Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

Heparin binds to Leishmania donovani promastigotes and inhibits protein phosphorylation.

We show that promastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar), possess heparin receptors on their surface. From a linear Scatchard plot of the binding data obtained using [3H]heparin and viable promastigotes, one derives a binding constant of 4.7 x 10(-7) M and an estimate of 860,000 receptors per parasite. The [3H]heparin bound to parasites could not be displaced by hyaluronic acid or by three other glycosaminoglycans (dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate). It was demonstrated that exponential phase promastigotes growing in medium 199 supplemented with fetal bovine serum incorporate 35SO4 into a cell-associated macromolecule that has the properties of heparin proteoglycan. Heparin inhibits the activity of the cell-surface histone-protein kinase; incubation of viable promastigotes with [gamma-32P]ATP and MgCl2 (10 mM) in the absence and presence of heparin (0.01-0.5 mg/ml) for 10 min, followed by analysis by SDS/polyacrylamide-gel electrophoresis and autoradiography, revealed that the phosphorylation of 12 or 13 parasite proteins was inhibited by the glycosaminoglycan. These data suggest that heparin may play a role in the host-parasite relationship.     

A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Observation of a sterically unfavorable side-chain conformation in a leucyl residue: Crystal and molecular structure of L-leucyl–L-leucine · DMSO solvate

The crystal structure of a dipeptide L -leucyl–L -leucine (C₁₂H₂₄N₂O₃) has been determined. The crystals are monoclinic, space group P2₁, with a = 5.434(4) Å, b = 15.712(7) Å, c = 11.275(2) Å, β = 100.41(1)°, and Z = 2. The crystals contain one molecule of dimethyl sulfoxide (DMSO) as solvent of crystallization for each dipeptide molecule. The structure has been solved by direct methods and refined to a final R index of 0.059 for 920 reflections (sinθ/λ ? 0.60 Å^?1) with I ? 2σ (I). The trans peptide unit shows substantial degree of non-planarity (Δω = 14°). The peptide backbone adopts an extended conformation with torsion angles of ψ₁ = 138(1)°, ω₁ = 166(1)°, ?₂ = ? 149.3(7)°, ψ₂₁ = 164.2(7)°, and ψ₂₂ = ? 15(1)°. For the first leucyl residue, the side-chain conformation is specified by the torsion angles ¹χ₁ = 176.7(7)°, ¹χ₂₁ = 62(1)°, ¹χ₂₂ = ? 177.4(8)°; the second leucyl residue adopts a Sterically unfavorable conformation with ²χ₁ = 61(1)°, ²χ₂₁ = 97(1)°, and ²χ₂₂ = ?151(1)°. The packing involves head-to-tail interaction of peptide molecules and segregation of polar and nonpolar regions. The DMSO molecule is strongly hydrogen bonded to the terminal NH group. © 1994 John Wiley & Sons, Inc.     

Response of excised embryos of rice (Oryza sativa L.) to X-rays

Summary The response of rice (Oryza sativa L.) embryos to X-rays (M₁ to M₃) was studied. By means of irradiating excised embryos, both chlorophyll and macromutation were successfully induced in three genotypes of rice. However, differential responses in terms of mutation frequency, mutation spectrum and optimal levels of X-rays required for induction of mutation (chlorophyll as well as morphological) were found to exist between cultivars. In Satika and Ashkhata, LD₅₀ values and maximum induced seed sterility are concomitant to optimum level of radiation required for triggering chlorophyll mutation. However, optimum dose for induction of macromutation in Satika and Kerangserang is independent of either LD₅₀ and/or induced seed sterility.Chances of obtaining both dominant and locus specific recessive mutations in the immediate X-ray treated generation (M₁) are large. This indicates the very high degree of effectiveness of the excised embryo irradiation technique with rice.     

Ryanodine-affinity chromatography purifies 106 kD Ca release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.     

Modelling expected trout ranges under current and future water temperature regimes in the Eastern Cape,South Africa

Different values have resulted in conflicts between anglers and conservation lobbies in the management of trout in South Africa. Key to the conflict is the demarcation of boundaries to areas in which brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss currently occur, or are likely to establish following stocking for angling. To provide a longer-term perspective on these areas, we developed models to link salmonid biological thermal thresholds to elevation. These, when applied spatially using a digital elevation model with a probability of occurrence model, provided the basis for estimating potentially available thermal habitat for these two cold water species. Here, we acknowledge that other variables (stocking history; river connectivity) also play a role in understanding trout distributions. Using a simple scenario of an increase in mean daily water temperatures of 2 °C, we demonstrated that both brown and rainbow trout are likely to exhibit considerable range reductions in the future. Because it is possible that these range restrictions will result in an increasing desire to introduce trout into areas above their current distribution limits for the maintenance of angling opportunities, conservation managers should prioritise these areas, with management interventions seeking to understand what will help to limit introductions.     

The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

Background  

Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin.     

Membrane-Active Macromolecules Resensitize NDM-1 Gram-Negative Clinical Isolates to Tetracycline Antibiotics

Gram-negative ‘superbugs’ such as New Delhi metallo-beta-lactamase-1 (bla _NDM-1) producing pathogens have become world’s major public health threats. Development of molecular strategies that can rehabilitate the ‘old antibiotics’ and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards bla _NDM-1 Klebsiella pneumonia and bla _NDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards bla _NDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.