Characterization of Caenorhabditis Elegans Lectin-Binding Mutants

We have identified 45 mutants of Caenorhabditis elegans that show ectopic surface binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA). These mutations are all recessive and define six genes: srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. Mutations in these genes fall into two phenotypic classes: srf-2, -3, -5 mutants are grossly wild-type, except for their lectin-binding phenotype; srf-4, -8, -9 mutants have a suite of defects, including uncoordinated movement, abnormal egg laying, and defective copulatory bursae morphogenesis. Characterization of these pleiotropic mutants at the cellular level reveals defects in the migration of the gonadal distal tip cell and in axon morphology. Unexpectedly, the pleiotropic mutations also interact with mutations in the lin-12 gene, which encodes a putative cell surface receptor involved in the control of cell fate. We propose that the underlying defect in the pleiotropic mutations may be in the general processing or secretion of extracellular proteins.     

Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, [gamma-32P]-gamma-[[(4-benzoylphenyl)methyl]amido]-7-methyl-GTP [Blaas et al. (1982) Virology 116, 339; abbreviated [32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP [( 125I]APTM), the monoanhydride of m7GDP with [125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for [125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with [125I]APTM. Like [32P]BPM, [125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP [( 32P]APGM), the monoanhydride of m7GDP with [32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike [32P]BPM and [125I]APTM, however, [32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.     

Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Characterization of surface alloantigens of murine neutrophils

Serological analysis of highly purified (>97%) mouse peritoneal exudate neutrophils using a protein-A rosetting technique, showed that these cells possessed the surface phenotype: Ig^–, Thy-1^–, Ly-1^–, Ly-2^–, Ly-3^–, Ly-4⁺, Ly-5⁺, Ly-6⁺, Ly-7^–, Ia^–, FcR⁺ and C3R⁺.     

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

Maternal transfer of strain-specific immunity in an invertebrate

The most celebrated component of the vertebrate immune system is the acquired response in which memory cells established during primary infection enhance the proliferation of antibodies during secondary infection. Additionally, the strength of vertebrate acquired immune responses varies dramatically depending on the infecting pathogen species or on the pathogen genotype within species. Because invertebrates lack the T-cell receptors and Major Histocompatibility Complex (MHC) molecules that mediate vertebrate adaptive immune responses, they are thought to lack adaptive immunity and be relatively unspecific in their interactions with pathogens. With only innate immunity, invertebrate hosts are believed to be nai;ve at each new encounter with pathogens. Nevertheless, some forms of facultative immunity appear to be important in insects; some individuals have enhanced immunity due to population density, and some social insects benefit when their nest-mates have been exposed to a pathogen or pathogen mimic (; see for a predation example.) Here we provide evidence for acquired strain-specific immunity in the crustacean Daphnia magna infected with the pathogenic bacteria Pasteuria ramosa. Specifically, the fitness of hosts was enhanced when challenged with a bacterial strain their mother had experienced relative to cases when mother and offspring were challenged with different strains.     

Reengineering Rate-Limiting, Millisecond Enzyme Motions by Introduction of an Unnatural Amino Acid

Rate-limiting millisecond motions in wild-type (WT) Ribonuclease A (RNase A) are modulated by histidine 48. Here, we incorporate an unnatural amino acid, thia-methylimidazole, at this site (H48C-4MI) to investigate the effects of a single residue on protein motions over multiple timescales and on enzyme catalytic turnover. Molecular dynamics simulations reveal that H48C-4MI retains some crucial WT-like hydrogen bonding interactions but the extent of protein-wide correlated motions in the nanosecond regime is decreased relative to WT. NMR Carr-Purcell-Meiboom-Gill relaxation dispersion experiments demonstrate that millisecond conformational motions in H48C-4MI are present over a similar pH range compared to WT. Furthermore, incorporation of this nonnatural amino acid allows retention of WT-like catalytic activity over the full pH range. These studies demonstrate that the complexity of the protein energy landscape during the catalytic cycle can be maintained using unnatural amino acids, which may prove useful in enzyme design efforts.     

Response of millet and sorghum to a varying water supply around the primary and nodal roots

Background and Aims

Cereals have two root systems. The primary system originates from the embryo when the seed germinates and can support the plant until it produces grain. The nodal system can emerge from stem nodes throughout the plant''s life; its value for yield is unclear and depends on the environment. The aim of this study was to test the role of nodal roots of sorghum and millet in plant growth in response to variation in soil moisture. Sorghum and millet were chosen as both are adapted to dry conditions.

Methods

Sorghum and millet were grown in a split-pot system that allowed the primary and nodal roots to be watered separately.

Key Results

When primary and nodal roots were watered (12 % soil water content; SWC), millet nodal roots were seven times longer than those of sorghum and six times longer than millet plants in dry treatments, mainly from an 8-fold increase in branch root length. When soil was allowed to dry in both compartments, millet nodal roots responded and grew 20 % longer branch roots than in the well-watered control. Sorghum nodal roots were unchanged. When only primary roots received water, nodal roots of both species emerged and elongated into extremely dry soil (0·6–1·5 % SWC), possibly with phloem-delivered water from the primary roots in the moist inner pot. Nodal roots were thick, short, branchless and vertical, indicating a tropism that was more pronounced in millet. Total nodal root length increased in both species when the dry soil was covered with plastic, suggesting that stubble retention or leaf mulching could facilitate nodal roots reaching deeper moist layers in dry climates. Greater nodal root length in millet than in sorghum was associated with increased shoot biomass, water uptake and water use efficiency (shoot mass per water). Millet had a more plastic response than sorghum to moisture around the nodal roots due to (1) faster growth and progression through ontogeny for earlier nodal root branch length and (2) partitioning to nodal root length from primary roots, independent of shoot size.

Conclusions

Nodal and primary roots have distinct responses to soil moisture that depend on species. They can be selected independently in a breeding programme to shape root architecture. A rapid rate of plant development and enhanced responsiveness to local moisture may be traits that favour nodal roots and water use efficiency at no cost to shoot growth.     

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6 g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4 h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP + ½ ADP)/(ATP + ADP + AMP)] when compared to controls. However, after 4 h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28 days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.