Papers in Microbiology

Giant Vacuoles Observed in Dictyostelium discoideum

Aidong Yuan et al. — Tue, 19 Jan 2010 07:17:31 PST

Large intracellular vacuoles, >4 μm in diameter and either round or oval-shaped, were observed infrequently in Dictyostelium discoideum amoebae of axenically-grown strain AX2 (only 1 in 10⁶–10⁸ cells). These previously unreported single or multiple “giant” vacuoles were more common, however, in newly germinated KAX3 cells (0.55% of the population) and AT-K_neg, a strain that lacks an esterase (0.47% of the population). A vacuolar H⁺-ATPase was enriched in their membranes of intracellular giant vacuoles, indicating that the vacuoles were related possibly to both endosomes and the contractile vacuole compartment. When monitored over time, giant vacuoles protruded from, and retracted back into cells under hyperosmotic conditions, suggesting an osmoregulatory role for these vacuoles. Some of the intracellular and protruded giant vacuoles harbored a fluid-phase marker, fluorescein-labeled dextran, implying a pinocytotic origin for the vacuoles.

Exogenous Farnesol Interferes with the Normal Progression of Cytokine Expression during Candidiasis in a Mouse Model

Dhammika H. M. L. P. Navarathna et al. — Mon, 10 Mar 2008 18:43:04 PDT

Candida albicans, a dimorphic fungus composed of yeast and mycelial forms, is the most common human fungal pathogen. Th1 cytokines such as interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), which are induced by macrophage IL-12, are critical to resistance against systemic candidiasis, while Th2 cytokines such as IL-4 and IL-5 are less critical. Farnesol is a quorum-sensing molecule produced by C. albicans that controls the formation of mycelia but is also a virulence factor. To determine whether farnesol enhances the virulence of C. albicans by modulating the production of Th1 and Th2 cytokines, mice were pretreated with farnesol prior to intravenous infection with a sublethal dose of farnesol-producing C. albicans. Production of IL-2, IL-4, IL-5, TNF-α, IFN-γ, and IL-12 was evaluated by bead-array flow cytometry and enzyme-linked immunosorbent assay. Mice exhibited an elevation in serum TNF-α levels at 48 h and an elevation in IFN-γ and IL-12 levels at 6 to 12 h after infection with C. albicans. Pretreatment with farnesol significantly reduced the elevation of both IFN-γ and IL-12 but not TNF-α. In contrast, mice pretreated with farnesol exhibited an unexpected elevation in IL-5 levels. To determine whether farnesol has a direct effect on macrophage production of IL-12, peritoneal macrophages were pretreated with farnesol prior to stimulation with IFN-γ plus lipopolysaccharide (LPS). Farnesol inhibited production of both IL-12 p40 and p70 from IFN-γ/LPS-stimulated macrophages. Therefore, the role of farnesol in systemic candidiasis is likely due to its ability to inhibit the critical Th1 cytokines IFN-γ and IL-12 and perhaps to enhance a Th2 cytokine, IL-5.

Phenotype of the Triplo-lethal locus of Drosophila melanogaster and its suppression by hyperoxia

Laura K. Smoyer et al. — Fri, 22 Feb 2008 09:50:15 PST

The Triplo-lethal locus (Tpl) of Drosophila is both triplo-lethal and haploinsufficient, but the function of the locus is unknown. We have examined Tpl-aneuploid embryos and find that, in both trisomics and monosomics, the midgut shows extensive cell death and the tracheae are abnormal. Shortly thereafter, all tissues die. PCR-based genotyping of individual embryos and larvae show that this phenotype occurs in the trisomics after hatching and in the monosomics before hatching. Weak alleles of the interacting gene Su(Tpl) delay the death of Tpl trisomics, but they still show the same tracheal and midgut phenotypes before dying. Hyperoxia (45% oxygen) partially suppresses the phenotype of Tpl aneuploids, even though the use of a hypoxia reporter strain shows that dying Tpl aneuploids are not hypoxic. This is the first report of a phenotype associated with the Tpl locus and the first report of an environmental condition that suppresses the phenotype.

