Structural and conformational features that affect the interaction of polylactosaminoglycans with immobilized wheat germ agglutinin

We examined the interaction between immobilized wheat germ agglutinin and the large, polylactosamine-containing glycans from human erythrocytes and human K-562 erythroleukemic cells. Three classes of interaction were identified. One class of glycan was merely retarded during chromatography. The other two classes were retained and could be distinguished by their ease of displacement with N-acetylglucosamine (GlcNAc); one was a moderate-affinity fraction displaced by 0.1 M GlcNAc and the other was a high-affinity fraction subsequently displaced by 1.0 M GlcNAc. A relatively small fraction of the K-562 polylactosamines were in the high-affinity class. We explored the role that fucose and sialic acid substitutions play in the strength of the lectin-glycan interaction. Although sialic acid is recognized by wheat germ agglutinin, sialylation was not required for the high-affinity interaction, and the presence of sialic acids actually prevented some glycans from binding with high affinity. In contrast, fucose is not part of the binding determinant, yet the removal of fucose resulted in decreased affinity. The possibility that some of these changes in affinity were the result of conformational changes was explored using matrices that had wheat germ agglutinin immobilized at different densities. At low wheat germ agglutinin densities, adult and fetal erythroglycans and K-562 glycophorin-like glycans were not retained by the matrix. As the density increased, the proportion of glycans that were retarded, and ultimately retained, increased. While these increases in the proportions retained occurred in parallel for the three different glycans, the apparent affinities of the glycan-lectin interactions differed. The glycophorin-like glycans were always readily displaced by 0.1 M GlcNAc, even at higher wheat germ agglutinin densities. In contrast, as the wheat germ agglutinin density increased, the proportion of erythroglycans that could be displaced by 0.1 M GlcNAc decreased; at 10 mg/ml immobilized wheat germ agglutinin, greater than 80% of the erythroglycans exhibited this tighter interaction. We suggest that this higher affinity interaction is the result of the large glycans spanning adjacent wheat germ agglutinin molecules, and is determined by the proximity of these molecules and the conformation of the glycans.     

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.     

Paramolybdate anion-exchange resin, an improved catalyst for the C-1-C-2 rearrangement and 2-epimerization of aldoses

Aqueous solutions of molybdate at 90 degrees bring about the inversion of the C-1-C-2 fragment of aldoses having four or more carbon atoms, generating thermodynamically equilibrated mixtures of the starting aldose and its 2-epimer. In some cases, notably with the aldopentoses, substantial proportions of the 3-epimers are produced, as well as 2-epimers that have not undergone inversion of the C-1-C-2 fragment. These side-reactions can be controlled by using the paramolybdate form of an anion-exchange resin (AG MP-1) together with the formate form of the same resin. The latter acts to scavenge unbound molybdate and paramolybdate anions that appear to be responsible for the side reactions.     

Characterization of the cellular basis for the inhibition of cytolytic effector cells by murine placenta

Direct suppression of cytolytic effector cell function by cells of the placenta may represent one mechanism that protects the "fetal allograft" from rejection by maternal transplantation immunity. Collagenase disaggregated murine placental cells block target cell lysis by natural killer, lymphokine-activated killer, and (CTL)-type killer cells. This inhibition is reversible and noncompetitive, similar to a previously described inhibitor of CTL found in spleens of mice undergoing an acute graft vs host (GVH) response. Velocity sedimentation separation of placental cells shows that the inhibitory activity is primarily associated with cells that cosediment with nucleated fetal erythrocytes. When these erythrocytes were lysed, an increased number of non-erythrocytic cells could be separated and under this circumstance, inhibitory activity was seen in association with either small white cells or fetal erythrocytes and with large white cells. There may be several cell populations in murine placenta that can inhibit cytolytic effector cells. The possible relevance of direct placental inhibition of cytolytic effectors to protection of the "fetal allograft" is discussed.     

Pollen as a chronometer and sediment tracer,Burrinjuck Reservoir,Australia

Pollen analysis is widely used to reconstruct vegetation and land use histories, but can also provide sedimentological information. At Burrinjuck Reservoir, in south-eastern Australia, annual grass pollen peaks are used to distinguish each year's sediment, even when there are no visible laminations. In conjunction with other dating methods, this allows the determination of year by year influxes of all sediment components. Pollen grains in the Burrinjuck sediments are shown to be predominantly waterborne so that they can be used to trace sediment to its source in particular vegetation stands. Pollen concentration and the proportion of damaged pollen might also distinguish sediment eroded from topsoils and that from subsoils. Pollen analysis can thus be used to locate specific erosion events in both time and space.     

The endoscopic diagnosis of gastroesophageal malignancy. A cytologic review

Cytologic reports were compared to final diagnoses for 1,157 gastroesophageal samples from an eight-year period in order to evaluate the diagnostic accuracy of endoscopic cytology and to determine the significance of a "suspicious" cytologic report. In the subgroup of patients with adenocarcinoma evaluated by paired endoscopic biopsy and cytology, the relative and combined sensitivities of the sampling methods were studied. Cytologic examination was reported as positive or suspicious in 85% of 229 cases of malignancy. There were three false-positive diagnoses of squamous-cell carcinoma of the esophagus, representing 0.3% of all submitted samples. Suspicious cytologic reports were issued in 5% of all cases. The majority (63%) of patients with a suspicious cytologic report had a final diagnosis of malignancy, with gastric adenocarcinoma present in almost half of the cases. Adenocarcinoma was diagnosed in 168 of the patients. Combined endoscopic biopsy and cytology was more sensitive (96%) than biopsy alone (90%) in making the initial diagnosis. Cytology may be of particular value in the diagnosis of gastroesophageal malignancy when the lesions are small and superficial or where stricture precludes adequate biopsy. Regardless of the biopsy findings, patients with "suspicious" cytologic reports require careful reevaluation since a high percentage of those cases in our series were subsequently verified as having malignancy.     

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.     

Influence of Meloidogyne incognita on Resistant and Susceptible Sweet Potato Cultivars

Effects of several population densities ofMeloidogyne incognita on the sweet potato cultivars Centennial (susceptible) and Jasper (moderately resistant) were studied. Field plots were infested with initial levels (Pi) of 0, 10, 100, 1,000, 5,000, and 10,000 eggs and juveniles/500 cm³ soil in 1980 and 0, 100, 1,000, 2,000, 3,000, 4,000, and 5,000 in 1981. M. incognita population development trends were similar on both cultivars; however, at high Pi, more eggs and juveniles were recovered from Centennial than from Jasper. The highest Pi did not result in the highest mid-season (Pm) counts. Pi was negatively correlated with the number of marketable roots and root weight but positively correlated with total cracked roots, percentage of cracked roots, and cracking severity. Jasper tolerated higher Pi with greater yields and better root quality than Centennial. Cracking of fleshy roots occurred with both cultivars at low Pi.     

Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype.

Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.