds with high organic loads in the tropical regions can provide cost-effective and efficient wastewater treatment and biofuel production. This work examines the ability of Euglena sp. growing in wastewater ponds for biofuel production and treatment of wastewater. The algae were isolated from the sewage treatment plants and were tested for their nutrient removal capability. Compared to other algae, Euglena sp. showed faster growth rates with high biomass density at elevated concentrations of ammonium nitrogen (NH₄-N) and organic carbon (C). Profuse growth of these species was observed in untreated wastewaters with a mean specific growth rate (μ) of 0.28 day^?1 and biomass productivities of 132 mg ?L^?1? day^?1. The algae cultured within a short period of 8 days resulted in the 98 % removal of NH₄-N, 93 % of total nitrogen 85 % of ortho-phosphate, 66 % of total phosphate and 92 % total organic carbon. Euglenoids achieved a maximum lipid content of 24.6 % (w/w) with a biomass density of 1.24 g ?L^?1 (dry wt.). Fourier transform infrared spectra showed clear transitions in biochemical compositions with increased lipid/protein ratio at the end of the culture. Gas chromatography and mass spectrometry indicated the presence of high contents of palmitic, linolenic and linoleic acids (46, 23 and 22 %, respectively), adding to the biodiesel quality. Good lipid content (comprised quality fatty acids), efficient nutrient uptake and profuse biomass productivity make the Euglena sp. as a viable source for biofuel production in wastewaters.     

FAF1, a gene that is disrupted in cleft palate and has conserved function in zebrafish

Cranial neural crest (CNC) is a multipotent migratory cell population that gives rise to most of the craniofacial bones. An intricate network mediates CNC formation, epithelial-mesenchymal transition, migration along distinct paths, and differentiation. Errors in these processes lead to craniofacial abnormalities, including cleft lip and palate. Clefts are the most common congenital craniofacial defects. Patients have complications with feeding, speech, hearing, and dental and psychological development. Affected by both genetic predisposition and environmental factors, the complex etiology of clefts remains largely unknown. Here we show that Fas-associated factor-1 (FAF1) is disrupted and that its expression is decreased in a Pierre Robin family with an inherited translocation. Furthermore, the locus is strongly associated with cleft palate and shows an increased relative risk. Expression studies show that faf1 is highly expressed in zebrafish cartilages during embryogenesis. Knockdown of zebrafish faf1 leads to pharyngeal cartilage defects and jaw abnormality as a result of a failure of CNC to differentiate into and express cartilage-specific markers, such as sox9a and col2a1. Administration of faf1 mRNA rescues this phenotype. Our findings therefore identify FAF1 as a regulator of CNC differentiation and show that it predisposes humans to cleft palate and is necessary for lower jaw development in zebrafish.     

Agarose as a suitable substrate for use in the study of Al dynamics in the rhizosphere

Because of experimental difficulties, few authors have studied the dynamics of aluminium in the rhizosphere. The aim of this paper is to present a suitable method for studying rhizosphere Al dynamics. It is based on the use of agarose as a substrate for plant growth. Agar and agarose gels are often used in rhizosphere studies, but most are poorly characterized and occasionally give rise to experimental artefacts, especially with low mobility elements like Al. The results reported here show that agarose is a relatively pure substrate, nearly devoid of phosphorus and other Al-complexing substances. Aqueous extracts of agarose also exhibit Al phytotoxicity equivalent to that of a nutrient solution. Since this substrate has the properties of a variable charge exchange complex, it can be considered as a physico-chemical model for organic matter. Finally, its Al adsorption capacity is high enough for the Al reserve in the substrate not to exert a limiting effect on plants and low enough to allow accurate measurement of Al depletion in the rhizophere.     

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.     

The solubility of the ribotoxin alpha-sarcin, produced as a recombinant protein in Escherichia coli, is increased in the presence of thioredoxin

The yield of purified recombinant alpha-sarcin increases approximately three- to fourfold when this toxin is co-expressed in Escherichia coli with thioredoxin. This increased production is attributed to the existence, in the presence of thioredoxin, of a reducing environment which allows rearrangement of incorrect disulphide bonds to produce the soluble native conformation. The protein thus produced retains the structural, spectroscopic and enzymatic features of the natural fungal alpha-sarcin.     

Oryzias curvinotus has DMY,a gene that is required for male development in the medaka,O. latipes

DMY is a Y-specific DM-domain gene required for male development and appears to be the sex-determining gene in the teleost fish medaka, Oryzias latipes. Although the genomic region containing DMY appears to have originated through duplication of the DMRT1 region, it is unknown when the duplication occurred. Here we show that O. curvinotus also has the DMY gene on the Y chromosome, which is homologous to the Y chromosome of medaka, and that DMY is expressed in XY embryos. A phylogenetic tree based on the amino acid sequence including the DM-domain shows that DMY was derived from DMRT1 immediately before speciation of O. latipes and O. curvinotus.     

The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia

Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.     

