ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.     

Effect of chronic ethanol and food deprivation on intestinal villus morphology and brush border membrane content of lipid and marker enzymes

Brush border membranes (BBM) were isolated from the jejunum and ileum of control, ad libitum (CAL); control, food-restricted (CFR); control, weight gain (CWG); and ethanol-fed (EF) rabbits. Jejunal alkaline phosphatase activity was similar among control groups, but higher in CAL than EF animals. Sucrase activity was higher in EF and CWG animals than in CAL and CFR. The alkaline phosphatase/sucrase ratio was lower in EF than control animals. Ileal enzyme marker activity was similar among EF and control animals. Sucrase (S) activity was lower in the ileum than in the jejunum. Jejunal free fatty acid and phospholipid/cholesterol (PL/C) were lower in EF than control animals, whereas ileal lipid content was generally similar among all animal groups. Total phospholipid content was similar between sites, but the cholesterol and free fatty acid content were lower in the ileum than the jejunum. The phospholipid/cholesterol ratio was increased only in the ileum of EF animals. The amount of lecithin was decreased in the jejunal BBM of EF animals resulting in a decreased choline/amine phospholipid ratio as compared with control animals. The ileal phospholipid composition was similar among all groups. A large increase in villus height is observed in the jejunum of EF animals. Villus surface area and mucosal surface area are altered with ethanol feeding and food deprivation. Thus, (i) there is a gradient of S and cholesterol between the BBM of jejunum and ileum; (ii) changes in food intake are associated with changes in the morphology as well as the enzyme marker and lipid content of BBM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Abstract. Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.     

Beak and radula growth in Octopus vulgaris

The dry weight and the crest length of the upper and lower beak, the length of the radula ribbon, the average width of the base of the six proximal and distal rachidian teeth as well as the total number of these teeth have all been related to the live body weight of octopuses between 1.1 and 4440 g. From any one of these parameters it is possible to estimate the size and approximate age of the animal.     

Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.