Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: effects on the components of transmembrane transport of indol-3yl-acetic acid

Phenylacetic acid (PAA), a naturally-occurring acidic plant growth substance, was readily taken up by pea (Pisum sativum L. cv. Alderman) stem segments from buffered external solutions by a pH-dependent, non-mediated diffusion. Net uptake from a 0.2 M solution at pH 4.5 proceeded at a constant rate for at least 60 min and, up to approx. 100 M, the rate of uptake was directly proportional to the external concentration of the compound. The net rate of uptake of PAA was not affected by the inclusion of indol-3yl-acetic acid (IAA) in the uptake medium (up to approx. 30 M) and, unlike the net uptake of IAA, was not stimulated by N-1-naphthylphthalamic acid (NPA) or 2,3,5-triiodobenzoic acid. At an external concentration of 0.2 M and pH 4.5, the net rate of uptake of PAA was about twice that of IAA. It was concluded that the uptake of PAA did not involve the participation of carriers and that PAA was not a transported substrate for the carriers involved in the uptake and polar transport of IAA. Nevertheless, the inclusion of 3–100 M unlabelled PAA in the external medium greatly stimulated the uptake by pea stem segments of [1-¹⁴C]IAA (external concentration 0.2 M). It was concluded that whilst PAA was not a transported substrate for the NPA-sensitive IAA efflux carrier, it interacted with this carrier to inhibit IAA efflux from cells. Over the concentration range 3–100 M, PAA progressively reduced the stimulatory effect of NPA on IAA uptake, indicating that PAA also inhibited carrier-mediated uptake of IAA. The consequences of these observations for the regulation of polar auxin transport are discussed.Abbreviations IAA indol-3yl-acetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - TIBA 2,3,5-triiodobenzoic acid     

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.     

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.     

Class I-like MHC molecules expressed by baboon placental syncytiotrophoblast

Human placental villous trophoblast is known to be unreactive with W6/32 and other monoclonal antibodies recognizing monomorphic determinants of human class I MHC heavy chains, whereas extravillous cytotrophoblast in the placental bed is W6/32-reactive by immunohistology. We have now demonstrated, in contrast, that syncytiotrophoblast is the only cellular component of baboon early placental villous tissue which is reactive with any of these antibodies. Radioimmunoprecipitation of detergent-solubilized baboon placental membrane preparations, and subsequent SDS-PAGE, has shown the W6/32-reactive component to have an m.w. of 41,000 and to be associated with beta 2-microglobulin, whereas baboon peripheral lymphocytes express 45,000 m.w. W6/32-reactive antigens comparable with the HLA-A,B,C heavy chains of human lymphocytes.     

Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances antibody production and protein kinase activity in murine B cells

Treatment of murine spleen cells with 30 nM TCDD resulted in an approximately 3 fold increase in unstimulated antibody production after 3 days in culture. This response was not accompanied by increased cellular proliferation and may represent an effect of TCDD on B cell activation or differentiation. Since PMA is capable of activating B cells, presumably via PKC, we have compared the effects of PMA and TCDD on protein kinase activation and phosphorylation of endogenous proteins in a highly purified preparation of B cells. In contrast to a reduction of cytosolic PKC activity, the expected effect of PMA, TCDD caused an increase in basal kinase activity with no effect on PKC activity. Addition of either PMA or TCDD resulted in enhanced phosphorylation of a similar profile of proteins, including proteins of Mr 12.2, 14.6, 29.2, 52.3 and 62.7 KDa. Addition of TCDD also resulted in the increased phosphorylation of a protein of Mr 45.2, which was unaffected by PMA. Combined treatment with PMA and TCDD resulted in additive responses. The additive effects of PMA and TCDD suggest an interaction at the level of protein phosphorylation which is mediated by different kinases. Therefore, TCDD may be stimulating B cells via an early effect on an unidentified protein kinase.     

Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.