Laminin synthesized by stationary and migrating rat liver epithelial cells lacks the A chain

The expression of laminin chains was analyzed in normal and Ha-ras1-transformed rat liver epithelial cells. The normal, nontumorigenic cells were induced to migrate by epidermal growth factor, whereas the Ha-ras1-transformed, malignant derivatives migrate constitutively. None of these cells express a typical (EHS-like) laminin A chain. Immunoprecipitation of [35S]-methionine-labeled liver cell lysates with an antibody against EHS-laminin revealed B1 and B2 chains and, in addition, two high Mr polypeptides. These polypeptides were not recognized by the antibody in immunoblots, suggesting that they might constitute alternative laminin A chains. Analysis of the expression of all three laminin chains at the RNA and protein level revealed that the pattern of expression of the stationary cells does not differ from that of the migratory ones and is also not influenced by epidermal growth factor. These results indicate that expression of a typical laminin A chain by rat liver epithelial cells is not required for the secretion and deposition of the protein in the extracellular matrix. The data also indicate that an EHS-like laminin A chain is not required for the migration of these epithelial cells.     

Promoter hypermethylation using 24-gene array in early head and neck cancer: Better outcome in oral than in oropharyngeal cancer

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.     

Relative accuracy of spatial predictive models for lynx Lynx canadensis derived using logistic regression-AIC,multiple criteria evaluation and Bayesian approaches

We compared probability surfaces derived using one set of environmental variables in three Geographic Information Systems (GIS) -based approaches: logistic regression and Akaike's Information Criterion (AIC),Multiple Criteria Evaluation (MCE),and Bayesian Analysis (specifically Dempster-Shafer theory). We used lynx Lynx canadensis as our focal species,and developed our environment relationship model using track data collected in Banff National Park,Alberta,Canada,during winters from 1997 to 2000. The accuracy of the three spatial models were compared using a contingency table method. We determined the percentage of cases in which both presence and absence points were correctly classified (overall accuracy),the failure to predict a species where it occurred (omission error) and the prediction of presence where there was absence (commission error). Our overall accuracy showed the logistic regression approach was the most accurate (74.51% ). The multiple criteria evaluation was intermediate (39.22%),while the Dempster-Shafer (D-S) theory model was the poorest (29.90%). However,omission and commission error tell us a different story: logistic regression had the lowest commission error,while D-S theory produced the lowest omission error. Our results provide evidence that habitat modellers should evaluate all three error measures when ascribing confidence in their model. We suggest that for our study area at least,the logistic regression model is optimal. However,where sample size is small or the species is very rare,it may also be useful to explore and/or use a more ecologically cautious modelling approach (e.g. Dempster-Shafer) that would over-predict,protect more sites,and thereby minimize the risk of missing critical habitat in conservation plans.     

Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity

Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.     

Photoperiod affects the growth and development of yam plantlets obtained by in vitro propagation

The effects of photoperiod on the development of in vitro grown plantlets of yam (Dioscorea alata L.), were investigated. Plantlets were transplanted into pots, acclimatizated until they reached vegetative stages V₁ (3 leaves) or V₂ (8 leaves), and then grown under 12-h or 16-h photoperiod. The formation and development of underground tubers was only induced under 12-h photoperiod. Tuber initiation was not related to the initial vegetative stage of plants, and the tubers were visible at about 18 – 24 d. On the contrary, a 16-h photoperiod inhibited tuber formation and stimulated vine and leaf growth. The total dry matter production and the number of leaves per plant of V₁ stage plants were 50 and 30 % lower respectively, after 44 d under 12-h compared to 16-h photoperiod. These parameters were not influenced by photoperiod in V₂ stage plants. Consequently, the effect of 12-h photoperiod on dry matter of V₁ plants was attributed to a source limitation related to the early initiation of tuberization. The transfer of plants grown under 12-h to 16-h photoperiod stopped tuber growth and starch accumulation. On the other hand, it stimulated the shoots and the roots to grow.Abbreviations

Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor alpha is associated with changes in the gene expression of secreted proteins

Summary Rat liver epithelial cells are induced to migrate by epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) in serum-free medium supplemented with insulin. Immunohistological staining of the migration tracks containing laminin and fibronectin has allowed a quantitative analysis of the process. The growth factor-induced migration is relatively slow, but very efficient. Between 24 and 48 h after exposure to EGF (or TGF-α), 50 to 70% of the cells have migrated away from their site of initial attachment and spreading. This delayed effect of the interaction of the receptor with its ligands is associated with changes in gene expression, but is not associated with a stimulation of cell proliferation. In serum-free medium supplemented with insulin, the cells secrete six major proteins, as revealed by SDS-polyacrylamide gel electrophoresis. The media of cultures supplemented with insulin plus EGF (or TGF-α) contain in addition two new proteins and an increased amount of fibronectin. One secreted protein is synthesized in significantly reduced amounts. The most conspicuously EGF-induced protein (EIP-1, Mr 47 000) is detected within 2 h, depends on the continued presence of the growth factor, and has not been detected as bound to the substratum. The stringent regulation of EIP-1 suggests that this gene product might participate in the modulation of the changes induced by the growth factor. The system is being used for the further analysis of the regulation of gene expression by EGF and of the migration of normal and neoplastically transformed epithelial cells. This paper is dedicated to the memory of Dr. Luis F. Leloir. A preliminary communication has been presented at the Cold Spring Harbor Meeting on Liver Gene Expression, May 1987. Editor's Statement Mitogen-stimulated gnees are an active area of study with fibroblastic systems. In this paper the approach is extended to epithelial cells and functional correlations are also made.     

Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea

Summary Embryogenic cell suspension cultures of Pinus caribaea var. hondurensis have been cryopreserved in liquid nitrogen for up to four months, using sucrose and dimethylsulfoxide as cryoprotectants. Post-thaw growth was obtained after a short lag phase. Removal of the remaining liquid around the cells using a filter disc favoured subsequent regrowth of the cells. These reestablished cultures maintained an embryogenic potential similar to non-frozen cultures. The embryos produced were able to regenerate into plants, which are now growing in a greenhouse.Abbreviations BA 6-benzyladenine - DMSO dimethylsulfoxide - FDA fluorescein diacetate - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - PAR photosynthetically active radiation