Role of Esterase gp70 and Its Influence on Growth and Development of Dictyostelium discoideum

Aidong Yuan et al. — Wed, 23 Jan 2008 12:13:41 PST

Gp70 is an esterase originally called crystal protein because of its presence in crystalline structures in aggregation-competent Dictyostelium discoideum cells. Although postulated to break down spore coats, the function of gp70 in vivo was incompletely investigated. Our immunolocalization and biochemical studies of vegetative D. discoideum amoebae show that gp70 was recruited to phagosomes and found in lysosomes. Purified gp70 was effective at hydrolyzing naphthyl substrates with acyl chains typical of lipids and lipopolysaccharides, indicating that the gp70 was involved in digesting endocytosed molecules. The activity of purified gp70 was inhibited by reductants that retarded its electrophoretic mobility and verified the presence of intramolecular disulfide bonds predicted by its amino acid sequence. Compared to wild-type cells, cells overexpressing gp70 were more phagocytically active, had shorter generation times, and produced more fruiting bodies per unit area, while cells lacking gp70 were phagocytically less active with longer doubling times, developed more slowly, and had significantly fewer fruiting bodies per unit area. Consistent with the phenotype of a disrupted metabolism, one-third of the gp70-minus cells were large and multinucleated. Together, these results indicated that despite its crystalline appearance, gp70 was an active esterase involved in both the growth and the development of D. discoideum.

Cytoskeletal Association of an Esterase in Dictyostelium discoideum

Catherine P. Chia et al. — Wed, 23 Jan 2008 12:09:59 PST

A 70-kDa glycoprotein, gp70, was found enriched in the detergentinsoluble cytoskeletal fraction of axenically grown Dictyostelium discoideum cells. Its N-terminal amino acid sequence identified it as “crystal protein” (L. Bomblies et al., 1990, J. Cell Biol. 110, 669– 679). This finding was corroborated when antibody to crystal protein cross-reacted with gp70 and its deglycosylated form. The postulated esterase activity of gp70/crystal protein was verified through comparative enzyme assays of extracts derived from cells that either overexpressed or lacked gp70. Gp70 cosedimented with cytoskeletons on sucrose gradients, suggesting an interaction with the cytoskeleton. Coisolation of gp70 with detergent-extracted cells, observed by immunofluorescence microscopy, also implied a gp70-cytoskeletal association. These data supported the idea that the localization or secretion of gp70, or both, was cytoskeletally mediated. Although axenically grown cells contained high levels of gp70, the same cell lines had reduced levels of gp70 when grown in bacterial suspension or in nutrient media containing bacteria. Bacterially grown cells, compared to axenically grown cells, had lower fluidphase uptake rates even when nutrient media was present, indicating that phagocytosis was a preferred mode of feeding. Thus, bacteria inhibited gp70 expression, which suggested a role for prestarvation factor, in regulating its synthesis.

Bioassay of Solubilized Bacillus thuringiensis var. israelensis Crystals by Attachment to Latex Beads

Danny J. Schnell et al. — Fri, 19 Oct 2007 08:21:04 PDT

Solubilized crystals of Bacillus thuringiensis var. israelensis were 7,000 times less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito larvae are filter feeders and selectively concentrate particles while excluding water and soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8- micrometer latex beads, with retention of toxicity. The latex bead assay should make it possible to analyze the structure and mode of action of the mosquito toxin.

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Bradley A. Plantz et al. — Fri, 19 Oct 2007 08:18:46 PDT

A growth-associated model was applied to the production of recombinant ovine interferon-τ (rOvIFN-τ) with Pichia pastoris for the purpose of manufacturing pre clinical and clinical active material. This model predicts that product yields will be the greatest when the specific growth of the culture is maintained at a steady and optimal rate. However, rOvIFN-τ yields did not meet the expected linear model but most closely corresponded to a polynomial relationship. After transitioning from glycerol to methanol, product accumulated for 31–45 h, and then the yield decreased. This production shift, which has been termed decoupling, was clearly related to time on methanol and not culture density. It was determined that a correlation exists between the decoupling point and a drop in energy state of the cell when expressing β-galactosidase. By assigning decoupling as a constraint that limits productivity and by reformulating the growth medium, the time prior to decoupling increased to 46.8 ± 2.4 h, product yield improved for rOvIFN-τ from 203 to 337 mg l−1, and the coefficient of variation for yield decreased from 67.9 to 23.3%.

