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Biomedical subjects

J E Varner

Publications and source records attributed to J E Varner.

At least 19 recordsLinked to original sources
PubMed1996

Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans.

The terminal process of xylogenesis, autolysis, is essential for the formulation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H2O2 challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.

Amino Acid Sequence↗
PubMed1995

Differential expression of two O-methyltransferases in lignin biosynthesis in Zinnia elegans.

Caffeic acid 3-O-methyltransferase (CAOMT) and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) are involved in different methylation pathways in lignin biosynthesis. We previously showed that only the CCoAOMT was markedly induced during lignification in in vitro differentiating tracheary elements (TEs) of Zinnia elegans. To further examine the expression patterns of CAOMT in lignification, we isolated a cDNA clone for Zinnia CAOMT. RNA gel blot analysis showed that the expression of the CAOMT gene did not correlate well with lignification during in vitro TE differentiation from Zinnia-isolated mesophyll cells. Tissue-print hybridization showed that, in the young internodes, the CAOMT mRNA signal was much more evident in phloem fibers than in xylem, whereas the CCoAOMT mRNA signal was predominantly present in differentiating xylem regions. In the older internode, both the CAOMT and CCoAOMT mRNAs markedly accumulated in phloem fibers and differentiating xylem regions. Immunocytochemical localization showed that the CAOMT protein staining was much more evident in phloem fibers and xylem fibers than in xylem TEs. These results indicate that the expression of these two O-methyltransferases is differentially regulated during lignification in different cell types in Zinnia. We suggest that all the intermediates in the CAOMT-mediated methylation pathway might become substrates for the CCoAOMT-mediated methylation pathway after coenzyme A ligation when these two pathways occur in the same lignifying cell types.

Cells, Cultured↗
PubMed1994

Tissue printing.

An image of the tissue distribution of specific mRNAs, proteins, enzymes, or antigens is conveniently obtained by "printing" the cut surface of a tissue section onto a suitable substrate film and developing the film with appropriate reagents. It is also possible to localize metabolites by tissue printing. Most plant tissues leave a physical print/impression with detailed anatomical information. Epidermal surfaces of growing organs can be printed without damaging the organ. We provide this review to encourage further use and further development of these convenient and helpful procedures.

Enzymes↗
PubMed1994

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.

Cell Division↗
PubMed1994

An alternative methylation pathway in lignin biosynthesis in Zinnia.

S-Adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is implicated in disease resistant response, but whether it is involved in lignin biosynthesis is not known. We isolated a cDNA clone for CCoAOMT in differentiating tracheary elements (TEs) induced from Zinnia-isolated mesophyll cells. RNA gel blot analysis showed that the expression of the CCoAOMT gene was markedly induced during TE differentiation from the isolated mesophyll cells. Tissue print hybridization showed that the expression of the CCoAOMT gene is temporally and spatially regulated and that it is associated with lignification in xylem and in phloem fibers in Zinnia organs. Both CCoAOMT and caffeic acid O-methyltransferase (COMT) activities increased when the isolated Zinnia mesophyll cells were cultured, whereas only CCoAOMT activity was markedly enhanced during lignification in the in vitro-differentiating TEs. The induction pattern of the OMT activity using 5-hydroxyferuloyl CoA as substrate during lignification was the same as that using caffeoyl CoA. Taken together, the results indicate that CCoAOMT is associated with lignification during xylogenesis both in vitro and in the plant, whereas COMT is only involved in a stress response in vitro. We propose that CCoAOMT is involved in an alternative methylation pathway in lignin biosynthesis. In Zinnia in vitro-differentiating TEs, the CCoAOMT mediated methylation pathway is dominant.

Acyl Coenzyme A↗
PubMed1993

Gene expression patterns associated with in vitro tracheary element formation in isolated single mesophyll cells of Zinnia elegans.

Tracheary element formation from isolated Zinnia leaf mesophyll cells is an excellent system for the dissection of patterned secondary cell wall thickening and lignification. We used mRNAs from cells cultured for 48 h in the induction medium to isolate differentially regulated genes. Thirteen unique cDNA clones were isolated using a subtractive hybridization method. These clones can be divided into three distinct groups according to their characteristic gene expression in different media. The first group includes those genes whose expression is induced in the basal medium without 1-naphthaleneacetic acid (NAA) and benzyladenine; this indicates that the expression of these genes is regulated by chemical and physical factors other than these hormones. Three of these clones, p48h-229, p48h-114, and p48h-102, show significant homology to a pathogenesis-related protein II, a serine proteinase inhibitor, and a sunflower anther-specific proline-rich protein, respectively. The second group includes those genes whose expression is mainly NAA induced. One of these clones, p48h-10, shows high protein sequence homology to a barley aleurone-specific cDNA, B11E. The p48h-10-encoded protein shares some common characteristics of plant nonspecific lipid transfer proteins (low molecular weight, the secretion signal peptide, eight conserved cysteine residues, and a basic protein), although no significant protein sequence homology is found between p48-10 and other plant nonspecific lipid transfer proteins. The third group includes those genes whose expression is induced primarily in the induction medium; this indicates that the expression of these genes is closely associated with the process of tracheary element formation. Two of these clones, p48h-107 and p48h-17, show high homology to adenylate kinase and papaya proteinase I, respectively. The possible roles of these differentiation-specific genes during tracheary element formation are discussed.

