Transfer and co-amplification of a gene encoding a 96-kDa immune IFN-inducible human melanoma-associated antigen. Preferential expression by mouse melanoma host cells

An immune IFN-inducible human melanoma-associated glycoprotein Ag, 96-kDa MAA, having preferential distribution on metastases has been defined by mouse mAb CL203.4. To initiate molecular genetic analysis of 96-kDa MAA, the gene encoding the Ag was transfected into mouse B16 melanoma cell clone B78H1. Formation of B78H1-transfectant colonies expressing a surface Ag reactive with mAb CL203.4 in an immunorosetting assay was dependent on addition of chromosomal DNA from human melanoma cells [primary (1 degree) transfer] or from Ag-expressing transfectant cells (2 degrees, 3 degrees, 4 degrees transfer). The mAb CL203.4-reactive species expressed by the transfectant cells is a glycoprotein with a molecular mass 93-kDa, within the range of 93 to 96-kDa observed for the endogenous human Ag. In the presence of tunicamycin, an inhibitor of N-linked glycosylation, both mouse melanoma transfectants and human melanoma cells express a 50- to 51-kDa antigenic species. Human alu family repeat sequences (h-alu) are present in the genomes of 3 degrees transfectant cells. Continued presence of these h-alu after dilution of extraneous human DNA by three cycles of transfection suggests their association with the transferred 96-kDa MAA gene. Use of a selective co-amplification procedure led to transfectant cells' increased expression of 96-kDa MAA and to commensurate increases in their content of presumed 96-kDa MAA gene-associated h-alu. Preferential DNA-mediated transferability of the 96-kDa MAA+ phenotype into B78H1 cells as compared with LMTK- mouse fibroblasts suggests host cell specificity of 96-kDa MAA gene expression.     

Coenzyme reorientations in the active sites of aspartate aminotransferase isoenzymes studied by linear dichroism method

Cytosolic and mitochondrial pig heart aspartate aminotransferases (cAspAT and mAspAT) and chicken heart cAspAT have been oriented in a compressed slab of polyacrylamide gel and their linear dichroism LD spectra have been recorded. The coenzyme's tilt angles in the active sites of chicken cAspAT and pig mAspAT and their quasisubstrate complexes imitating catalytic intermediates have been computed. The computations are based on reduced linear dichroism values (delta A/A), the known directions of the transition dipole moments in the coenzyme ring and atomic coordinates of the coenzyme obtained by X-ray crystallography. It has been found that formation of the enzyme complex with glutarate and protonation of the internal pyridoxal-lysine aldimine induce reorientations of the coenzyme. As a result of protonation, the coenzyme ring tilts by 27 degrees in cAspAT and 13 degrees in mAspAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAspAT and 39 degrees in mAspAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAspAT and 53 degrees in mAspAT. It is inferred that the basic features of the active site dynamics are similar in the three AspAT's studied. The differences in the coenzyme tilt angles between cAspAT and mAspAT may be linked to catalytic and structural peculiarities of the isoenzymes.     

Self-organizing maps: stationary states,metastability and convergence rate

We investigate the effect of various types of neighborhood function on the convergence rates and the presence or absence of metastable stationary states of Kohonen's self-organizing feature map algorithm in one dimension. We demonstrate that the time necessary to form a topographic representation of the unit interval [0, 1] may vary over several orders of magnitude depending on the range and also the shape of the neighborhood function, by which the weight changes of the neurons in the neighborhood of the winning neuron are scaled. We will prove that for neighborhood functions which are convex on an interval given by the length of the Kohonen chain there exist no metastable states. For all other neighborhood functions, metastable states are present and may trap the algorithm during the learning process. For the widely-used Gaussian function there exists a threshold for the width above which metastable states cannot exist. Due to the presence or absence of metastable states, convergence time is very sensitive to slight changes in the shape of the neighborhood function. Fastest convergence is achieved using neighborhood functions which are "convex" over a large range around the winner neuron and yet have large differences in value at neighboring neurons.     

Loading and transfer control for Northern hybridization.

We report a simple and inexpensive method to quantitate loading and transfer of RNA and to examine RNA integrity for use with Northern hybridization. This technique involves quantitation by computer-assisted video densitometry of a negative photograph of 28S and 18S rRNA from ethidium bromide-stained RNA fixed to nylon membranes. This method eliminates the issue of variability of expression of "housekeeping" genes and the need for a second round of hybridization to quantitate control probes.     

The effects of monensin on membrane lipids of cultured human skin fibroblasts

We have investigated the effects of monensin, a monovalent cationophore, on the metabolism of neutral lipids, fatty acids, ceramide and phospholipids in cultured human skin fibroblasts. Treatment with 1 microM monensin for 18 h reduced the cellular cholesterol ester content to less than one-third of untreated cells, and incorporation of [3H]acetate into cholesterol ester was also reduced, to less than one-fifth. Concomitantly, a greater conversion of [3H]acetate into free cholesterol occurred. There was a moderate increase in free fatty acids, but no change in triacylglycerol content, although the content of the latter appeared to increase in the presence of fetal calf serum in the culture medium. Phosphatidylcholine decreased in content and phosphatidylserine increased among the phosphatides, but ceramide remained unchanged after monensin treatment. These findings suggest that monensin influences the metabolic interrelationships of structural lipids in fibroblasts.     

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.