Nonspecific interactions alter lipopolysaccharide patterns and protein mobility on sodium dodecyl sulfate polyacrylamide gels

Aidong Yuan et al. — Tue, 09 Oct 2007 10:39:30 PDT

In testing whether bacterial lipopolysaccharide (LPS) was a natural substrate for an esterase from the soil amebae Dictyostelium discoideum, we observed altered banding patterns of the LPS and changed protein mobility on sodium dodecyl sulfate (SDS) polyacrylamide gels after incubation of LPS with the enzyme. The initial interpretation of these results was that the enzyme had removed ester-linked acyl chains from the LPS, leading to a change in its migration on gels. However, esterase inactivated by treatment with either dithiothreitol (DTT), heat, or SDS generated the same mobility shifts. Bovine serum albumin (BSA) also induced the same change in the electrophoretic pattern. We conclude that the altered LPS patterns and protein mobility on SDS gels were caused by nonspecific interactions between LPS and protein.

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

Aidong Yuan et al. — Tue, 09 Oct 2007 10:35:11 PDT

1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[35S]methionine into the lumbar spinal cord.
2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.
3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.
4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.

Phagosomal Proteins of Dictyostelium discoideum

Betsy L. Rezabek et al. — Wed, 26 Sep 2007 15:36:51 PDT

In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradients. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H+-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.

Integrity of the actin cytoskeleton required for both phagocytosis and macropinocytosis in Dictyostelium discoideum

Aidong Yuan et al. — Fri, 21 Sep 2007 08:15:13 PDT

Filamentous (F-) actin is enriched in cellular extensions, such as phagocytic cups and macropioocytic crowns, of Dlctyostelium discoideum amebae. Previous studies of actin-disrupting agents that implicated the involvement of the actin cytoskeleton in Dictyostelium phagocytosis and pinocytosis, however, have yielded conflicting results. We show that the integrity of the actin cytoskeleton is required for both phagocytosis and macropinocytosis in D. discoideum with latrunculin A (IatA), which binds to monomeric actin, and cytochalasin A (cytA), which caps the plus end of actin filaments. Using rhodamine-phalloidin to visualize F-actin, cells treated for 30 min. with 1 to 4 pM of latA displayed an increasing dissolution of the cortical actin cytoskeleton that was accompanied by the appearance of numerous cytoplasmic dots of F-actin. In parallel, phagocytosis of fluorescently labeled yeast and macropinocytosis of the fluid-phase marker fluorescein isothiocyanate-dextran both were inhibited in a dose-dependent manner. Cells were nearly devoid of F-actin at latA concentrations greater than 5pM whereas the uniform distribution of monomeric actin appeared unaffected. Cells gradually recovered their intact actin cytoskeleton and concomitantly, their phagocytic and macropinocytic activities when latA was removed by washing. To achieve 50% inhibition of phagocytosis or macropinocytosis, five-fold more cytA than latA was required. Unlike latA-treated cells, cytAtreated cells stained with rhodamine phalloidin retained an actin cytoskeleton even at high concentrations (>25 μM), but were smaller and rounder than untreated cells. The cortical F-actin, however, appeared irregular, and almost discontinuous, which made the cells seem stiff and rigid in comparison to normal cells that looked more fluid and plastic. The distinctive alterations in the cytoskeletal patterns reflected the specific modes of action of the drugs on the actin network that was vital for both phagocytosis and macropinocytosis.

A role for calcineurin in Dictyostelium discoideum phagocytosis

Aidong Yuan et al. — Fri, 21 Sep 2007 08:11:13 PDT

The Ca2+/calmodul1n-dependent protein phosphatase calcinewin is involved in the development of the cellular slime mold Dictyostelium discoideum. Because of its interactions with Ca2+, which appear to influence D. discoideum phagocytosis (Yuan and Chia, 1999, Mol. Biol. Cell 10, 220a), we undertook studies to test whether calcineurin also plays a role in Dictyostelium phagocytosis. The immunosuppressants cyclosporin A and FK506, through the formation of cyclosporin A-cyclophilin A and FK506- FK506-binding protein complexes, respectively, inhibited calcineurin activity. These two calcineurin inhibitors suppressed phagocytosis of fluorescently labeled yeast in a dose-dependent manner. Although it inhib~ted phagocytosis, cyclosporin A had an insignificant effect on the macropinocytosis of the fluid-phase marker fluorescein isothiocyanatedextran. Furthermore, trifluoperazine, a calmodulin antagonist that indirectly inhibits calcinewin, also suppressed phagocytosis in a dosedependent fashion and induced the formation of giant intracellular vacuoles Fluorescence microscopy of cyclosporin A-treated (for 30 min.) cells stained with rhodamine-phalloidin had cytoplasmic chunks of F-actin that were not present in control cells, while cells treated with FK506 and trifluoperazine (also for 30 min.), displayed less cortical but more cytoplasmic F-actin staining than normal cells. Typically, drug-treated cells were smaller and rounder than untreated cells. Our data suggest calcineurin may play a role in D. discoideum phagocytosis, either through the dephosphorylation of actinregulating proteins or other cytoskeletal proteins such as the heavy chain subunit of nonmuscle myosin I1 since dephosphorylation of the latter promotes filament assembly.

