Abscisic acid binding to subcellular fractions from leaves of Vicia faba

A centrifugation binding assay has been used to demonstrate the binding of [³H] (±) abscisic acid to membrane-rich fractions prepared from leaves of Vicia faba L. Kinetic analysis of this binding shows evidence of saturation of binding sites with increasing concentration of ligand. Scatchard analysis of these data yields a biphasic plot possibly indicating the presence of two types of binding sites. The dissocation constant for the high affinity site has been calculated to be 3.5×10^-8 mol 1^-1.     

Vascular smooth muscle cell detachment from elastin and migration through elastic laminae is promoted by chondroitin sulfate-induced “shedding” of the 67-kDa cell surface elastin binding protein

Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in AN-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a threedimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580.

Two non-peptide substance P antagonists exhibit opposite rank orders of potency for the human and rat neurokinin-1 receptors. CP-96,345 shows selectivity for the human receptor, whereas RP67580 shows selectivity for the rat receptor. Amino acid sequence comparison of the two receptors reveals 22 divergent residues. To elucidate the molecular basis for the species selectivity of these antagonists, divergent residues in the human neurokinin-1 receptor were substituted by the rat homologs. Analysis of mutant receptors revealed that substitution of 2 residues (V116L and I290S) in the transmembrane domain of the human neurokinin-1 receptor is both necessary and sufficient to reproduce the antagonist binding affinities of the rat receptor. The nature of these substitutions and the magnitude of the changes in binding affinity suggest that residues 116 and 290 do not interact directly with the antagonist molecules. The present results support a model in which phylogenetically conserved residues interact directly with the antagonists, while phylogenetically divergent residues affect the local helical packing of the receptor. Such a change in local structure would lead to increased binding affinity for one class of antagonists and decreased affinity for another.     

A gymnosperm extensin contains the serine-tetrahydroxyproline motif

The extensin family is a diverse group of hydroxyproline-rich glycoproteins located in the cell wall and characterized by repetitive peptide motifs glycosylated to various degrees. The origin of this diversity and its relationship to function led us earlier to compare extensins of the two major groups of angiosperms from which we concluded that the highly glycosylated Ser-Hyp₄ motif was characteristic of advanced herbaceous dicots, occurring rarely or not at all in a representative graminaceous monocot (Zea mays) and a chenopod (Beta vulgaris) representative of primitive dicots. Because these results could arise either from loss or acquisition of a characteristic feature, we chose a typical gymnosperm representing seed-bearing plants more primitive than the angiosperms. Thus, salt eluates of Douglas fir (Pseudotsuga menziesii) cell suspension cultures yielded two monomeric extensins differing in size and composition. The larger extensin reported earlier lacked the Ser-Hyp₄ motif, was rich in proline and hydroxyproline, and contained peptide motifs similar to the dicot repetitive proline-rich proteins. The smaller extensin monomer reported here (Superose-6 peak 2 [SP2]) was compositionally similar to typical dicot extensins such as tomato P1, mainly consisting of Hyp, Thr, Ser, Pro, Val, Tyr, Lys, His, abundant arabinose, and a small but significant galactose content. A chymotryptic peptide map (on Hamilton PRP-1) of anhydrous hydrogen fluoride-deglycosylated SP2 yielded eight peptides sequenced after further purification on a high-resolution fast-sizing column (polyhydroxyethyl aspartamide; Poly LC). Significantly, two of the eight peptides contained the Ser-Hyp₄ motif, consistent both with the SP2 amino acid composition as well as the presence of hydroxyproline tetraarabinoside as a small (4% of total Hyp) component of the hydroxyproline arabinoside profile; thus, hydroxyproline tetraarabinoside corroborates the presence of Ser-Hyp₄, in agreement with our earlier observation that Hyp contiguity and Hyp glycosylation are positively correlated. Interestingly, other peptide sequences indicate that SP2 contains motifs such as Ser-Hyp₃-Thr-Hyp-Tyr, Ser-Hyp₄-Lys, and (Ala-Hyp)_n repeats that are related to and typify dicot extensins P1, P3, and arabinogalactan proteins, respectively. Overall, these peptide sequences confirm our previous prediction that Ser-Hyp₄ is indeed an ancient motif and also strongly support our suggestion that the extensins comprise an extraordinarily diverse, but nevertheless phylogenetically related, family of cell wall hydroxyproline-rich glycoproteins.     

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli

The integral membrane protein bacterioopsin, found in the extremely halophilic archaebacterium Halobacterium halobium, was expressed in Escherichia coli as a fusion protein containing 13 heterologous amino acids at the amino terminus. The expressed protein was localized primarily to the E. coli cytoplasmic membrane (greater than 80%) and had an in vivo half-life of 26 min. The amount of bacterioopsin in E. coli crude lysates was quantitated immunologically from Western blots and was expressed at 10-20-fold higher levels than seen previously (i.e., 17 mg/L; 5.6% of the total protein). Three distinct forms of the protein were detected immunologically: two of the forms were generated by the removal of either one or four amino acid residues at the amino terminus; the third form remained unaltered.     

Activation and inhibition of human alcohol dehydrogenase by monoclonal antibodies

The human class I alcohol dehydrogenase isozyme beta 2 beta 2 was used as the antigen to raise monoclonal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was isozyme specific and all of them exhibited a similar affinity against all isozymes of the human class I ADH. Five out of the seven monoclonal antibodies had no effect on beta 2 beta 2 activity. Antibody G3 acted as a non-competitive inhibitor with a KI of 3 micrograms/ml at pH 7.5. Increasing pH was effective in reducing the level of inhibition. On the other hand, antibody 1D4 exhibited a pH-dependent activation of ADH activity. In the presence of this antibody, the pH optimum of beta 2 beta 2 was shifted from 9 to 8.5 and total activity was increased by 70% at this optimal pH. Kinetic analysis indicates that 1D4 probably acts as a non-competitive activator and may exert its action by interacting with the coenzyme binding site.     

A review of the bioavailability of petroleum constituents

Exposure to petroleum constituents at contaminated sites may occur through a variety of pathways, including inhalation of vapors and particulates, ingestion of water and soils, and dermal contact with water and soils. Accurately assessing the human health risks from such exposures requires information on the medium‐ and route‐specific bioavailability of petroleum constituents (e.g., how well these chemicals enter the body via the gastrointestinal tract and skin). For example, when the medium or exposure route in an animal toxicity assay (e.g., ingestion of water) differs from the actual route of human exposure at the petroleum contaminated site (e.g., dermal contact with soil), adjustments should be made that reflect the relative bioavailability of the chemical in the different media. The focus of this article is on (1) the availability of oral and dermal absorption data for one PAH (benzo[a]pyrene, (B[a]P) and three VOCs in soil (benzene, toluene, and xylene); (2) factors affecting the uptake of these PAHs and VOCs from soil; and (3) ways to incorporate bioavailability data into human health risk assessments. Based on our review, we recommend the following default values for the oral and dermal absorption of B[a]P, benzene, toluene, and xylene from soil:

A review of the bioavailability of petroleum constituents

All authors

Jennifer Bramard & Barbara D. Beck

https://doi.org/10.1080/15320389209383417

Published online:

02 December 2008

Display Table

Site‐specific information such as chemical concentrations in soil, soil characteristics, soil loadings on the skin, contact site, and contact time could result in modifications of these numbers. As shown, our default absorption values are generally less than those recommended by the U.S. EPA (1991a,b,c). The implications of these estimates of bioavailability for risk assessment and for the selection of soil cleanup levels at petroleum‐contaminated sites are discussed.