Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with ¹²⁵I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Intracellular cAMP levels in normal rat mammary gland and adenocarcinoma. In vivo vs in vitro.

Intracellular cAMP levels determined by radioimmunoassay technique were compared in normal rat mammary gland and DMBA-induced mammary adenocarcinoma as well as in epithelial cells derived from these tissues and grown in monolayer cultures. It was found that cAMP levels were higher in mammary tumors (0.643 p mole/mg wet weight) than in normal gland (0.158 p mole/mg). In contrast, cAMP levels in cultured adenocarcinoma cells were lower than those in normal mammary epithelial cells. The apparent contradiction may be a consequence of the fact that $\text{in vivo}$ cAMP values represent the average value of the composite cell types and not the epithelial components in question.     

The permeability of liposomes to nonelectrolytes. II. The effect of nystatin and gramicidin A.

The process of selective permeation of nonelectrolytes across liposomes of different lipid composition and amount of cholesterol has been studied. The extent of the selectivity for diffusion within the membranes has been found to be related to the physical state of the hydrocarbon chains. It has been also found that incorporation of cholesterol into egg-lecithin membranes decreases the overall permeability by affecting the dehydration step more than the subsequent diffusion of the solute. The incorpporation into liposomes of the antibiotics nystatin and gramicidin A produces changes in the selective permeation of nonelectrolytes that are consistent with the formation by these molecules of aqueous pores of fixed dimensions. Finally, comparisons are made between the process of permeation in biological membranes and in liposomes with and without antibiotics.     

Migration inhibitory activity in serum and cell supernatants in patients with Sezary syndrome.

The abnormal lymphocytes from patients with the Sezary syndrome produce macrophage migration inhibitory factors (MIF) both in vitro and in vivo. Five of six individuals studied had significant serum MIF activity and one had borderline activity. In contrast, in this study 47 normal individuals and 9 of 10 patients with extensive skin disease other than that of the Sezary syndrome had no such activity. Since the neoplastic T cells in patients with the Sezary syndrome localize in the skin, their production of chemical mediators in that site may be responsible for the generalized exfoliative erythroderma seen in that disease.     

Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants

The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with Ki values below 0.1 nM. Protein phosphatase 2B is inhibited 1000-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.     

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells

BACKGROUND: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. RESULTS: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1(-/-) ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1(+/+) cells. Other AGC kinases - namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) - had normal activity or were activated normally in PDK1(-/-) cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1(-/-) cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1(-/-) cells. CONCLUSIONS: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.     

Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.