Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

Lithium induces autophagy by inhibiting inositol monophosphatase

Macroautophagy is a key pathway for the clearance of aggregate-prone cytosolic proteins. Currently, the only suitable pharmacologic strategy for up-regulating autophagy in mammalian cells is to use rapamycin, which inhibits the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Here we describe a novel mTOR-independent pathway that regulates autophagy. We show that lithium induces autophagy, and thereby, enhances the clearance of autophagy substrates, like mutant huntingtin and alpha-synucleins. This effect is not mediated by glycogen synthase kinase 3beta inhibition. The autophagy-enhancing properties of lithium were mediated by inhibition of inositol monophosphatase and led to free inositol depletion. This, in turn, decreased myo-inositol-1,4,5-triphosphate (IP3) levels. Our data suggest that the autophagy effect is mediated at the level of (or downstream of) lowered IP3, because it was abrogated by pharmacologic treatments that increased IP3. This novel pharmacologic strategy for autophagy induction is independent of mTOR, and may help treatment of neurodegenerative diseases, like Huntington's disease, where the toxic protein is an autophagy substrate.     

Enumeration of anaerobic oxalate-degrading bacteria in the ruminal contents of sheep

Abstract Concentrations of oxalate-degrading anaerobes in ruminal contents of sheep were determined from counts of colonies producing clear zones on a calcium oxalate medium (D agar with 7 mM CaCl₂). Viable counts of oxalate degraders from a 55-kg sheep fed a diet containing 32% halogeton (4.6% oxalate) averaged 2.6 × 10⁶/ g (dry weight). When the halogeton concentration in the diet was reduced to 16%, counts of oxalate degraders decreased nearly 300-fold. Oxalate-degrading isolates from this sheep were similar to OxB, the type strain of Oxalobacter formigenes . When a 45-kg sheep was fed diets containing 2.2, 1.5, and 0.8% oxalate, viable counts of oxalate degraders (enumerated on D agar with 14 mM CaCl₂ and 20% filter-sterilized ruminal fluid) represented 0.85, 0.52, and 0.06% of the total viable population, respectively; total viable counts were essentially unchanges by these concentrations of dietary oxalate. Similar percentages of oxalate degraders were also observed when a 23-kg sheep was fed diets containing 1.5 or 0.8% oxalate. This report presents the first direct measurements of the concentrations of oxalate-degrading bacteria in the rumen and supports the concept that the availability of oxalate in the diet influences the proportion of oxalate-degrading bacteria in the rumen     

Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases

Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Below-ground herbivory promotes soil nutrient transfer and root growth in grassland

Extremely little is known about the ecosystem-level implications of below-ground herbivory, which often represents the dominant form of consumption of primary productivity. We provide the first empirical evidence that low levels of below-ground herbivory may promote soil nutrient flux and root growth of both host plants and companion plants. Low levels of white clover ( Trifolium repens L.) root infection by clover cyst nematodes ( Heterodera trifolii Goffart) increased root growth by 141% and 219% in the host plant and the uninfected neighbouring grass ( Lolium perenne L.), respectively. Root infection increased the size of the soil microbial biomass in the root zone and the transfer of ¹⁵N from the host plant to soil and the neighbouring grass. These data suggest that low amounts of below-ground herbivory may increase the transfer of plant carbon and nitrogen below-ground, leading to increases in root growth and soil nutrient recycling in grasslands. Presumably, such interactions will influence the competitive interactions between plant species, altering plant community structure in grasslands.     

Phagocytosis by Lymnaea stagnalis haemocytes: a potential role for phosphatidylinositol 3-kinase but not protein kinase A

The molecular events that regulate phagocytosis, an important innate immune response, in invertebrate defence cells (haemocytes) are poorly understood. Lymnaea stagnalis haemocytes were used as a model to elucidate the role of cell signalling pathways in phagocytosis by molluscan defence cells. The phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, significantly impaired haemocyte phagocytic activity in a dose-responsive manner with 10 microM LY294002 reducing internalization of fluorescent-conjugated Escherichia coli by 62% (P < or = 0.001). In contrast, the protein kinase A (PKA) inhibitor KT5720 was without effect. Therefore, PI3-K, but not PKA, appears to control phagocytosis by haemocytes in these gastropod molluscs.     

Optimum activity of the phosphofructokinase from Ascaris suum requires more than one metal ion

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (FBP) using MgATP as the phosphoryl donor. As the concentration of Mg(2+) increases above the concentration needed to generate the MgATP chelate complex, a 15-fold increase in the initial rate was observed at low MgATP. The effect of Mg(2+) is limited to V/K(MgATP), and initial rate studies indicate an equilibrium-ordered addition of Mg(2+) before MgATP. Isotope partitioning of the dPFK:MgATP complex indicates a random addition of MgATP and F6P at low Mg(2+), with the rate of release of MgATP from the central E:MgATP:F6P complex 4-fold faster than the net rate constant for catalysis. This can be contrasted with the ordered addition of MgATP prior to F6P at high Mg(2+). The addition of fructose 2,6-bisphosphate (F26P(2)) has no effect on the mechanism at low Mg(2+), with the exception of a 4-fold increase in the affinity of the enzyme for F6P. At high Mg(2+), F26P(2) causes the kinetic mechanism to become random with respect to MgATP and F6P and with MgATP released from the central complex half as fast as the net rate constant for catalysis. The latter is in agreement with previous studies [Gibson, G. E., Harris, B. G., and Cook, P. F. (1996) Biochemistry 35, 5451-5457]. The overall effect of Mg(2+) is a decrease in the rate of release of MgATP from the E:MgATP:F6P complex, independent of the concentration of F26P(2).     

Isotope partitioning in the adenosine 3',5'-monophosphate dependent protein kinase reaction indicates a steady-state random kinetic mechanism

Isotope partitioning beginning with the binary E.MgATP and E.N-acetyl-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ser-peptide) complexes indicates that the kinetic mechanism for the adenosine 3',5'-monophosphate dependent protein kinase is steady-state random. A total of 100% of the initial radioactive E.MgATP complex is trapped as phospho-Ser-peptide at infinite Ser-peptide concentration at both low and high concentration of uncomplexed Mg2+, suggesting that the off-rate of MgATP from the E.MgATP.Ser-peptide complex is slow relative to the catalytic steps. Km for Ser-peptide in the trapping reaction decreases from 17 microM at low Mg2+ to 2 microM at high Mg2+, indicating that Mg2+ decreases the off-rate for MgATP from the E.MgATP complex. A total of 100% of the radioactive E.Ser-peptide complex is trapped as phospho-Ser-peptide at low Mg2+, but only 40% is trapped at high Mg2+ in the presence of an infinite concentration of MgATP, suggesting that the off-rate for Ser-peptide from the central complex is much less than catalysis at low but not at high Mg2+. In support of this finding, the Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly (Ala-peptide) increases from 0.27 mM at low Mg2+ to 2.4 mM at high Mg2+. No trapping was observed at either high or low Mg2+ for the E.MgADP complex up to a phospho-Ser-peptide concentration of 5 mM. Thus, it is likely that in the slow-reaction direction the kinetic mechanism is rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.