CRYSTAL PROTEIN EXPRESSION DURING VEGETATIVE GROWTH OF DICTYOSTELIUM

Kristy K. Taylor et al. — Fri, 21 Sep 2007 08:02:10 PDT

We are studying interactions between membrane proteins and the cytoskeleton of Dictyostelium discoideum. Detergent (Triton X-100) -insoluble cytoskeletons from vegetative (AX2) amebae are enriched in a 70 kDa Concanavalin A (Con A)-binding protein (gp70). An enriched fraction of gp70 was prepared by Con A affinity chromatography of cytoskeletons from axenically-grown cells. N-terminal amino acid sequence of gel-purified gp70 established its identity to a 69 kDa 'crystal protein' previously characterized by Bomblies, et al. (1990, J. Cell Biol. 110:669 -679). Enzymatic deglycosylation of gp70 resulted in a 65 kDa product that did not bind Con A, verifying the predicted number of N-glycosylation sites. Bomblies, et a1 showed crystal protein (gp70) to be developmentally regulated, and with its homology to esterases, suggested to be involved in spore case degradation. We find crystal protein expression to be influenced by growth conditions. As determined by immunoblotting, axenically-grown, vegetative cells express moderate levels of crystal protein. When bacteria are the sole food source for both wild type and over-expressor cell lines grown in suspension, crystal protein levels are reduced significantly in vegetative cells. However, in these same cell lines grown on bacteria, crystal protein is expressed late in development. The differences in crystal protein expression may indicate that it is regulated during vegetative growth and during development.

A Circular Dichroic Study of Cu (II) -Ribonuclease Complexes

Kenneth Nickerson et al. — Fri, 21 Sep 2007 07:59:55 PDT

The visible and ultraviolet circular dichroic (CD) spectra resulting from the interaction of ribonuclease with successive Cu(I1) ions have been recorded under a variety of conditions. At pH 7 in the presence of 0.16 M KC1 a broad, negative band was found in the visible region. This band increased in intensity and changed in shape as successive coppers were added. The circular dichroic spectra could be analyzed in terms of two kinds of binding sites: a single strong site with CD minimum at about 710 nm, and four weaker sites with CD minimum at about 600 nm. The binding constants observed are close to those obtained by more conventional means. Carboxymethylation of one histidine results in loss of one of the weaker sites. In 0.01 M salt, only the 600-nm band is seen.

Binding at pH 9.6 differed in that saturation did not occur until about 33 sites had been filled. The presence of tetra coordination at this pH was indicated by the shift of the primary d-d transition down to 530 nm. Additional structure in the visible and near ultraviolet CD was now present in the form of a negative band at 355 nm and, for the first two Cu(II)‘s added, a positive one at 480 nm.

Strong positive bands were observed at 251 and 305 nm for all pH values ≥7. These are tentatively ascribed to charge transfer complexes between Cu(I1) and the peptide backbone. The relationship of the Cu(II)-ribonuclease CD spectra to those of natural, copper-containing metalloproteins, both “blue” and “non-blue”, is discussed, with special emphasis on the oxyhemocyanins.

A Receptor-Like Glycoprotein from Dictyostelium discoideum: Functions in Phagocytosis and Cell Adhesion?

P. Christopher LaRosa et al. — Fri, 21 Sep 2007 07:51:07 PDT

The molecular mechanisms for the initial recognition and subsequent internalization of food and unicellular pathogens by phagocytes are incompletely understood We have hypothesized that a surface-exposed, glycosylated I30 kDa protein, gp130, that 1s concentrated on the plasma membrane and found In phagosomes, has a role In phagocytosis by D. discoideum amoebae. GpI30 appears to have a cytoskeletal association and has extracellular domains susceptible to proteolytic digestion. It is tightly bound to the plasma membrane probably via a carboxyterminal hydrophobic anchor predicted from the cDNA. Gp130 may be the same as a similarly sized protein, gp126, that was implicated as a phagocytosis receptor and a cell adhesion protein during starvation-induced development (Chadwick et al, 1984, Nature, 307, 646) Primers for DNA polymerase chain reaction (PCR) were designed from internal amino acid sequences of proteolyzed gp130 and its gene sequence was determined by PCR and cycle sequencing.

