Penetration and establishment of Phakopsora pachyrhizi in soybean leaves as observed by transmission electron microscopy.

For over 30 years, it has been known that Phakopsora pachyrhizi is unusual in that it penetrates from urediniospores directly through the leaf cuticle without entering stomates. This unusual mode of penetration suggests that disease resistance mechanisms might exist for soybean rust that do not exist for most rust diseases. As a result, we decided to conduct a histological study using transmission electron microscopy to further elucidate the mechanisms of penetration and early establishment of P. pachyrhizi in soybean leaves. Based on our study, it was concluded that P. pachyrhizi utilizes primarily mechanical force, perhaps with the aid of digestive enzymes, to penetrate the cuticle on the leaf surface. However, the lack of deformation lines in micrographs indicated that digestive enzymes, without mechanical force, are used by the penetration hypha to penetrate the outer and inner epidermal cell walls. Digestive enzymes, again indicated by the lack of deformation lines, are used by haustorial mother cells to breach the walls of mesophyll cells to form haustoria. The possibility exists for eventual determination of the precise roles of pressure and digestive enzymes in the development of soybean rust and elucidation of some of the determinants of resistance and susceptibility to this important plant disease.

Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine.

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol (EtOH) as a dehydrating solvent and propylene oxide (PO) as a transition fluid. Both solvents have some undesirable properties: EtOH solubilizes lipids; PO is highly flammable, volatile, toxic, and potentially carcinogenic. Their replacement by a compound devoid of these characteristics is therefore desirable. Acetonitrile (AN) appears to be such a solvent. It is freely miscible with water, alcohols, acetone, and epoxy resins; it does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam stability. AN is also an excellent dehydrating agent whose use does not necessitate modification of current techniques. Most importantly, the low solubility of phospholipids (PL) in AN limits the loss of membrane lipids and, hence, leads to a better preservation of tissue features.

The Wistar Furth rat: an animal model of hereditary macrothrombocytopenia.

The mechanisms that determine and regulate platelet size are unknown. By phase microscopy, we observed that Wistar Furth (WF) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes of WF rats with those of Wistar, Long-Evans hooded (LE), and Sprague-Dawley rats. In addition, we have examined the mode of inheritance of this WF rat platelet abnormality. The average platelet count of WF rats was only one-third that of the other three rat strains. In contrast, the mean platelet volume (MPV) of adult WF rats was twice that of the other rat strains; however, the average megakaryocyte diameter and DNA content distribution of WF rats were not significantly different from those of LE rats. The average megakaryocyte concentration was 30% lower in the WF strain compared with that of LE rats. Mazelike membrane formations were observed in WF platelets and megakaryocytes by electron microscopy. Reciprocal crosses of WF and LE rats resulted in offspring with MPVs and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the WF and LE strains resulted in MPVs like those of the donor strain, demonstrating that the macrothrombocytopenia is an intrinsic marrow abnormality of the WF strain. Splenectomy did not alter the MPV of WF rats. The response of WF megakaryocytes and platelets to severe, acute thrombocytopenia was similar to that of LE rats except that the shift to higher megakaryocyte DNA contents was muted and platelet recovery was slower in the WF rats. In summary, the WF rat has a hereditary macrothrombocytopenia that is recessive in nature and not due to differences in megakaryocyte size or DNA content. These results suggest that the macrothrombocytopenia of WF rats results from the formation of fewer platelets per megakaryocyte, possibly resulting from a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.

Quantitative subcellular localization of calmodulin-dependent phosphatase in chick forebrain.

Using a radioimmunoassay, we have measured the level of calmodulin-dependent phosphatase (calcineurin) in various subcellular fractions from chick forebrain. Our results revealed high levels of the enzyme in the cytoplasm and microsomes. A considerable amount was also observed in synaptosomes, where it was found exclusively in the synaptoplasm, comprising 0.32% of the total synaptoplasmic protein. Immunocytochemical localization of the phosphatase in isolated synaptosomes supported the biochemical finding. Phosphatase was not detected in nuclei, myelin, synaptic vesicles, and mitochondria. These results suggest that myelin basic protein and histone H1, widely used in biochemical characterization studies of the phosphatase, may not be physiological substrates, and that the cytoplasm, microsomes, and synaptoplasm may prove to be useful sources for the identification of physiological substrates.

