Computer modelling of neural tube defects

Neurulation, the curling of the neuroepithelium to form the neural tube, is an essential component of the development of animal embryos. Defects of neural tube formation, which occur with an overall frequency of one in 500 human births, are the cause of severe and distressing congenital abnormalities. However, despite the fact that there is increasing information from animal experiments about the mechanisms which effect neural tube formation, much less is known about the fundamental causes of neural tube defects (NTD). The use of computer models provides one way of gaining clues about the ways in which neurulation may be compromised. Here we employ one computer model to examine the robustness of different cellular mechanisms which are thought to contribute to neurulation. The model, modified from that of Odell et al (Odell, G.M., Oster, G., Alberch, P. and Burnside, B., (1981)) mimics neurulation by laterally propagating a wave of apical contraction along an active zone within a ring of cells. We link the results to experimental evidence gained from studies of embryos in which neurulation has been perturbed. The results indicate that alteration of one of the properties of non-neural tissue can delay or inhibit neurulation, supporting the idea, gained from observation of embryos bearing genes which predispose to NTD, that the tissue underlying the neuroepithelium may contribute to the elevation of the neural folds. The results also show that reduction of the contractile properties of a small proportion of the neuroepithelial cell population may have a profound effect on overall tissue profiling. The results suggest that the elevation of the neural folds, and hence successful neurulation, may be vulnerable to relatively minor deficiencies in cell properties.     

Chemotaxis and nod Gene Activity of Bradyrhizobium japonicum in Response to Hydroxycinnamic Acids and Isoflavonoids

For Bradyrhizobium japonicum, the chemotactic and the nod gene-inducing effects of hydroxycinnamic acids and two of their derivatives were compared with those of isoflavonoids. Only the hydroxycinnamic acids were strong chemoattractants, while the other substances tested were chemotactically inactive. Besides the known nod gene induction by isoflavonoids, a weak nod gene induction by coniferyl alcohol, chlorogenic acid, and ferulic acid was found.     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

In vitro correlation between force and energy metabolism in porcine malignant hyperthermic muscle studied by 31P NMR.

A comparative study of mechanical and energetic parameters of superfused muscle strips from normal pigs and malignant hyperthermia susceptible (MHS) pigs has been conducted. Phosphorus nuclear magnetic resonance spectroscopy at 80.9 MHz and mechanical measurements were used to assess muscle metabolic state. At rest, biceps femoris biopsies of MHS pigs displayed reduced phosphocreatine level, higher inorganic phosphate, and a more acidic internal pH. In normal stimulated fibers, caffeine infusion (8 or 16 mM) induced twitch potentiation and contracture while twitch tension was reduced and contracture more pronounced in malignant fibers. In normal and malignant fibers, calcium ionophore A23187 produced effects similar to those of caffeine, with the exception of twitch potentiation, which was not observed. With caffeine or A23187, the ATP level remained constant throughout the rest-stimulation-recovery protocol for normal and malignant fibers but phosphocreatine dropped to undetectable levels upon stimulation of malignant fibers. In both treatments some heterogeneity in the resonances of inorganic phosphate was observed in malignant fibers together with a more severe acidosis which might play a role in the impairment of the excitation-contraction process.     

Human placental sialidase complex: characterization of the 60 kDa protein that cross-reacts with anti-saposin antibodies

Sialidase isolated from human placenta is associated with several proteins including acid beta-galactosidase, carboxypeptidase, N-acetyl-alpha-galactosaminidase, and others. These proteins are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. (Biochem. Biophys. Res. Comm. 173, 449-456, 1990) as a sialidase protein: this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposins A, C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie (gamma 1).     

Fusogenic activity of SIV (simian immunodeficiency virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.     

Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complexcis-acting loci: Evidence fromDrosophila hybrids

Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.     

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.     

Growth and thiophene accumulation by hairy root cultures of Tagetes patula in media of varying initial pH

Summary Hairy roots of Tagetes patula were grown for 24 days in modified Murashige and Skoog's liquid medium at different initial pH levels of 4.0, 5.0, 5.7, 6.0 and 7.0. Irrespective of the initial pH, after 12 days, the pH of the culture medium was approximately 4.5. However the final pH, after 24 days of growth, did depend weakly on the initial pH of the medium. The biomass yield was lowest at an initial pH of 4.0, possibly due to lower utilization of ammonium at this pH. Similar patterns of thiophene accumulation was observed at all pH levels tested. Maximum thiophene accumulation occurred in root cultures which were 12–16 days old.Abbreviations BBTOH 5-(4-hydroxy-1-butenyl)-2,2-bithienyl - BBTOAc 5-(4-acetoxy-1-butenyl)-2,2-bithienyl - BBT 5-(3-buten-1-ynyl)-2,2-bithienyl - MS Murashige and Skoog's nutrient medium - B5 Gamborg's B5 nutrient salts - HPLC High pressure liquid chromatography