Membrane glycoprotein gp130 of Dictyostelium discoideum is lipid-linked and its fate altered in the presence of tunicamycin

Betsy L. Barent et al. — Fri, 21 Sep 2007 07:41:01 PDT

We are studying the structure and role in phagocytosis of gp130, a glycoprotein located on the extracellular surface of D. discoideum amoebae. Predictions from the protein sequence of gp130 deduced from the cDNA indicate its attachment to the membrane via a glycosylphosphatidylinositol (GP1)-anchor. This was confirmed when radiolabeled palmitic acid was incorporated into gp130 of axenically grown cells, and when nitrous acid deamination released gp130 from purified membranes. The GPI-anchor of gp130 resisted cleavage by bacterial and mammalian phosphoinositol-specific phospholipases. However, in the presence of protease inhibitors, we detected in vitro a time-dependent cleavage of gp 130 that was effectively inhibited by ZnClz. This activity and its inhibition were analogous to the release documented for two known GPI-linked proteins, gp80 of D. discoideum and VSG of Trypanosoma brucei. Cells treated with tunicamycin (0.5 μglmlT; M), an inhibitor of N-linked glycosylation, produced an immunoreactive 85 kDa version of gp130 that was apparently unanchored and soluble rather than membrane-associated. The 85 kDa species did not bind Concanavalin A, and thus presumed to be devoid of (N-linked) carbohydrate. Also detected in TM-treated cells but absent in untreated, control cells were small quantities of an 81 kDa species that was either a second unglycosylated form of gp130 or a proteolytic product. TM-treated cells had two-fold less mature gp130 than control cells, as judged by densitometric analyses of immunoblots of cell lysates and sucrose gradient-purified plasma membranes. The totaled signals from the gp130- immunoreactive species of TM-treated cells were comparable to the 9~130si gnal of control cells. We conclude that the TM-inhibited glycosylation interfered with the attachment of the GPI-anchor to gp130. Consequently, a soluble and unglycosylated form of gp130 accumulated, presumably in the lumen of the ERIGolgi network.

Detergent (Sodium Dodecyl Sulfate) Shock Proteins in Escherichia coli

Michael Adamowicz et al. — Fri, 07 Sep 2007 08:33:16 PDT

The protein composition of Escherichia coli W3110 grown in the presence and absence of 5% sodium dodecyl sulfate (SDS) was examined by two-dimensional gel electrophoresis. In SDS-grown cells, at least 4 proteins were turned on, 13 were turned off, 15 were elevated, and 15 were depressed. The 19 unique and elevated SDS-induced spots constituted 7.91% of the total ³⁵S-labeled protein. There was no apparent overlap between these 19 detergent (SDS) stress proteins and those of other known bacterial stress responses. The detergent stress stimulon is a distinct and independent stimulon. Its physiological relevance probably derives from the presence of bile salts in animal gastrointestinal tracts.

Structural Disulfide Bonds in the Bacillus thuringiensis subsp. israelensis Protein Crystal

Graham A. Couche et al. — Fri, 07 Sep 2007 08:28:03 PDT

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 x 10^-8 mol of free sulfhydryls and 3.24 x 10-^-8 mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Non-reduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.

Amino Sugars in the Glycoprotein Toxin from Bacillus thuringiensis subsp. israelensis

Mary Ann Pfannenstiel et al. — Fri, 07 Sep 2007 08:24:49 PDT

The carbohydrate content of purified Bacillus thuringiensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The anmino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.

Calmodulin Levels in the Yeast and Mycelial Phases of Ceratocystis ulmi

Ganapathy Muthukumar et al. — Fri, 07 Sep 2007 08:19:51 PDT

The calmodulin content of the yeast and mycelial phases of Ceratocystis ulmi was determined by radioimmunoassay. Calmodulin levels increased at the G1-S boundary of the cell cycle, coinciding with the first visible appearance of buds or germ tubes. However, in both phases the cellular calmodulin levels were equivalent. No differential synthesis was observed.