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.     

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.     

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.     

In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.     

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.     

Heterogeneity of Lactobacillus plantarum isolates from feta cheese throughout ripening

Thirty-two Lactobacillus plantarum strains isolated from Feta cheese throughout ripening were studied for their phenotypic characteristics, protein profile of cell-free extracts, enzyme profiles, plasmid profiles, proteolytic and acidifying abilities and ability to grow at low pH and in the presence of bile. Results showed that some biotechnologically important characteristics, such as acidifying and proteolytic activities, can differ between strains. In addition, different plasmid profiles suggest the presence of different Lact. plantarum strains in Feta cheese throughout ripening. The results suggest the possibility of choosing strains with specific biotechnologically interesting properties.