Preinoculation of lettuce and onion with VA mycorrhizal fungi reduces deleterious effects of soil salinity

The hypothesis that inoculation of transplants with vesicular-arbuscular mycorrhizal (VAM) fungi before planting into saline soils alleviates salt effects on growth and yield was tested on lettuce (Lactuca sativa L.) and onion (Allium cepa L.). A second hypothesis was that fungi isolated from saline soil are more effective in counteracting salt effects than those from nonsaline soil. VAM fungi from high- and low-salt soils were trap-cultured, their propagules quantified and adjusted to a like number, and added to a pasteurized soil mix in which seedlings were grown for 3–4 weeks. Once the seedlings were colonized by VAM fungi, they were transplanted into salinized (NaCl) soil. Preinoculated lettuce transplants grown for 11 weeks in the saline soils had greater shoot mass compared with nonVAM plants at all salt levels [2 (control), 4, 8 and 12 dS m^–1] tested. Leaves of VAM lettuce at the highest salt level were significantly greener (more chlorophyll) than those of the nonVAM lettuce. NonVAM onions were stunted due to P deficiency in the soil, but inoculation with VAM fungi alleviated P deficiency and salinity effects; VAM onions were significantly larger at all salt levels than nonVAM onions. In a separate experiment, addition of P to salinized soil reduced the salt stress effect on nonVAM onions but to a lesser extent than by VAM inoculation. VAM fungi from the saline soil were not more effective in reducing growth inhibition by salt than those from the nonsaline site. Colonization of roots and length of soil hyphae produced by the VAM fungi decreased with increasing soil salt concentration. Results indicate that preinoculation of transplants with VAM fungi can help alleviate deleterious effects of saline soils on crop yield.     

Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity

Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor. Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF. In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF. In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold. Combination of domain substitutions to generate the [Glu107,123]bFGF and [Arg19,Lys123,126]bFGF mutants did not show any additivity of the mutations on biological activity. Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification. Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency. The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin. We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor.     

Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup⁺ phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase EcoRI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn5-mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.     

Unusual fungal niches

Fungi are found in all aerobic ecosystems, colonizing a diversity of substrates and performing a wide diversity of functions, some of which are not well understood. Many spices of fungi are cosmopolitan and generalists or habitats. Unusual fungal niches are habitats where extreme conditions would be expected to prevent the development of a mycobiota. In this review we describe five unusual fungal habitats in which fungi occupy poorly understood niches: Antarctic dry valleys, high Arctic glaciers, salt flats and salterns, hypersaline microbial mats and plant trichomes. Yeasts, black yeast-like fungi, melanized filamentous species as well as representatives of Aspergillus and Penicillium seem to be dominant among the mycobiota adapted to cold and saline niches. Plant trichomes appear to be a taxa. The advent of new sequencing technologies is helping to elucidate the microbial diversity in many ecosystems, but more studies are needed to document the functional role of fungi in the microbial communities thriving in these unusual environments.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Rapid Colony Screening Method for Identifying Hydrogenase Activity in Rhizobium japonicum

A method has been developed for the rapid screening of Rhizobium japonicum colonies for hydrogenase activity based on their ability to reduce methylene blue in the presence of respiratory inhibitors and hydrogen. Hydrogen uptake-positive (Hup⁺) colonies derepressed for hydrogenase activity were visualized by their localized decolorization of filter paper disks impregnated with the dye. Appropriate responses were seen with a number of Hup⁺ and Hup⁻ wild-type strains of R. japonicum as well as Hup⁻ mutants. Its specificity was further confirmed in selected strains on the basis of comparisons with chemolithotrophic growth and the presence of other genetic markers. Utilization of the method in identifying Hup⁺ colonies among 16,000 merodiploid derivatives of the Hup⁻ mutant strain PJ17nal containing cloned DNA fragments of the Hup⁺ strain 122 DES has demonstrated its applicability as a screening procedure in the genetic analysis of the R. japonicum hydrogen uptake system.     

An analysis of the role of guanine nucleotide binding proteins in antigen receptor/CD3 antigen coupling to phospholipase C.

In permeabilized human T lymphocytes, phospholipase C (PLC)-mediated metabolism of polyphosphatidylinositols can be stimulated by triggering the T cell antigen receptor/CD3 antigen complex (Ti/CD3) with the CD3 antibody UCHT1 or by activation of G proteins with the non-hydrolyzable guanine nucleotide analogue, guanosine 5'-O-(3-thiotrisphosphate) (GTP[S]). Ti/CD3 induction of inositol phosphate production demonstrated no dependence on exogenous guanine nucleotides. Furthermore, Ti/CD3 stimulation did not influence the kinetics or dose-response of GTP[S]-induced inositol phosphate production, suggesting that the Ti/CD3 complex does not regulate guanine nucleotide exchange on the G protein pool stimulated by GTP[S]. These data indicate that the Ti/CD3 complex is not G protein-linked to PLC in a manner analogous to the G protein linkage of receptors to adenylate cyclase. However, the inhibitory guanine nucleotide, GDP, antagonizes not only GTP[S]-induced polyphosphatidylinositol hydrolysis but also UCHT1-induced inositol phosphate production. These data infer that a G protein can modulate the coupling of the Ti/CD3 complex to PLC and that there may be some "cross-talk" between Ti/CD3 and G protein PLC coupling mechanisms.     

Phorbol esters regulate CD2- and CD3-mediated calcium responses in peripheral blood-derived human T cells

The purpose of the present study was to examine the effect of protein kinase C (pkC) activation on calcium responses generated through the CD3 and CD2 Ag in both normal peripheral blood-derived T lymphocytes and the leukemic T cell line Jurkat. The data reveal a major difference with respect to the regulation of receptor-mediated calcium responses in these two cells. Thus, the pkC activator phorbol-12,13-dibutyrate (Pdbu) enhances calcium responses induced via CD3 and CD2 molecules in normal T cells by accelerating the rate of elevation of intracellular calcium levels and increasing the maximum change in calcium concentration achieved. In contrast, Pdbu inhibits both CD3- and CD2-induced calcium responses in Jurkat cells. Pdbu does not influence calcium responses generated by the guanine nucleotide-binding protein activator, aluminium fluoride, indicating that the effect of pkC occurs at a point proximal to a guanine nucleotide-binding protein regulation of T cell calcium responses.     

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.