A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif

Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes.     

The effect of 4-week streptozotocin-induced diabetes on responses to endothelin-1 in rat isolated atria

The effect of endothelin-1 has been examined on isolated spontaneously beating right atria and electrically driven left atria from diabetic rats and age-matched controls. Diabetes was induced by a single i.v. injection of streptozotocin (65 mg/kg) 4–5 weeks before the experiments. Endothelin-1 (0.01–100 nM) caused concentration-dependent increases in atrial rate and force; the increases were not different between atria from diabetic and control rats. The ability of endothelin-1 to reduce chronotropic and inotropic responses to noradrenaline was also not different between the two groups. Endothelin-1 (10 nM) decreased the chronotropic response to sympathetic nerve stimulation (2 Hz, 10 s) in atria from control rats by 68 ± 5% (n = 8), but this decrease was slightly smaller (45 ± 6%, N = 8) in atria from diabetic rats.

The results provide no evidence to suggest that the diabetic state markedly alters cardiac responses to endothelin-1.     

Transformation of Tobacco, Tomato, Potato, and Arabidopsis thaliana Using a Binary Ti Vector System

Using a binary tumor-inducing (Ti) plasmid vector system, several plant species were transformed with a kanamycin resistance marker (neomycin phosphotransferase gene). Four Nicotiana species, seven tomato cultivars, two potato cultivars, and Arabidopsis thaliana were transformed by the binary vector transformation method. In this method, various plant organ pieces were co-cultivated with Agrobacterium tumefaciens cells carrying the binary vector, pGA472, and a helper Ti plasmid. We have also demonstrated that a wild type Ti plasmid can be used as a helper to obtain a transformed plant.