Localization of acyl carrier protein in Escherichia coli.

Acyl carrier protein was localized by immunoelectron microscopy in the cytoplasm of Escherichia coli. These data are inconsistent with the previous report of an association between acyl carrier protein and the inner membrane (H. Van den Bosch, J.R. Williamson, and P.R. Vagelos, Nature [London] 228:338-341, 1970). Moreover, bacterial membranes did not bind a significant amount of acyl carrier protein or its thioesters in vitro. A thioesterase activity specific for long-chain acyl-acyl carrier protein was associated with the inner membrane.

Does cell density correlate with red cell age?

We have examined age-related changes in red cells by employing a serial hypertransfusion protocol to generate populations of in vivo-aged mouse red cells. Studies of these old cells have revealed alterations in membrane components; namely, a conversion of band 4.1b to 4.1a and an increase in the amount of membrane-associated, Triton-insoluble globin. Furthermore, we have found that the erythrocyte does change in density, but only during the earliest stages of its life-span. As the cell continues to age, there is no longer a correlation between age and density.

Changes in the cytoskeletal structure of human platelets following thrombin activation.

The temporal changes in human platelet actin polymerization, cytoskeletal morphology, and protein content that occur during thrombin-induced platelet activation were investigated by analysis of Triton-extracted platelets. Measurement of the DNase inhibitory activity of control platelets immediately after adding an equal volume of 2% Triton X-100, 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.1 M Tris, pH 7.4, showed that approximately 50% of the actin in unstimulated platelets was filamentous and unable to inhibit DNase-catalyzed hydrolysis of DNA. Activation of platelets with thrombin for 15 s caused the amount of actin in the filamentous form to increase to approximately 65%. Examination of the morphology and protein composition of these filamentous structures showed that the cytoskeletal structures from control platelets consisted of a random array of filaments which contained 14% of the total platelet myosin and 6% of the total actin-binding protein. In contrast, the cytoskeletal structures of thrombin-activated platelets appeared as cytoskeletal structures of individual platelets. The composition of these cytoskeletons varied depending on the time of thrombin activation. Those from platelets activated with thrombin for 15 to 30 s contained 90% of the platelet myosin and 20% of the platelets with thrombin before Triton addition resulted in a decreased association of myosin to 60% with no change in either the actin or actin-binding protein content of the cytoskeletal structures. Since these changes are rapid and precede serotonin secretion, it is suggested that they are involved in the physiological response of the platelet.

Interaction of platelet plasma membranes with thrombin-activated platelets.

This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin-activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.

Protein architecture of the erythrocyte membrane.

The normal human erythrocyte is comprised of as many as 90 polypeptides. These membrane polypeptides are organized asymmetrically within the membrane. In a well-washed erythrocyte, all the polypeptides exposed on the outside surface are transmembrane proteins. Six such polypeptides have been identified. They are the anion transport component protein 3, the glucose transport protein 4.5 and the sialoglycoproteins PAS 1, 2', 2 and 3. The major membrane protein, protein 3, which comprises 25% of the total membrane peptide, interacts on the cytoplasmic surface with the cytoskeletal components. The sialoglycoprotein PAS 2 has also been shown to interact with cytoskeleton and has been named glycoconnectin. Employing a monolayer freeze-fracture technique, the transmembrane proteins have been shown to distribute asymmetrically. For example, the major sialoglycoproteins distribute with the outer half of the bilayer (E-face) while protein 3 is found exclusively on the inner half of the bilayer (P-face). The anchoring of the transmembrane proteins determines on which half of the bilayer the protein will be found. Well-defined fragments of the sialoglycoprotein are produced by the freeze-fracture procedure indicating that selected covalent bonds of these transmembrane proteins were broken. Correlation of these results with the appearance of intramembrane particles on the E- and P-faces indicate that protein 3 may account for most of the intramembrane particles found on the P-face, while the sialoglycoproteins probably account for little, if any, of the particles. Although the evidence is not conclusive, protein 4.5 may account for the particles on the E-face of the freeze-fractured human erythrocyte membrane.

