Differential localization of Acanthamoeba myosin I isoforms

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.     

Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.     

Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro

Altered lysosomal function in the visceral yolk sac can result in abnormal development. As proteolysis is an important function of the rodent visceral yolk sac during early and mid-gestation, we characterized the lysosomal proteolytic enzyme activity of this extraembryonic membrane and determined the effects of inhibitors of protein degradation on embryonic development. Constituent activities of cysteine and aspartic acid proteinases were measured in rat visceral yolk sac on gestation day 12, and the effects of the cysteine proteinase inhibitors leupeptin, E-64 [trans-epoxysuccinyl-l-leucylamido(4-guanido)butane] and N-ethylmaleimide and the aspartic acid proteinase inhibitor pepstatin were determined in Sprague-Dawley rat embryos cultured in vitro from gestation days 10-12. It was determined that only cysteine proteinases, primarily cathepsins B and L, are active in the mid-gestation visceral yolk sac. The cysteine proteinase inhibitors leupeptin and E-64 both produced a concentration-related decrease in embryonic growth, as measured by crown-rump length, somite number, and embryonic protein content, and a concentration-related increase in incidence of abnormalities. A characteristic pattern of abnormalities was produced which involved a decrease in neural tube volume and the formation of a subectodermal blister opposite the point of attachment of the vitelline vessels. At high concentrations, anophthalmia was also observed. The decreased neural tube volume was associated with increased osmolality of the exocoelomic fluid, the major extraembryonic fluid compartment. It is possible that the osmotic change decreased neural tube volume by causing water to move to the compartment with a higher solute concentration, out of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

The herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells.

Earlier studies have shown that the UL11 gene of herpes simplex virus encodes a myristylated virion protein and that the UL11 gene enables efficient virion envelopment and export from infected cells. A rabbit polyclonal antibody directed against an affinity-purified UL11-glutathione-S-transferase fusion protein was made and used to study the properties of the UL11 protein and its distribution in infected cells. We report the following: (i) UL11 protein formed up to five bands (apparent M(r)s, 17,000 to 22,000) in denaturing polyacrylamide gels; (ii) fluorescent-antibody studies revealed the presence of UL11 protein in the perinuclear space and in sites within the nucleus; (iii) immune electron microscopic studies indicated that the UL11 gene products were associated with the inner nuclear membrane, with cytoplasmic membranes and ribbon-like cytoplasmic structures resembling membranous organelles, with nuclear bodies shown by fluorescence microscopy to be different from nucleoli in which US11 protein accumulates, and with enveloped virions but not with nuclear capsids; and (iv) the nuclear bodies containing UL11 protein were reminiscent both of type IV morphotypes consisting of an electron-dense core containing the UL11 proteins surrounded by a more electron-transluscent core and of type V morphotypes consisting of material homogenous in electron opacity. We conclude that (i) the UL11 protein is processed after synthesis; (ii) the localization of UL11 protein with virions and membranes is consistent with the hypothesis that UL11 plays a role in the transport of virions to the extracellular space; and (iii) although the significance of the association of UL11 proteins with nuclear bodies is unknown, the results indicate that nuclear bodies differ with respect to their morphologies and contents of viral protein and suggest that UL11 protein may have more than one function in the infected cell.     

Comparison of the response of midgut epithelial cells and cell lines from lepidopteran larvae to CryIA toxins from Bacillus thuringiensis

The cytotoxic responses of midgut epithelial cells (MEC) from spruce budworm (SBW), gypsy moth (GM) and silkworm (SW) larvae were compared with the cytotoxic response of lepidopteran cell lines (SF-9, SE-1a, and CF-1) to CryIA toxins from Bacillus thuringiensis. The MEC from SBW, SW and GM had binding proteins for CryIA(a,b,c) toxins, whereas the lepidopteran cell lines had binding proteins for CryIA(c). Single MEC exposed to CryIA(a,b,c) toxins in a qualitative lawn assay were equally susceptible to the toxins with a threshold response at about 1ng. The cell lines were not susceptible to CryIA(a,b) toxins in the dose range tested, but had threshold responses for CryIA(c) of 3.4ng for SF-9, 50.2ng for SE-1a and 5.9ng for CF-1. In the quantitative Live/Dead assay, MEC were equally susceptible to CryIA(a,b,c) toxins with a threshold effect at about 1ng and a maximum effect at about 10ng. CF-1 was most sensitive to CryIA(c) with a threshold effect at 0.39ng and a maximal effect at about 1ng. In contrast, a 25-50 times greater dose of CryIA(a) or CryIA(b) was required to elicit a similar response as CryIA(c) for CF-1. SF-9 and SE-1a were most susceptible to CryIA(c) with a threshold effect observed at about 0.5ng and maximal effects at about 2ng. SF-9 cells have a threshold and maximum response to CryIA(a,b) of about 10ng and 20ng, respectively. SE-1a cells have a threshold and maximal response to CryIA(a,b) of 5ng and 10ng, respectively. Intact midgut epithelium exposed to CryIA(a,b,c) toxins had a threshold dose of 2ng for CryIA(b), 10-30ng for CryIA(a) and 2-30ng for CryIA(c). This study has shown that MEC are affected by a broader spectrum of toxins compared to the lepidopteran larvae and insect cell lines.     

Definition of a Sequence Unique in βII Spectrin Required for Its Axon-Specific Interaction with Fodaxin (A60)

Abstract: Spectrin isotypes segregate in neurons and are differentially distributed between axons and somatodendritic compartments. Their functions in those compartments are likely to be mediated by proteins that interact selectively with one or other isotype. Fodaxin (an axon-specific protein previously termed A60) colocalizes in CNS neurons with axonal spectrin and in vitro binds brain spectrin (a mixture of αI, βI, αII, and βII polypeptides) but not erythrocyte spectrin (αI and βI). Because αII and βII spectrin polypeptides are enriched in axons, we investigated a possible binding of fodaxin to the types of spectrin found in axons. Fodaxin did not bind to isolated brain α chains. Bacterially expressed C-terminal segments 18–19 of βII spectrin bound to fodaxin and inhibited the binding of fodaxin to whole brain spectrin. By contrast, recombinant segments 18–19 of the somatodendritic βIΣ2 spectrin showed no interaction with fodaxin. Within βII, fodaxin binding activity was localized to residues 2,087–2,198, which are unique to βII and link between the end of segment 18 and the pleckstrin homology domain in segment 19. The divergent regions of sequence in segments 19 of βII and βIΣ2 are candidates to mediate the isotype-specific functions of spectrin. Fodaxin is the first protein to be described that discriminates between the unique regions of β spectrin isoforms.     

Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes.

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.     

The Effect of Soil Type on Movement and Infection Rate of Larvae of Tylenchulus semipenetrans

Most of the Tylenchulus semipenetrans larvae applied on the surface of four soils in pots 14.5-cm deep moved no further downward than 6.5 crn, and remained in the upper half of the pot. The percentage of second-stage larvae that developed into adult females on ''Homosassa'' sweet orange in the soils were: sandy loam, 6.8% in the same soil with inoculation holes, 8.6%; loamy sand, 5.4%; coarse sand, 0.2%; and in sand-peat (2:1, v/v)mixture 0.04%. Low percentage infection in coarse sand and sand-peat mixture may have been caused by restriction of larval migration in the coarse sand, and by low pH or toxic organic acids in the sand-peat mixture. Good root development occurred throughout all soils.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. VII. The effect of immunodeficiency disease.

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseases—immunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients' lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients' or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.