Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the binding of the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.     

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

Heterogeneity of Lethals in a "Simple" Lethal Complementation Group

Of 24 ethyl methanesulphonate-induced, recessive-lethal mutations in the region 9E1-9F13 of the X chromosome of Drosophila melanogaster, eight fall into a typically homogeneous lethal complementation group associated with the raspberry (ras) locus. Mutations in this group have previously been shown to be pleiotropic, affecting not only ras but also two other genetic entities, gua 1 and pur 1, which yield auxotrophic mutations.--The eight new mutations have been characterized phenotypically in double heterozygotes with gua 1, pur 1 and ras mutations. Despite their homogeneity in lethal complementation tests, the mutations prove quite diverse. For example, two mutations have little or no effect on eye color in double heterozygotes with ras2. The differences between the lethals are allele-specific and cannot be explained as a trivial outcome of a hypomorphic series.--Taken alone, the lethal complementation studies mask the complexity of the locus and the diversity of its recessive lethal alleles. By extension, we argue that the general use of lethal saturation studies provides an unduly simplified image of genetic organization. We suggest that the reason why recessive lethal mutations rarely present complex complementation patterns is that complex loci tend to produce mutations that affect several subfunctions.     

Determination of impurities in nucleoside 3'-phosphoramidites by fast atom bombardment mass spectrometry

Negative-ion fast atom bombardment mass spectrometry is quite useful for the identification of products and by-products formed during the synthesis of nucleoside 3'-phosphoramidites. The data show that detritylation and oxidation are side reactions which occur during the synthesis of monomeric units used in the construction of oligodeoxyribonucleotides by the phosphite triester method.     

Molecular analysis of gene deletion in aniridia-Wilms tumor association

Summary Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, ^A, ^G, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.     

TOR and aging: less is more

Metabolism and mitochondrial activity are thought to be important determinants of life span. A new study in this issue of Cell Metabolism (Bonawitz et al., 2007) suggests that the TOR pathway controls mitochondrial respiration in yeast and that the harder mitochondria work, the longer yeast live.     

Transgenic Plant Monitor (TPM): a relational database for the management and analysis of information in plant tissue culture and transformation laboratories

Increased public concern and strict statutory regulations relating tothe generation and exploitation of genetically modified organisms, make itimperative to track accurately individual plants through DNA transformationprogrammes. The ability to rapidly retrieve information associated withspecifictransgenic events and to provide accurate reports on demand is an increasinglyimportant feature for public research laboratories. Transgenic Plant Monitor(TPM) has been developed as a database structured to allow efficient recording,monitoring and analysis of the extensive and complex data generated in planttissue culture and transformation experiments. TPM is built upon the widelyavailable Microsoft Access database engine and can be readily adoptedand/or adapted by other users. The key features and the utility of TPM as aresearch tool are discussed in this article.     

Genes associated with the end of dormancy in grapes

A grape bud EST library was constructed and 4,270 ESTs sequenced. The library clones were arrayed for the purpose of investigating the level of gene expression over time, particularly leading up to the buds release from dormancy. The arrays were hybridized with P³³-labeled probes produced from samples of buds collected at weekly intervals. These probes covered the time from 9 weeks prior to bud burst until just after the emergence of the shoots. Expression patterns from these genes have been examined. It was found that 74% of the genes in the data set were homologous to known proteins. Genes were then assigned to functional categories according to their primary BLAST match. Of these 13% were involved with photosynthesis, 13% with disease resistance and defense, 5% energy, 12% metabolism, 20% protein production and processing, 25% cell structure and plant growth and the remaining 12% were unclassified The expression pattern of a selection of candidate genes retrieved from literature previously reporting an association with dormancy changes was assessed. On closer examination most of these genes relate to the oxidative processes and stress responses within the cell. The results of this study show that even in the dormant state, gene expression in the buds is high.