Adhesion Pad Formation and the Involvement of Cutinase and Esterases in the Attachment of Uredospores to the Host Cuticle

We have investigated the basis of adhesion of uredospores of the obligately parasitic rust fungus Uromyces viciae-fabae to leaves of its broad bean host. Upon contact with an aqueous environment, spores form a structure that we have termed an adhesion pad. The adhesion pad is formed by both living and autoclaved spores, but only adhesion pads formed by living spores adhered to the cuticle of leaves of the host plant. Treatment of living spores with the serine-esterase inhibitor diisopropyl fluorophosphate prevented the adhesion of the pad to the leaf surface, suggesting a functional role for esterase or cutinase in the process of adhesion. A cutinase and two nonspecific serine-esterases were found to be localized on the surface of spores. These enzymes were released rapidly from the spore surface upon contact with an aqueous environment. The addition of the cutinase and the nonspecific esterases to autoclaved spores restored their ability to adhere to the host cuticle. Thus, whereas pad formation appears to be a passive response to the aqueous environment, the actual adhesion of pads to the host cuticle appears to depend on the cutinase and esterases associated with the spore surface. These results suggest a new role for cutinases and serine-esterases in the fungal infection process.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.     

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.     

Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.     

Identification of the Low Molecular Weight Copper Protein from Copper-intoxicated Mung Bean Plants

Mung bean plants (Wilczek) accumulate increasingly greater amounts of buffer-extractable copper in both their shoots and roots when grown in liquid medium containing greater than 2 micrograms per milliliter copper (31.4 micromolar) as cupric sulfate. This increase in soluble copper is accompanied by an increase in the relative amount of low molecular weight (7,000 to 20,000) macromolecular-bound copper and a decrease in the relative amount of high molecular weight (greater than 20,000) copper. The major low molecular weight copper protein has been isolated from copper-intoxicated mung bean plants by a combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. It was identified as mung bean plastocyanin on the basis of its molecular weight, optical behavior, and amino acid composition. No evidence was found for a low molecular weight copper-binding protein corresponding to mammalian thionein or chelatin.     

Cell junctions and intercellular communication

Summary We have compared intercellular communication in normal and regenerating rat liver. Gap junctions are greatly reduced in size and numbers 29 to 35 hr after hepatectomy, but we still find some 90% of hepatocytes coupled by electrophysiological criteria. The spread of dyes such as carboxyfluorescein however is very limited in the regenerating organs as compared to the situation in the controls. We show how the apparent discrepancies between morphological and physiological data can be reconciled. We also present a summary of preliminary findings on the biosynthesis of gap junction protein and some of the conclusions one can draw from the sequence of 58 amino acids at the amino terminal of the protein. Presented in the symposium on Molecular and Morphological Aspects of Cell-Cell Communication at the 31st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. The original research described was supported by Grants GM 06965 and RR 07003 from the National Institute of Health, and funds from the North-west Area Foundation. David Meyer and Barbara Yancey were the recipients of NIH postdoctoral fellowships (NS 06240 and AM05700). This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center.     

Cytosolic phospholipase A2 from U937 cells: size of the functional enzyme by radiation inactivation.

We have studied the cytosolic phospholipase A2 (cPLA2) of human U937 cells by radiation inactivation in order to characterize the functional form of the native enzyme by a method that was independent of the discrepancies observed by SDS-PAGE and cDNA cloning. The Radiation Inactivation Size of cPLA2 was reproducible and gave a value of 76,800-80,100 daltons. We eluted the active enzyme from polyacrylamide-gradient gel electrophoresis at a molecular weight of 77,000, confirming the irradiation result. We conclude that cPLA2 is active as the monomeric enzyme and is composed of a single major functional domain that is sensitive to irradiation.