Identification of membrane proteins mediating the interaction of human platelets.

Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that are observable after Triton extraction. The cytoskeletal structures from thrombin-aggregated platelets were similar to those from thrombin-activated platelets, except that the structural elements from individual platelets remained aggregated rather than randomly dispersed in the actin filaments. This suggested that the membrane components that mediate the direct interaction of platelets were in Triton residue from aggregated platelets. Only a small percentage of the membrane surface proteins and glycoproteins were found in the cytoskeletal structures from either washed platelets or thrombin-activated platelets. In contrast, the aggregated cytoskeletal structures from thrombin-aggregated platelets contained membrane glycoproteins IIb (26 percent of the total in pre-extracted platelets) and III (14 percent), suggesting that one or both of these glycoproteins participate in the direct interaction of platelets during aggregation.

Distribution of transmembrane polypeptides in freeze fracture.

Human erythrocytes have been freeze-fractured, and the polypeptides associated with the separate halves of the membrane bilayer have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The transmembrane proteins were differentially separated by the fracture process. Although sialoglycoproteins associated with the outer half of the membrane, the anion transport protein (band 3) mainly remained with the inner half of the membrane. Well-defined fragments of the sialoglycoproteins were produced by the freeze-fracture procedure, indicating that selected covalent bonds of these transmembrane proteins were broken.

Comparison of solubility properties of alpha-paramyosin, beta-paramyosin and acid-extracted paramyosin.

The solubility properties of paramyosin in the zones of pH and ionic strength in which aggregation occurs were initially studied using preparations isolated by a method originally described by Bailey (Bailey, K. (1956), Pubbl. Stn. Zool. Napoli 29, 26). Other preparations yielding apparently different protein components have been described by Hodge (Hodge, A.J. (1952), Proc. Natl. Acad. Sci., U.S.A. 38, 850) using acid conditions, and Stafford and Yphantis (Stafford, W.F., AND Yphantis, D. (1972), Biochem. Biophys. Res. Commun. 49, 848) have identified alpha-, beta-, and gamma-paramyosin using various times and temperatures of extraction with or without ethylenediaminetetraacetic acid. We have found that acid-extracted paramyosin is very similar if not identical to alpha-paramyosin, but that both acid and alpha forms differ considerably from beta- and gamma-paramyosin. Beta-Paramyosin precipitates abruptly from solution in narrow zone of pH below neutrality, and increases in ionic strength shift the zone of precipitation toward lower pH values. In contrast, both acid and alpha-paramyosin show gradual aggregation with changing pH at lowerionic strength (less than 0.3) but sharp transitions similar to beta-paramyosin at higher ionic strength (greater than 0.3). Transitions were also found at lower pH (ca. 4.0) which were not mirror images of transitions at higher pH (ca. 7.0). Viscosity measurements show that acid extracted paramyosin is close in behavior to a native extract obtained by extraction in mild, nondenaturing media containing mixed antibiotics. Each of these extracts differed considerably from beta-paramyosin. Mild, nonhydrolytic procedures employed by others to remove small, noncovalent bonded components or to separate protein complexes were not effective in converting alpha- to beta-paramyosin. Comparison of extraction procedures strongly supports the suggestion of Stafford and Yphantis that beta- and gamma-paramyosin are hydrolytic products of alpha-paramyosin and that the proteases responsible may be of bacterial origin.

Localization of thyroid peroxidase and the site of iodination in rat thyroid gland.