Adenine↗
PubMed1991

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding.

Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in lambda gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion. Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I-V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1-3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2-6-Tyr-Pro and (Gly)2-6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2-5-Arg repeats.

Amino Acid Sequence↗
PubMed1991

Comparative localization of three classes of cell wall proteins.

The localization of the cell wall proline-rich proteins (PRPs), and the gene expression of the cell wall glycine-rich proteins (GRPs) and the hydroxyproline-rich glycoproteins (HRGPs) were examined in several dicot species. The PRPs are accumulated in the corner walls of the cortex where several cells are joined together and in the protoxylem cell walls of 3-day-old soybean root. In 1-month-old soybean plants, the PRPs are specifically deposited in xylem vessel elements of the young stem, and they are accumulated in both phloem fibers and xylem vessel elements and fibers of the older stem. Likewise, the PRPs are localized in xylem vessel elements and fibers in tomato, petunia, potato and tobacco stems. They are also found in outer and inner phloem fiber cell walls of tomato stem and in outer phloem fiber cell walls of petunia stem. The gene expression of the HRGPs and the GRPs is developmentally regulated in tomato, petunia and tobacco stems. HRGP mRNAs are abundant in outer and inner phloem regions, while GRP mRNAs are present mostly in primary xylem and in the cambium region. Immunocytochemical localization showed that the GRPs have a localization pattern similar to that of the PRPs in tomato, petunia and tobacco stems.

Amino Acid Sequence↗
PubMed1991

Justification for routine cholangiography during laparoscopic cholecystectomy.

Laparoscopic cholecystectomy has been accepted by surgeons in the United States with unprecedented rapidity. Since introduction it has become, in many areas, the standard of care for treating patients with cholelithiasis. However, as with all new surgical procedures, complications are being recognized. Bile duct injuries are a complication of laparoscopic cholecystectomy, perhaps with greater incidence than with traditional cholecystectomy. Routine cholangiography may minimize the incidence of common bile duct injury. We review our experience with laparoscopic cholangiography and suggest methods to avoid common bile duct injury.

Catheterization↗
PubMed1989

Histochemical localization of cysteine-rich proteins by tissue printing on nitrocellulose.

A rapid technique for the histochemical localization of cysteine-rich proteins in plant tissues was developed. It is based on the immediate transfer of proteins to nitrocellulose membranes when a fresh cut organ is pressed against the membrane surface. The print was labeled for cysteine-rich proteins by reduction and alkylation of cysteinyl residues with dansylated iodoacetamide [N-iodoacetyl-N'-(-5-sulfo-1-naphthyl)ethylenediamine]. The S-carboxymethylated proteins were visualized by their fluorescence when excited with 360 nm light.

Antimicrobial Cationic Peptides↗
PubMed1987

Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining and by tissue printing on nitrocellulose paper.

In soybean seed coats the accumulation of the hydroxyproline-rich glycoprotein extensin is regulated in a developmental and tissue-specific manner. The time course of appearance of extensin during seed development was studied by Western blot analysis and by immunogold-silver localization. Using these techniques extensin was first detected at 16-18 d after anthesis, increasing during development to high levels at 24 d after anthesis. Immunogold-silver localization of extensin in the seed coat showed marked deposition of the glycoprotein in the walls of palisade epidermal cells and hourglass cells. The immunolocalization of extensin in developing soybean seeds was also made by a new technique--tissue printing on nitrocellulose paper. It was found that extensin is primarily localized in the seed coat, hilum, and vascular elements of the seed.

Antibodies↗
PubMed1983

Insolubilization of hydroxyproline-rich cell wall glycoprotein in aerated carrot root slices.

The hydroxyproline-rich glycoprotein of plant cell walls is secreted from the cytoplasm as a soluble monomer which slowly becomes insolubilized. A tyrosine derivative, isodityrosine, is formed in the cell wall during this insolubilization and could serve as a protein-protein crosslink. Glycoprotein insolubilization is inhibited by peroxidase inhibitors and free radical scavengers, the most effective of which is L-ascorbate. These data support a hypothesis that the hydroxyproline-rich cell wall glycoprotein forms a covalently crosslinked wall network under the control of an extracellular peroxidase/ascorbate oxidase system.

Biopolymers↗
PubMed1981

Estimation of protein turnover in soybean leaves using magic angle double cross-polarization nitrogen 15 nuclear magnetic resonance.

Magic angle cross-polarization 15N nuclear magnetic resonance spectra of intact lyophilized soybean leaves have been obtained at 9.12 MHz. The leaves were harvested over a 6-week period from 15N-uniformly labeled plants exposed to 13CO2 for 1 to 7 days. The concentration of 13C-15N double labels in the main chains of leaf protein was determined nondestructively by double cross-polarization techniques. Both incorporation and turnover rates of these double labels were estimated and together lead to the conclusion that the protein of a mature, fully expanded soybean leaf is fairly long lived, with a half-life of about 30 days.

Kinetics↗