Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.     

Integrins in morphogenesis and signaling

Integrins are a family of heterodimeric transmembrane receptors that provide a physical and biochemical bridge between components of the extracellular matrix and the intracellular physiological environment. Binding of integrins to their ligands results in the formation of cytoplasmic multi-protein assemblies composed of both cytoskeletal and signaling molecules. The composition and activity of these assemblies is regulated by the nature of integrin-ligand interactions, as well as by intracellular regulators that include tyrosine kinases and phosphatases, PKC, and small GTPases. Integrin-mediated cellular physiological responses include the activation of signal transduction, cytoskeletal rearrangements, and co-regulation of growth factor activities. These responses, combined with integrin-mediated cell adhesion, play a major role in tissue morphogenesis and developmental processes.     

Abrogation of Smad3 and Smad2 or of Smad4 gene expression positively regulates murine embryonic lung branching morphogenesis in culture

Smad genes are recently identified intracellular effectors for receptor signaling in the BMP/activin/TGF-beta pathway. Since TGF-beta ligands are known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad genes negatively regulate lung organogenesis. Antisense oligodeoxynucleotides were designed to attenuate Smad3 and Smad2 gene expression in embryonic (E11) mouse lungs over 4 days in culture. Endogenous Smad3 and Smad2 mRNA levels were suppressed by 97 and 91%, respectively, in cultured embryonic lungs when antisense oligodeoxynucleotide (40 microM) to Smad was added, compared to scrambled and sense sequence controls. The corresponding Smad3 and Smad2 protein amounts were also decreased respectively by 86 and 90% in lungs treated with Smad3 and Smad2 antisense oligodeoxynucleotide. Phenotypically, Smad antisense oligodeoxynucleotides resulted in a concentration-dependent increase in lung branching: embryonic lung branching was stimulated by up to 53% in culture with 40 microM antisense oligodeoxynucleotide, whereas both scrambled and sense controls showed no stimulatory effect. Thus, inhibition of endogenous Smad3 and Smad2 gene expression resulted in stimulation of embryonic lung branching similar to that caused by inhibition of TGF-beta type II receptor expression and signaling (J. Zhao et al., 1996, Dev. Biol. 180, 242-257). Abrogation of Smad4 (DPC4), the downstream mediator of Smad3 and Smad2 proteins, with antisense oligodeoxynucleotide, also resulted in increased branching morphogenesis. Furthermore, while TGF-beta alone inhibited lung branching morphogenesis in culture, addition of exogenous TGF-beta 1 could not overcome the stimulatory effect on lung branching of Smad antisense oligodeoxynucleotide treatment. By immunohistochemistry, Smad proteins were localized mainly to the epithelial cells lining the branching distal airways, indicating that Smad genes could regulate lung morphogenesis through mesoderm-endoderm interaction. Our results demonstrate, for the first time, that abrogation of Smad2 and Smad3 or of Smad4 gene expression stimulated early mouse embryonic lung branching morphogenesis in culture, possibly through reversing the negative influence of endogenous TGF-beta signaling upon lung branching morphogenesis.     

Viral determinants of pea early browning virus seed transmission in pea

Pea early browning virus (PEBV) is a member of the genus, Tobravirus. It is transmitted by soil-inhabiting trichodorid nematodes and through seeds from diseased plants. By introducing mutations into the PEBV genome, we have studied the viral determinants of seed transmission in pea. Neither deleting a portion of the genome containing the three nonstructural genes in RNA2 nor the interuption of any of the three genes individually prevented PEBV seed transmission. However, a comparison of two PEBV isolates indicated a minor role for RNA2 or its products. In contrast, the removal of the coding sequence of the 12K gene in RNA1 almost completely abolished viral seed transmission. The virus lacking the 12K gene caused more severe symptoms on leaves and pods, and accumulated to a higher level than the wild-type virus in both types of tissues. However, the 12K deletion mutant accumulated poorly in anthers and carpels, and could not be detected in pollen grains and ovules. These results suggest that the 12K gene is involved in the infection of the gametic cells and hence the seed transmission of PEBV in pea.     

Biotin synthesis in higher plants: purification and characterization of bioB gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells

Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E. coli overexpression strain. The purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe-2S] clusters. Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.     

Ultrastructure and morphogenesis of human immunodeficiency virus

The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.     

Human cervico-facial morphogenesis. Evaluation of acquired data and current outlook. Second part: cervical morphogenesis

After having recalled the formation of the so-called "branchial" organisation, each component of the segmentary units constituting this organisation is analysed, as well as their particularities. This lead us to recognize the existence of only five branchial arches in the human embryo, without an intermediary arch between the fourth arch and the pulmonary arch. This question is moreover linked to the signification of the so-called "ultimobranchial" body, which must be connected with the fourth pharyngeal pouch. The question of cervical segmentation is inseparable from the question of cephalic metamerisation. Two segments are individualised in front of the mandibular process: the fronto-nasal process and the maxillary process, corresponding to premandibular arches, which existence is well established in paleontology. In addition to the peripheral expression of this cervico-cephalic segmentation marqued by primitive characters. We observe the paraxial expression of segmentation by the somitomeres and the somites. Recent data provided by the developmental genes confirm that only one process is at the origin of these two expressions which appear distinct, but lead to a unitary organisation. The mutation of the gene Pax 6 affecting in the same time the nasal placode and the optic vesicle confirms the unity of the fronto-nasal process. The pre-eminence of genetic expression on skeletal, muscular and nervous tissues with respect to the vascular system confirms the inadequacy of the criterion given by the aortic arches for the analysis of the cervico-cephalic development, although it is classically linked to the concept of an embryonic "branchial apparatus".     