The iodinated protein was localized in thyroid tissue slices by using radioautography. In unfixed tissue, the labelled protein was localized in the colloid, whereas, in tissue that was fixed before the 125I addition, the label was within the follicular cell. This localizes thyroid peroxidase largely on the endoplasmic reticulum of the cell.

Accidental persistent infection of cell lines by Newcastle disease virus, showing three unusual features--defective neuraminidase, temperature sensitivity and intranuclear inclusions.

A persistent, defective infection by an unknown strain of Newcastle disease virus (NDV) appeared accidentally in established lines of pig, ox and sheep kidney cells. Virus particles released from the persistently infected cells were not infectious and were deficient in neuraminidase activity. Synthesis of some of the virus-specified proteins in the persistently infected cells was temperature-sensitive. Co-cultivation of mixed populations of carrier cells and healthy chick embryo cells induced cell fusion with the formation of multinucleate heterokaryons and intra-nuclear inclusions. The development of inclusions in the chicken nuclei was not accompanied by 'rescue' of infectious NDV.

Translocation of carbon in powdery mildewed barley.

This paper compares translocation in healthy and powdery mildew (Erysiphe graminis f. sp. hordei, race CR3) infected barley (Hordeum vulgare, variety Manchuria). The sink-like properties of the powdery mildew infection were used to determine what effect imposing a sink in the midst of normal source tissue (mature primary leaf) had on the translocation process. The pattern of translocation was determined by monitoring the movement of (14)C which was photosynthetically incorporated from (14)C either by the primary or second leaf. In the healthy primary leaf of barley, (14)C fixed in the tip section of the blade was preferentially translocated to the root, whereas (14)C fixed in the basal section was primarily translocated to the shoot. When a sporulating powdery mildew infection was present in the mid-section of the primary leaf, (14)C fixed in that section or in the acropetal healthy tip section readily accumulated in the infection area. Labeled carbon fixed in the healthy basal section was translocated into the other parts of the plant with only a small fraction moving acropetally into the infected mid-section. The (14)C fixed by the second leaf was translocated to the root and younger shoot with very little entering the primary leaf. The presence of the mildew infection did not alter this pattern.

Biphasic inhibition of photosynthesis in powdery mildewed barley.

A careful restudy of the photosynthetic process of barley (Hordeum vulgare) infected with powdery mildew (Erysiphe graminis f. sp. hordei) is reported. Unlike previous reports, which indicated a stimulation in infected host tissue photosynthesis during early stages of the disease followed by a rapid decline in activity, this study observed no stimulation but instead a biphasic inhibition in infected host photosynthesis under physiological concentrations of CO(2). Under high CO(2) (1.0%) a stimulation was observed during early stages of infection. The inhibition in low CO(2) and stimulation in high CO(2) of infected host photosynthesis was shown to occur in healthy host tissue when in the presence of 10(-2)m alpha-hydroxy-2-pyridinemethanesulfonic acid. The possible significance of these similar results is discussed.

Distribution of the Products of Photosynthesis between Powdery Mildew and Barley.

The photosynthetic assimilation of (14)CO(2) has been studied in healthy and mildew-infected barley. The parasite was separated from the host by removing the mycelium with a camel's hair brush. The ethanol soluble metabolites of the parasite, infected host and healthy host were extracted, separated by paper chromatography and individually identified. From this work it appears that there is a rapid movement of label from host to parasite mainly in the form of sucrose which is then quickly metabolized into many compounds. The majority is converted into mannitol, and lesser amounts are converted into trehalose, arabitol, aspartic acid, and glutamic acid. In conidia the major carbon reserve is arabitol instead of mannitol, with lesser amounts of trehalose and mannitol.Photosynthetic uptake of (14)CO(2) by the complex decreases steadily after inoculation as compared with healthy leaves. However, the ethanol soluble metabolites of the infected host tissue differ only slightly from those of healthy host tissue. The major differences are a slight decrease in the amount of sucrose and increases in malic acid and serine.