Affinity-dependent cross-linking to neurotoxin sites of the acetylcholine receptor mediated by catechol oxidation

The choline homologue 3-[(trimethylammonio)methyl]catechol (TMC) has been synthesized, and the controllable features of its complex oxidation have been examined spectroscopically and correlated with its toxin binding inactivating reactions with the acetylcholine receptor (AcChR) from Torpedo californica electroplax. Affinity-dependent reactions of early intermediates in the oxidation of TMC are suggested to intercede covalently in this inactivation. At pH 7.4, where the oxidative polymerization of catechols proceeds spontaneously, pyrocatechol produced no effect on the toxin binding function of AcChR, whereas comparable concentrations of TMC led to inactivation of half of all available sites. Lower concentrations of TMC converted via oxidation with ceric salts to an in situ mixture of monohydroxylated catechols were shown to be effective in short-term incubations in inactivating approximately half of the toxin binding sites by covalent labeling of the receptor. Mixtures of dihydroxycatechol intermediates, hydroxy-p-quinones, and polymeric products led to nonspecific toxin binding site inactivation of AcChR in excess of half of all available sites. Collectively, the results suggest that both covalent labeling and oxygen reduction product inactivating mechanisms are operative in these model macromolecular site reactions and that catechol-containing affinity reagents may be useful in elucidating the molecular features of sites to which they are directed.     

Intracellular calcium and calmodulin involvement in protoplast fusion

(45)Ca(2+) uptake was compared between fusogenic and nonfusogenic Daucus carota L. protoplasts. Fusogenic protoplasts took 10 minutes to reach calcium equilibrium compared to 5 minutes in the nonfusogenic protoplasts. Intracellular stores of calcium were manipulated by isolating protoplasts in different calcium regimes. Lowering of intracellular calcium lowered fusion potential, while raising intracellular stores of calcium enhanced fusion potential. Regardless of the amount of calcium sequestered in a store, mobilization with A23187 increased fusion levels within 10 minutes. Calmodulin antagonists were potent inhibitors of protoplast fusion. This inhibition was obtained by treating cells with the calmodulin antagonists during protoplast isolation. A23187, however, only allowed a partial recovery from this inhibition, indicating that calcium flux alone was not sufficient for maximum fusion potential. On the basis of the evidence presented, we propose that calcium fluxes during protoplast isolation activate a calmodulin-mediated biochemical process that is necessary for the formation or maintenance of a fusion permissive state.     

A 62-kD sucrose binding protein is expressed and localized in tissues actively engaged in sucrose transport.

Sucrose transport from the apoplasm, across the plasma membrane, and into the symplast is critical for growth and development in most plant species. Phloem loading, the process of transporting sucrose against a concentration gradient into the phloem, is an essential first step in long-distance transport of sucrose and carbon partitioning. We report here that a soybean 62-kD sucrose binding protein is associated with the plasma membrane of several cell types engaged in sucrose transport, including the mesophyll cells of young sink leaves, the companion cells of mature phloem, and the cells of the developing cotyledons. Furthermore, the temporal expression of the gene and the accumulation pattern of the protein closely parallel the rate of sucrose uptake in the cotyledon. Molecular cloning and sequence analysis of a full-length cDNA for this 62-kD sucrose binding protein indicated that the protein is not an invertase, contains a 29-amino acid leader peptide that is absent from the mature protein, and is not an integral membrane protein. We conclude that the 62-kD sucrose binding protein is involved in sucrose transport, but is not performing this function independently.     

Chemical crosslinking and enzyme kinetics provide no evidence for a regulatory role for the 53 kDa glycoprotein of sarcoplasmic reticulum in calcium transport.

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was used to cross-link the protein components of rabbit skeletal muscle sarcoplasmic reticulum. Analysis of cross-linked material by SDS-polyacrylamide gel electrophoresis showed that both the (Ca(2+)-Mg2+)-ATPase and the 53 kDa glycoprotein could be cross-linked, since the amount of protein at the locations on the gel corresponding to uncross-linked material was reduced in the presence of 1.0 mM MBS. Cross-linked products of 130 kDa, 200-260 kDa and approx. 300 kDa were identified. Probing the cross-linked products with monoclonal antibodies against ATPase, 53 kDa glycoprotein and calsequestrin revealed no cross-linked products containing the ATPase and either calsequestrin or the 53 kDa glycoprotein over the range of molecular weights examined here. Possible interactions between the ATPase and calsequestrin or the 53 kDa glycoprotein were also investigated by studying the ATPase activity for the purified ATPase and for the ATPase in sarcoplasmic reticulum vesicles made permeable to Ca2+ with A23187. Effects of Ca2+ and ATP on the two systems were indistinguishable, providing no evidence for a major modulatory role of calsequestrin or the 53 kDa glycoprotein on the ATPase.     

Antibodies to tumor necrosis factor: a component of B cell immune responses with a role in tumor/host interaction

Infiltrating B lymphocytes are found within tumors, where their role and the antigens they recognize are poorly defined. After in vitro expansion of these cells, we were able to detect the production of antibodies to tumor necrosis factor (TNF) in 13 of 17 human tumors studied. These antibodies were detected by both enzyme-linked immunosorbent assay and by neutralization. Anti-TNF antibodies were not produced by resting peripheral blood B cells of normal subjects. However, anti-TNF antibodies were produced by B cells obtained from healthy individuals, after either in vivo or in vitro antigenic stimulation. This suggests that anti-TNF antibody production may constitute part of the overall B cell response to antigens. The intratumoral production of anti-TNF antibody may play a role in tumor/host interactions.This work was supported by grants from FAPESP (90/1844-4) and from the Share Foundation and the Concern Foundation     

The mouse Cat4 locus maps to Chromosome 8 and mutants express lens-corneal adhesion

Cat4 is the second largest allelism group in the collection of mouse dominant eye mutations recovered in Neuherberg and carriers express anterior polar cataract, central corneal opacity, and lens-corneal adhesions. We have mapped the Cat4 locus of the mouse to central Chromosome (Chr) 8 at position cM 31. Histological characterization of Cat4 ^a heterozygotes and homozygotes indicates failure of separation of the lens vesicle from the surface ectoderm. Human anterior segment ocular dysgenesis (ASOD) is autosomal dominant, carriers express an eye phenotype similar to that of Cat4 ^a carriers, and it has been mapped to a region of 4q homologous to mouse central Chr 8. Thus, on the basis of phenotype and map position, Cat4 may be a mouse model of human ASOD. The genes Junb, Jund1, Mel, and Zfp42 are discussed as possible candidates for Cat4. Received: 31 October 1996 / Accepted: 20 January 1997     

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.     

Stable transformation of maize: the impact of feeder cells on protoplast growth and transformation efficiency

Summary The importance of cell culture conditions, including the use of feeder cells, on protoplast growth and transformation in maize (Zea mays L.) was investigated. Total GUS activity, measured two days after transformation, was five-fold higher in protoplasts cultured on feeder cells compared to those grown in the absence of feeder cells. Since the specific activity of GUS was only slightly higher in the transformed protoplasts plated over feeder cells, the stimulation in transient gene expression resulted mainly from the improved environment provided by the feeder system. For stable transformation, either PEG treatment or electroporation of protoplasts was used to introduce the neo gene. When PEG was used, over 85% of the putative transformants (resistant to kanamycin) contained the neo gene. The combination of PEG transformation and the optimized cell culture protocol using feeder cells enabled the selection of about 100 stably transformed lines per gFW of cells. Electroporation was less efficient.