Architectural patterns in branching morphogenesis in the kidney

During kidney development, several discrete steps generate its three-dimensional pattern including specific branch types, regional differential growth of stems, the specific axes of growth and temporal progression of the pattern. The ureteric bud undergoes three different types of branching. In the first, terminal bifid type, a lateral branch arises and immediately bifurcates to form two terminal branches whose tips induce the formation of nephrons. After 15 such divisions (in humans) of this specifically renal type of branching, several nephrons are induced whose connecting tubules fuse and elongate to form the arcades. Finally, the last generations undergo strictly lateral branching to form the cortical system. The stems of these branches elongate in a highly regulated pattern. The molecular basis of these processes is unknown and we briefly review their potential mediators. Differential growth in three different axes of the kidney (cortico-medullary, dorsoventral and rostro-caudal) generate the characteristic shape of the kidney. Rapid advances in molecular genetics highlight the need for development of specific assays for each of these discrete steps, a prerequisite for identification of the involved pathways. The identification of molecules that control branching (the ultimate determinant of the number of nephrons) has acquired new urgency with the recent suggestion that a reduced nephron number predisposes humans to hypertension and to progression of renal failure.     

VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo

Three approximate free energy calculation methods are examined and applied to an example ligand design problem. The first of the methods uses a single simulation to estimate the relative binding free energies for related ligands that are not simulated. The second method is similar, except that it uses only first derivatives of free energy with respect to atomic parameters (most often charge, van der Waals equilibrium distance, and van der Waals well depth) to calculate free energy differences. The last method PROFEC (Pictorial Representation of Free Energy Components), generates contour maps that show how binding free energy changes when additional particles are added near the ligand. These three methods are applied to a benzamidine/trypsin complex. They each reproduce the general trends in the binding free energies, indicating that they might be useful for suggesting how ligands could be modified to improve binding and, consequently, useful in structure-based drug design.     

Genetic control of flower shape in Antirrhinum majus

The asymmetric shape of the Antirrhinum corolla is determined by genes acting differentially along the dorsoventral axis of the flower. Genes so far identified determine asymmetry by acting in dorsal regions. We describe a gene, divaricata, which in contrast to previously identified genes acts in ventral regions of the flower. We show by the analysis of mutant combinations that the divaricata gene is negatively regulated by the dorsal genes cycloidea and dichotoma. In addition, we show by the analysis of chromosomal duplications that the divaricata gene acts in a dosage-dependent manner, suggesting that the level of its product is critical for determining ventral identities.     

Fine structure and morphogenesis of Borna disease virus

Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with n-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.     

Primer-induced labeling of pea and field bean chromosomes in situ and in suspension

A protocol for primed in situ DNA labeling (PRINS) was optimized for pea (Pisum sativum L.) and field bean (Vicia faba L.) chromosomes attached to coverslips. Cloned DNA or synthetic oligonucleotides were used as probes for repetitive DNA sequences (rDNA, Fok-element) and different reaction conditions were tested to achieve the highest specific signal-to-background ratio. A procedure based on direct labeling by fluorescein-dUTP was compared with an indirect one using digoxigenin detected by fluorescently labeled antibody. Under optimal conditions, strong and specific signals were obtained exclusively on chromosome regions known to contain respective DNA sequences. Compared to the direct labeling, significantly stronger signals were obtained when the indirect procedure was used. Both types of labeling were successfully applied to chromosomes in suspension and were shown to produce signals comparable to that obtained with chromosomes attached to coverslips. It is expected that primed in situ DNA labeling en suspension (PRINSES) will provide a basis for flow-cytometric discrimination and sorting of otherwise indistinguishable chromosomes according to their specific fluorescent labeling.     

Association between ion fluxes and defense responses in pea and cowpea tissues

The glycopeptide elicitor from a pea pathogen, Mycosphaerella pinodes, induced rapid alkalinization and increases in levels of Na+ and K+ ions in the extracellular solution upon contact with pea and cowpea tissues. The presence of monensin, nigericin, lidocaine, quinidine or phenytoin together with the elicitor markedly inhibited these changes, whereas the presence of valinomycin, gramicidin D, tetraethylammonium, CsCl and aminopyridine did not. The production of phytoalexins in pea and cowpea tissues was also strongly inhibited by the simultaneous presence of the former reagents but not of the latter reagents. Inhibitory effects on the production of phytoalexins were diminished when monensin, nigericin or a Na(+)-channel blocker was applied 3 h after the start of treatment with elicitor. Furthermore, orthovanadate and neomycin, which suppress defense responses in both tissues, also inhibited the above mentioned changes. By contrast, the species-specific suppressor from M. pinodes inhibited the elicitor-induced release of Na+ and K+ ions from pea tissues, but, conversely, by itself it elicited either the defense response or the release of Na+ and K+ ions from cowpea tissues. The results indicate that these ion-related changes, in particular the efflux of Na+ and K+ ions, might be closely associated with the signal transduction system for defense responses at the tissue level.