Locomotory behavior, contact inhibition, and pattern formation of 3T3 and polyoma virus-transformed 3T3 cells in culture

The social behavior of 3T3 cells and their polynoma virus-transformed derivative (Py3T3 cells) was examined by time-lapse cinemicrography in order to determine what factors are responsible for the marked differences in the patterns formed by the two cell lines in culture. Contrary to expectations, both cell types have been found to exhibit contact inhibition of cell locomotion. Therefore, the tendency of 3T3 cells to form monolayers and of Py3T3 cells to form crisscrossed multilayers cannot be explained on the basis of the presence versus the absence of contact inhibition. Morevover, with the exception of cell division control, the social behavior of the two cell types is qualitively similar. Both exhibit cell underlapping and, after contact between lamelliopodia, both show inhibition of locomotory activity and adhesion formation. Neither cell type was observed to migrate over the surface of another cell. The two cell types do show quantitative differences in the frequency of underlapping, the frequency with which contact results in inhibition of locomotion, and the proportion of the cell margin that adheres to the substratum. The increased frequency pf Py3T3 underlapping is correlated with the reduced frequency of substratum adhesions, which in turn favors underlapping. On the basis of these observations, it is concluded that the differences in culture patterns are the result of differences in the shapes of the individual cells, such that underlapping, and hence crisscrossing, is favored in Py3T3 cell interactions and discouraged in 3T3 cells.     

Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae

Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7p's COOH terminus shifts Pex7p's intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the "extended shuttle" model of peroxisome import receptor function and open the way to future studies of receptor export.     

Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes.

Acyl-CoA oxidase is a major induced enzyme in peroxisomes of Candida tropicalis grown on fatty acids. The gene, POX4, encoding acyl-CoA oxidase was expressed in vitro, and the resulting polypeptide was imported into purified peroxisomes in a temperature-dependent fashion. Plasmids containing fragments of POX4 were prepared, expressed and the polypeptides tested for import into peroxisomes. We identified two regions of acyl-CoA oxidase (amino acids 1-118 and 309-427) that contained information that specifically targeted fragments of acyl-CoA oxidase to peroxisomes. The corresponding regions of the gene were fused to cDNA encoding the cytosolic enzyme dihydrofolate reductase (DHFR), and the expressed fusion proteins were likewise imported into peroxisomes. DHFR itself neither bound to, nor was imported into peroxisomes. Thus, there are at least two regions of peroxisomal targeting information in the acyl-CoA oxidase gene.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.     

Suramin prevents import of acyl-CoA oxidase into rat liver peroxisomes.

The inhibitory effect of suramin on the import of [35S]acyl-CoA oxidase into purified rat liver peroxisomes was investigated in vitro. The import of acyl-CoA oxidase was inhibited completely by 10 microM suramin, whilst the latency of catalase remained unchanged. The important value decreased 60% by pretreatment of peroxisomes with 10 microM suramin, but it did not decrease by pretreatment of translation products. Polysulfonate compounds which have two clusters of negative charges, such as Cibacron blue F3GA and Trypan blue, as well as suramin, inhibited the import, whilst mono- and disulfonate compounds did not.     

Rat liver peroxisomes catalyze the beta oxidation of fatty acids

Peroxisomes were purified by differential and equilibrium density centrifugation from the livers of rats treated with clofibrate to enhance their peroxisomal system of fatty acid oxidation. These purified peroxisomes were tested for the presence of crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase using spectroscopic techniques that utilize the characteristic absorption bands of the appropriate 4-carbon acyl-CoA substrates. All three enzymes were found. Analysis of the fractions from equilibrium density centrifugation revealed major peaks of these enzyme activities in peroxisomes and excluded contamination by mitochondria as an explanation of the results. In the presence of excess CoA the purified peroxisomes oxidized palmitoyl-CoA to acetyl-CoA, and reduced NAD, with a 1:5:5 stoichiometry. The peroxisomes were inactive with butyryl-CoA and less active with octanoyl-CoA than with lauroyl-CoA or palmitoyl-CoA; they appear specialized for the beta oxidation of long chain fatty acids.     

The import receptor Pex7p and the PTS2 targeting sequence

This chapter concerns one branch of the peroxisome import pathway for newly-synthesized peroxisomal proteins, specifically the branch for matrix proteins that contain a peroxisome targeting sequence type 2 (PTS2). The structure and utilization of the PTS2 are discussed, as well as the properties of the receptor, Pex7p, which recognizes the PTS2 sequence and conveys these proteins to the common translocation machinery in the peroxisome membrane. We also describe the recent evidence that this receptor recycles into the peroxisome matrix and back out to the cytosol in the course of its function. Pex7p is assisted in its functioning by several species-specific auxiliary proteins that are described in the following chapter.     

Short-term fasting, seizure control and brain amino acid metabolism

The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.