Biological activity of extracts isolated from a virulent strain of Sh. flexneri grown in the presence of calcium ions]

The authors carried out a comparative study of the genetically connected Sh. flexneri cultures (3 virulent strains, 3 clones of an avirulent mutant selected in the flux of an oblique light from the virulent strain, and lac+ Kcp A-hybrids obtained by crossing the initial virulent cultures with the E. coli K12 Hfr strains). The absence of any correlation between the virulence of the strains under study and the lipopolysaccharide (by rhamnose) content in the extracts from them in growing the cultures in the presence of calcium ions was noted. Toxicity of the extracts from the virulent cultures was demonstrated on a model of developing chick embryos. No such property was possessed by the extracts from avirulent strains. The extracts from the virulent cultures in nontoxic doses possessed the capacity to decrease LD50 of shigella strains used for the infection. The biologically active factor determined in the extracts from the virulent cultures apparently was not lipopolysaccharide.     

Evidence for self-association of prothrombin fragment 1 in the absence of calcium ions. Implications for the interpretation of cooperativity of calcium binding

Sedimentation equilibrium studies have demonstrated that prothrombin fragment 1 from either human or bovine plasma reversibly dimerizes in the absence of Ca2+ with an equilibrium constant of 1,000 M-1. In the presence of 10 mM Ca2+ this association constant increased to 10,000 M-1. A model for preferential binding of Ca2+ to the pre-existing dimer has been found capable of accounting quantitatively for the cooperative Ca2+ binding to this prothrombin fragment, and for the dependence of its sedimentation coefficient on protein concentration in the presence and absence of metal ion. Sedimentation equilibrium studies of intact bovine and human prothrombins have confirmed previous reports that these prothrombins dimerize. For both prothrombins the association constant is 10,000 M-1, both in the absence and presence of Ca2+.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.     

AHNAK, a protein that binds and activates phospholipase C-gamma1 in the presence of arachidonic acid.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.     

Functional evidence for the presence of a phosphoramidon-sensitive enzyme in rat brain that converts big endothelin-1 to endothelin-1

Endothelin-1 (ET-1) is produced from its precursor, big endothelin-1 (BigET-1), by a putative endothelin-converting enzyme (ECE), but it is not known whether the enzyme is present in the brain. This study was conducted to examine the central hemodynamic effects of BigET-1 and to indirectly determine the presence of an ECE in rat brain. Cardiovascular effects of centrally administered BigET-1 and ET-1 were examined in anesthetized, ventilated rats. BigET-1 (100 pmol) or ET-1 (10 pmol) applied to the IV ventricle produced similar prolonged decreases in mean arterial pressure (MAP) and renal blood flow (RBF). Thus, peak decreases with BigET-1 were (mean +/- S.E.): MAP = -35 +/- 4%; RBF = -27 +/- 5%, while those with ET-1 were: MAP = -36 +/- 5%; RBF = -29 +/- 9%. Pretreatment with phosphoramidon, a metalloprotease inhibitor (90 nmol), abolished the hemodynamic responses elicited by BigET-1 (MAP = -9 +/- 2%; RBF = -3 +/- 2%) but not those produced by ET-1. These data indicate that; i) conversion of BigET-1 to ET-1 in the brain is essential for the expression of hemodynamic actions and ii) a metalloprotease capable of converting BigET-1 to ET-1 is present in rat brain.     

Dye binding probes of lipid-binding structures. An investigation of 2-p-toluidinylnaphthylene-6-sulfonate binding to human and bovine prothrombin and fragment 1 in the presence and absence of calcium and magnesium ions

TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.     

Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor.

The amino acid L-glutamate is a major neurotransmitter at excitatory synapses within the central nervous system. Neuronal responses to glutamate are mediated by at least three receptor types, one of which is the NMDA subtype, named for its specific ligand N-methyl-D-aspartic acid. Neurotransmitter receptors are transmembrane proteins that can form ion channels upon binding a specific ligand and are involved in many physiological activities of the brain and in some neurological disorders. Elucidating the mechanisms of the formation of transmembrane receptor-channels and of receptor regulation and inhibition is necessary for understanding nervous system function and for designing potential therapeutic agents. This has been hampered by the lack of rapid reaction techniques suitable for investigating protein-mediated reactions on cell surfaces. Recently a laser-pulse photolysis technique was developed to study the chemical reactions of channel-forming receptor proteins in the microsecond-to-millisecond time region. To apply the technique to NMDA1 receptors a photolabile NMDA precursor (beta-DNB NMDA) was synthesized. In this precursor the side chain carboxylate was protected as a photosensitive 2,2'-dinitrobenzhydryl ester. Photolysis with 308 nm laser light generated free NMDA with a time constant of 4.2 +/- 0.1 microseconds at pH 7 and a photolysis quantum yield of 0.18 +/- 0.05. In rat hippocampal neurons the beta-DNB NMDA (250 microM) neither activated endogenously expressed receptors nor potentiated or inhibited the NMDA response. Equilibration of hippocampal neurons in the whole-cell current recording mode with 250 microM caged precursor followed by a pulse of 333 nm laser light resulted in a rapid current rise with a rate constant of 100 s-1 due to opening of NMDA-activated receptor-channels. The caged NMDA precursor described here now makes it possible to investigate the mechanism of NMDA receptors in the micro- to millisecond time region.