The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.     

Attachment of Pasteuria penetrans Endospores to the Surface of Meloidogyne javanica Second-stage Juveniles

Pasteuria penetrans spore adhesion to Meloidogyne javanica second-stage juveniles (J2) was examined following several different pretreatments of the latter. The detergents sodium dodecyl sulfate and Triton X-100, the carbohydrates fucose and α-methyl-D-mannoside, and the lectins concanavalin A and wheat germ agglutinin reduced spore attachment. Spores exposed to M. javanica surface coat (SC) extract exhibited decreased adherence to the J2 surface. Second-stage juveniles that had been treated with antibodies recognizing a 250-kDa antigen of J2 SC extract had fewer spores attached to their surfaces, as compared to nontreated J2, except in the head region. This inhibition pattern was similar to that of antibody-labelling on M. javanica J2 as observed by electron microscopy. It is suggested that several SC components, such as carbohydrate residues, carbohydrate-recognition domains, and a 250-kDa antigen, are involved in P. penetrans spore attachment to the surface of M. javanica.     

Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.     

Members of two gene families encoding ubiquitin-conjugating enzymes, AtUBC1-3 and AtUBC4-6, fromArabidopsis thaliana are differentially expressed

Covalent attachment of ubiquitin to other intracellular proteins is essential for many physiological processes in eukaryotes, including selective protein degradation. Selection of proteins for ubiquitin conjugation is accomplished, in part, by a group of enzymes designated E2s or ubiquitin-conjugating enzymes (UBCs). At least six types of E2s have been identified in the plantArabidopsis thaliana; each type is encoded by a small gene family. Previously, we described the isolation and characterization of two three-member gene families, designatedAtUBC1-3 andAtUBC4-6, encoding two of these E2 types. Here, we investigated the expression patterns, of theAtUBC1-3 andAtUBC4-6 genes by the histochemical analysis of transgenicArabidopsis containing the corresponding promoters fused to the -glucuronidase-coding region. Staining patterns showed that these genes are active in many stages of development and some aspects of cell death, but are not induced by heat stress. Within the two gene families, individual members exhibited both overlapping and complementary expression patterns, indicating that at least one member of each gene family is expressed in most cell types and at most developmental stages. Different composite patterns of expression were observed between theAtUBC1-3 andAtUBC4-6 families, suggesting distinct biochemical and/or physiological functions for the encoded E2s inArabidopsis.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

A comparison of calcium binding inCallinectes sapidus premolt and postmolt cuticle homogenates: Implications for regulation of biomineralization

Cuticle tissue homogenates (CTHs) fromCallinectes sapidus premolt cuticle bound approximately 367% more Ca²⁺ ions than did those from the postmolt cuticle. ThepH-stat assay which was used to comparein vitro CaCO₃ nucleation times confirmed that the premolt CTHs had greater inhibitory activity than did the postmolt CTHs. This inhibitory activity was indicated by CaCO₃ nucleation times in excess of control values. Premolt nucleation times exceeded those of postmolt samples by approximately 340%. A positive correlation was observed between Ca²⁺ binding and calcification inhibitory activity for both premolt and postmolt CTHs. Heat pretreatment of CTHs at 70°C for a 24-hr period had no significant effect on their Ca²⁺ binding. However, this heat pretreatment decreased their calcification inhibitory activity. Pretreatment of CTHs with Ca²⁺ diminished their calcification inhibitory activity. These results are consistent with a mechanism for inhibition of biocalcification by these proteins which involves their initial reversible binding to nascent calcite nuclei growth steps and kinks, rather than theirin vivo interaction with free Ca²⁺ ions in solution.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Oscillatory reaction-diffusion equations on rings

We study the behavior of traveling waves in - systems on both homogeneous and inhomogeneous rings. The stability regions in parameter space of - waves were previously known [15, 19]; the results are extended here. We show the existence of Hopf bifurcations of traveling waves and the stability of the limit cycles born at the Hopf bifurcation for some parameter ranges. Using a Lindstedt-type perturbation scheme, we formally construct periodic solutions of the - system near a Hopf bifurcation and show that the periodic solutions superimposed on the original traveling wave have the effect of altering its overall frequency and amplitude. We also study the - system on an annulus ofvariable width, which does not possess reflection symmetry about any axis. We formally construct traveling waves on this variable-width annulus by a perturbation scheme, and find that perturbing the width of the annulus alters the amplitude and frequency of traveling waves on the domain by a small (order ²) amount. For typical parameter values, we find that the speed, frequency, and stability are unaffected by the direction of travel of the wave on the annulus, despite the rotationally asymmetric inhomogeneity. This indicates that the - system on a variable-width domain cannot account for directional preferences of traveling waves in biological systems.     

Unidirectional waves on rings: Models for chiral preference of circumnutating plants

Twining plants exhibit a striking oscillation of their stems in their quest for a support. The oscillations, called circumnutation, have periods generally of 1–5 hr, and virtually all species have a preferred direction of twining. I seek to explain these chiral asymmetries in plant behavior by hypothesizing a chiral asymmetry in plant anatomy. Such asymmetries already exist, for example, in phyllotaxis. I explore wave phenomena on asymmetric but isotropic rings, and seek systems which will only support (stable) waves in one direction around the ring, and not in the other. Simulations indicate that (1) oscillatory reaction-diffusion systems do not support unidirectional waves on rings; (2) excitable reaction-diffusion systems do support unidirectional waves on rings; and (3) unidirectional phase-locking (discrete unidirectional waves) occurs in rings of coupled oscillators. Thus, chiral asymmetries of circumnutating plants cannot be explained by continuum oscillator phenomena, but can be explained by general discrete oscillators, or excitable phenomena on the continuum.