Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

In vitro effects of arachidonic acid on the prostaglandin synthesizing system in gastric mucosa

Self-destruction of the prostaglandin cyclooxygenase has been suggested to be an important factor in the regulation of endogenous prostaglandin synthesis. The present study was done in order to define the role of this substrate-induced inactivation in the regulation of prostaglandin synthesis in gastric mucosa. In tissue homogenate, the prostaglandin synthesizing capacity is rapidly inactivated at 37 degrees C, even in the absence of exogenous arachidonic acid. It was shown that this inactivation can be prevented both by EDTA as a chelator of calcium-ions and by tetracaine, a specific inhibitor of the phospholipase A2. Additional exogenous arachidonic acid again inactivated prostaglandin synthesis in a dose dependent manner. In contrast, the prostaglandin synthesizing capacity in organ cultured mucosal biopsies is well preserved, although the release of endogenous substrate was activated by extracellular calcium and Ca-ionophore A23187. Furthermore, even at high concentrations of exogenous arachidonic acid present in the culture medium, the synthesizing capacity in intact biopsies was only slightly and reversibly reduced. These large differences between intact biopsies and cell free tissue preparations point to very efficient mechanisms controlling the substrate availability for the cyclooxygenase system both from endogenous and exogenous sources in intact gastric mucosa.     

Mouse soleus muscle Na-K pump activity: direct correlation with in vitro and in vivo oxygen consumption

To evaluate the contribution of active sodium-potassium transport to energy utilization, measurements of sodium-potassium pump activity in isolated soleus muscles of normal mice were carried out and related to measurements of oxygen consumption by the tissue. A highly significant positive correlation (r = 0.89, p less than 0.001) was found between these two parameters. In addition, both the in vitro oxygen consumption and Na-K pump activity of the muscles were found to correlate with whole body oxygen utilization by the experimental animals. The results support a role for the sodium-potassium pump in the determination of energy turnover.     

In vitro selective effect of melphalan on human T-cell populations

Summary In vitro treatment with 2 g/2×10⁶ cells melphalan (l-PAM: l-phenylalanine mustard) significantly decreased the total number of T lymphocytes from peripheral blood (PBL) of healthy human donors and of the OKT₄ population (precursor suppressor/helper/inducer) T cells as defined by monoclonal antibodies OKT₃ and OKT₄, respectively. No changes in the OKT ₈ ⁺ lymphocyte population (cytotoxic/mature suppressor cells) were observed following the same treatment. Preincubation of PBL with l-PAM at concentrations that do not affect the rate of DNA synthesis in PHA-stimulated lymphocytes inhibited the generation of T suppressor lymphocytes by ConA, as shown by their effect on PHA stimulation. Treatment of allogeneic PBL with l-PAM had no effect on mature suppressor T cells already induced by Con A, as shown by incubation of PBL with l-PAM after incubation with ConA.     

Inhibitory effects of somatostatin on growth and differentiation in cultured intestinal mucosa

The effect of somatostatin on mucosal DNA, protein and brush border enzymes was studied in organ cultured rabbit jejunum and ileum. Compared to control cultures, somatostatin reduced the biopsy DNA and protein content in parallel in the jejunum, but was ineffective in the ileum. This was probably due to a direct growth inhibition, since DNA and brush border enzyme activity from desquamated cells in the postculture medium were unaffected. In addition, a direct inhibition of jejunal villous cell differentiation by somatostatin was reflected in a significant decrease of sucrase, maltase and alkaline phosphatase activity. In the ileum, only the specific activity of alkaline phosphatase was reduced. The key enzyme of cholesterol synthesis, HMG-CoA-reductase, was measured as an intracellular enzyme control and was not influenced by the hormone. The high somatostatin concentrations necessary to achieve the effects are not an artefact of hormone degradation during culture, as shown by radioimmunoassay, and suggest a local or "paracrine" rather than systemic, inhibitory action of somatostatin on intestinal growth and differentiation.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

In a previous report we documented an increased Na⁺-dependent transport of inorganic phosphate (P _i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P _i -uptake stimulator) lead to a 3-4-fold stimulation of Na⁺-dependent P _i -uptake (10ng cRNA injected, 3–5 days of expression). Na⁺-independent uptake of P _i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K _m -values for the induced Na⁺-dependent uptake were 0.26 ± 0.04 mm for P _i and 14.8 ± 3.0 mm for Na⁺. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P _i -uptake into Xenopus laevis oocytes, but which is not a P _i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996