The cellular pathway of sucrose transport in developing cotyledons ofVicia faba L. andPhaseolus vulgaris L.: a physiological assessment

The cellular pathway of sugar uptake in developing cotyledons of Vicia faba L. and Phaseolus vulgaris L. seed was evaluated using a physiological approach. The cotyledon interface with the seed coat is characterised by a specialised dermal cell complex. In the case of Vicia faba cotyledons, the epidermal component of the dermal cell complex is composed of transfer cells. Sucrose is the major sugar presented to the outer surface of both cotyledons and it is taken up from the apoplasm unaltered. Estimated sucrose concentrations within the apparent free space of Vicia and Phaseolus cotyledons were 105 and 113 mM respectively. Rates of in-vitro uptake of [¹⁴C]sucrose by cotyledon segments or by whole cotyledons following physical removal or porter inactivation of the outer cells demonstrated that, for both Vicia and Phaseolus cotyledons, the dermal cell complexes are the most intense sites of sucrose uptake. Accumulation of [¹⁴C]sucrose in the storage parenchyma of whole cotyledons was directly affected by experimental manipulation of uptake by the outer cell layers and plasmolytic disruption of the interconnecting plasmodesmata. These findings indicated that sucrose accumulated by the dermal cell complexes is transported symplasmically to the storage parenchyma. Overall, it is concluded that the dermal cell complexes of the developing legume embryo, irrespective of the presence or absence of wall ingrowths, are the major sites for the uptake of sucrose released from the maternal tissues to the seed apoplasm. Thereafter, the accumulated sucrose is transported radially inward through the symplast to the storage parenchyma.Abbreviations AFS apparent free space - CF 5-(6)-carboxyfluorescein - CFDA 5-(6)-carboxyfluorescein diacetate - Mes 2-(N-morpholino)ethanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SRG sulphorhodamine G The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are grateful to Stella Savoury for preparing the photomicrographs.     

Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis.

Cell-free systems are valuable tools for the dissection of complex cellular processes. Here we show that cytoplasmic extracts from cells exposed to anti-Fas antibody or UV radiation contain an activity capable of reproducing morphological changes typical of apoptosis in nuclei added to these extracts, as well as internucleosomal cleavage of DNA and proteolysis of a protein known to be cleaved during the apoptosis of intact cells. Extracts from control cell populations were inactive in this respect. These effects were partly blocked by the addition of purified Bcl-2 protein or a competitive inhibitor peptide of interleukin-1 beta-converting enzyme to the extracts. Furthermore, apoptotic activity was induced in cytoplasmic extracts from untreated cells by the addition of ceramide, a lipid second messenger implicated recently in apoptosis signaling. These extracts should prove highly useful in the dissection of molecular events that occur during apoptosis.     

Interferon-γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development

Interferon-γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF-γ addition was delayed after culture initiation. The effects of INFγ on BFU-E did not require the presence of interleukin-1α (IL-1α), tumor necrosis factor-α (TNFα), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1α, TNFα, or GM-CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL-1α- or TNFα-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INFγ antibody. In contrast, GM-CSF—induced suppression of BFU-E was negated by the simultaneous addition of anti-INFγ. We have previously shown that the addition of TNFα does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM-CSF—induced inhibition of BFU-E colony growth. © 1995 Wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.     

Synthesis and antiplatelet, antiinflammatory, and antiallergic activities of 2-substituted 3-chloro-1,4-naphthoquinone derivatives

A series of 2-substituted 3-chloro-1,4-naphthoquinones was synthesized, and the antiplatelet, antiinflammatory, and antiallergic activities of these compounds were evaluated. The structure-activity relationships in this series were also examined. Most of the 2-alkyl/arylcarboxamido derivatives of 3-chloro-1,4-naphthoquinone showed potent activities with similar trends in each of the activities evaluated.     

cAMP-stimulated termination of vitellogenesis in Hyalophora cecropia: formation of a diffusion barrier and the loss of patency

Activation of cAMP-dependent protein kinase (PKA) by cell-permeable analogs of cAMP causes early and mid-vitellogenic follicles of Hyalophora cecropia to terminate vitellogenin uptake [[Wang and Telfer, 1996], Insect Biochem. Mol. Biol. 26, 85-94 (1996)]. The response is shown here to entail the formation of an epithelial diffusion barrier. Follicle cells that have been loosely organized to provide intercellular pathways for the movement of vitellogenin to the oocyte surface transform into a tight epithelium within 1-2h of exposure to PKA activators. The follicle cells can now prevent the escape of Lucifer yellow CH that has been iontophoresed into the space surrounding the oocyte, and the entry of labeled vitellogenin from the medium. As they form this functional equivalent of a tight junction, the follicle cells further reduce the intercellular spaces by enlarging and pressing against each other, and by slowing the secretion of the sulfated glycosaminoglycan matrix that separates them during vitellogenesis. The activation of PKA in early and mid-vitellogenic follicles thus appears to trigger prematurely a set of changes that do not normally occur until the follicle has grown to a length of about 2.0mm.     

Structural basis for the RNA binding selectivity of oligonucleotide analogues containing alkylsulfide internucleoside linkages and 2'-substituted 3'-deoxyribonucleosides.

In this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)sdT, rT(2'OMe)sdT, dTsrU(2'OMe) and dT(2'OMe)srU(2'OMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the S/P-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5'sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp (or rT(2'OMe)sdTp) units into an oligodeoxynucleotide sequence was sufficient to abolish binding to complementary DNA but not RNA. The same three substitutions with dTsrU(2'OMe)p and dT(2'OMe)srU(2'OMe)p did not abolish binding to DNA but the resulting complexes had poor thermal stability. The RNA-binding 'selectivity' exhibited by these oligomers is attributed to the tendency of the 2'-substituted (branched) furanoses to adopt the C3'-endo pucker, a conformation that is inconsistent with the B-form structure of helical DNA. The preference of these sugars to exist often exclusively in the C3'-endo form is attributed to stereoelectronic effects, namely gauche and anomeric effects. Our findings support the hypothesis that nucleoside analogues puckered exclusively in the C3'-endo form may result in them being especially good binders of targeted mRNA [S.H. Kawai (1991), Ph.D. Thesis, McGill University; Kawasaki et al. (1993) J. Med. Chem. 36, 831-841].