GABA-immunoreactive neurons in the photosensory pineal organ of the rainbow trout: two distinct neuronal populations

Summary The distribution of putative GABA-ergic neurons in the photosensory pineal organ of the rainbow trout was investigated by use of a specific antiserum against -aminobutyric acid (GABA). GABA-immunoreactive (GABA-IR) neurons were located in the rostral portion of the pineal end-vesicle, presumably constituting a population of interneurons. GABA-IR neurons were also found in the pineal stalk. The axons of these neurons were traced along the pineal stalk toward the brain. The terminal areas of these axons could not be established. GABA-IR glial cells were observed in the pineal end-vesicle, but not in the pineal stalk.     

Determination of the partial specific volume of cytochrome P450 LM2

The partial specific volume (v) of highly purified membrane protein cytochrome P450 LM2 monooxygenase from rabbit liver endoplasmatic reticulum has been estimated by various independent methods. The values of v obtained through our experiments are practically equal to the value calculated from the amino acid composition of the protein (0.75 cm3/g).     

Characterization of adrenergic receptors on proximal tubular basolateral membranes

Alpha adrenergic receptors are identified on basolateral plasma membranes derived from proximal tubular epithelial cells. The density of alpha 2 receptors was over two-fold greater than alpha 1 receptors. The basolateral membranes were devoid of beta receptors. These results support previous demonstrations of alpha adrenergic receptors in rat renal cortex and concur with studies which suggest a limited presence of beta receptors on rat proximal tubules.     

Differential reactions of wheat and pea genotypes to root inoculation with growth-affecting rhizosphere bacteria

Spring wheat (Triticum aestivum L.) and pea (Pisum spp.) genotypes were tested for reaction to root inoculation with rhizosphere bacteria affecting plant growth. Plant response was studied in greenhouse experiments after treatment of seedlings with bacteria suspended in nutrient broth. Significant genotype variation was found in both wheat and pea in terms of shoot dry weight and severity of bacteria-induced leaf symptoms. For most bacterial isolates tested, there was good correlation between ratings of leaf symptoms 7 to 14 days after inoculation and growth inhibition measured after four weeks. Interactions between isolates and plant genotypes were significant in both wheat and pea (P=0.0024 and 0.0001, respectively), but genotypes with sensitivity or tolerance to most isolates could be distinguished. In an outdoor pot experiment, two of the bacterial isolates caused delayed plant development and differential decreases in grain yield of wheat genotypes. The hypothesis that the reaction of wheat genotypes to the tested becteria was related to their influence on bacterial establishment in the rhizosphere could not be substantiated.     

Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex

Interaction of von Willebrand factor (vWF) with its platelet receptor only occurs in vitro in the presence of a modulator such as ristocetin. We have recently confirmed that the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor involved in the ristocetin-dependent binding of vWF by reconstitution with the purified components [Berndt, M.C., Du, X., & Booth, W.J. (1988) Biochemistry 27, 633-640]. We have now developed a similar solid-phase reconstitution assay using an alternate modulator, botrocetin, for the competitive analysis of functional domains in both vWF and the GP Ib-IX complex. Botrocetin was purified from Bothrops jararaca venom by ammonium sulfate fractionation and subsequent DEAE-cellulose and hydroxylapatite chromatography. The purified protein was a 25-kilodalton (kDa) disulfide-linked dimer with apparent subunit molecular weights of 14,000 and 14,500. Binding studies with immobilized botrocetin demonstrated that botrocetin bound to vWF and to a 52/48-kDa region of vWF that contains the GP Ib binding domain, but not to glycocalicin, a proteolytic fragment of GP Ib that contains the vWF binding site. Binding of 125I-labeled vWF to GP Ib-IX complex coated beads and to platelets was strictly botrocetin-dependent with half-maximal binding at a botrocetin concentration of congruent to 0.27 microM. Botrocetin-dependent binding of vWF was specific, saturable, and comparable to that observed with ristocetin. An anti-vWF monoclonal antibody, 3F8, inhibited ristocetin- but not botrocetin-dependent binding of vWF, suggesting the presence of distinct ristocetin and botrocetin modulator sites on vWF. The botrocetin reconstitution assay was at least an order of magnitude more sensitive than the corresponding ristocetin assay for the competitive analysis of functional domains on both vWF and the GP Ib-IX complex and has confirmed the localization of the vWF-binding domain to the 45-kDa N-terminal region of GP Ib.     

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.     

Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane

Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (means +/- SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib alpha (22 000 +/- 2700, n = 3), WM 23, epitope on GP Ib alpha (21 000 +/- 3400, n = 3), FMC 25, epitope on GP IX (20 100 +/- 2700, n = 3), and FMC 25 (Fab')2 (27 100 +/- 800, n = 2).(ABSTRACT TRUNCATED AT 400 WORDS)     

Guaianolides and chromenes from Calea species

Chemical analysis of Calea berteriana yielded, besides the known flavonoid acacetin and the sesquiterpene lactone calbertolide C, three new guaianolides, desacyl-8-tiglylsubcordatolide A and 8-epi-8-tiglylrupicolins A and B. C. prunifolia provided acacetin, calbertolide C, desacyl-8-tiglylsubcordatolide A, and two new chromenes, prunichromenes A and B. From C. solidaginea acacetin, heliangine, calbertolide C, 8-epi-8-tiglylrupicolin A and desacyl-8-tiglylsubcordatolide A were isolated. The structures of the new compounds were established by chemical and spectral methods.     

A novel centromeric repetitive DNA from human chromosome 22

A recombinant DNA clone localized in the centromeric region of chromosome 22 was isolated from a flow-sorted human chromosome 22 DNA library. When the original insert of about 1.9 kb was used to probe Southern blots of EcoRI-digested genomic DNA it revealed at least 40 fragments. A comparable pattern was obtained with each of the three subclones (800, 700, and 380 bp). In situ hybridization showed signals clustered in the region 22cen. DNA sequence analysis using the 380 bp fragment subcloned in pTZ18/19 (p22hom48.4) revealed eight copies of a 48 bp repeat and the size of hybridizing restriction fragments indicated that this tandemly repeated sequence is spread over a region of a few hundred kilobases. Whereas this novel DNA, termed D22Z3, displayed no sequence homology to rodent and monkey genomes cross-homology was discernible for DNA from two great ape species.     

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.