Molecular epitopes of the ankyrin-spectrin interaction

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.     

Electrostatic potentials in membrane systems

A membrane with an arbitrary distribution of fixed charges inside and on its surfaces is considered. A procedure for calculating the local electrostatic potential at an arbitrary point of the system is described and its validity discussed. This procedure is based on the linearization of the 3-dimensional Poisson-Boltzmann equation around an exact 1-dimensional solution.     

The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'

The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the `empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl<sub>2</sub>, or by pretreatments up to 20 minutes with p-chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H<sub>2</sub>O<sub>2</sub>, by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The `steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Influence of assimilate demand on photosynthesis, diffusive resistances, translocation, and carbohydrate levels of soybean leaves

Rates of net photosynthesis and translocation, CO₂ diffusive resistances, levels of carbohydrates, total protein, chlorophyll, and inorganic phosphate, and ribulose 1,5-diphosphate carboxylase activity were measured in soybean (Glycine max L. Merrill) leaves to ascertain the effect of altered assimilate demand. To increase assimilate demand, the pods, stems, and all but one leaf (the “source leaf”) of potted plants were completely shaded for 6 or 8 days and the responses of the illuminated source leaf were monitored. Rate of net photosynthesis in the source leaf of the shaded plants was found to increase curvilinearly to a maximum on the 8th day. The source leaf of the control plants (no sink shading) maintained a constant photosynthetic rate during this period. Vapor-phase resistance to CO₂ diffusion did not vary with treatment, but mesophyll (liquid phase) resistance was significantly lower in the source leaf of the shaded plants.     

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer''s disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer''s disease.     

Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus

Maize dwarf mosaic disease is one of the most important viral diseases of maize (Zea mays L.) throughout the world. It is caused by several virus species in the family Potyviridae, genus Potyvirus, including Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Johnsongrass mosaic virus (JGMV) and Sorghum mosaic virus (SrMV). Resistance to another member of the family Potyviridae, Wheat streak mosaic virus (WSMV), is conferred by three alleles (Wsm1, Wsm2, Wsm3) in the maize inbred line Pa405, and these or closely linked genes were previously shown to confer resistance to the potyviruses MDMV and SCMV. In this study, we assessed whether Wsm alleles are linked to resistance to JGMV and SrMV. Near isogenic lines (NILs) carrying one or two of the Wsm alleles introgressed into the susceptible line Oh28 and F1 progeny from NIL × Oh28 were tested for their response to JGMV and SrMV. Our results indicate that Wsm1 provides resistance to both JGMV and SrMV in a dose-dependent manner. Wsm2 and Wsm3 each provide limited resistance, and combining Wsm alleles enhances that resistance.     

Inhalation anesthesia in adult cattle

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.