The Ras/Erk pathway induces primitive endoderm but prevents parietal endoderm differentiation of F9 embryonal carcinoma cells

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.     

Assessment of total body stores of vitamin A in Guatemalan elderly by the deuterated-retinol-dilution method

BACKGROUND: Deuterated retinol dilution (DRD) gives quantitative estimates of total body stores of vitamin A. OBJECTIVES: In elderly people, we studied 1) the time when an oral dose of deuterated vitamin A equilibrates with body stores, 2) whether serum ratios of deuterated to nondeuterated retinol (D:H) at 3 or 6 d postdosing predicted body stores, and 3) the ability of DRD to detect changes in the size of the body vitamin A pool. DESIGN: A 10-mg oral dose of [2H4]retinyl acetate was administered to 60-81-y-old Guatemalans (n = 47); percentage enrichment of serum retinol with deuterated retinol was determined at 1-3 time points per subject at 3, 6, 7, 14, 20, 21, and 54 d. In subjects from whom blood was obtained at 3 and 21 d (n = 15) and at 6 and 20 d (n = 9), total body stores were calculated by using the formula of Furr et al (Am J Clin Nutr 1989;49:713-6) with 21- or 20-d data and correlated with serum D:H at 3 or 6 d postdosing. Nine subjects received diets containing 982+/-20 microg RE (x+/-SEM) plus 800 microg RE as retinyl acetate supplements for 32 d. DRD, serum retinol, and relative dose response were used to assess vitamin A status before and after the intervention. RESULTS: Deuterated retinol equilibrated with the body pool by 20 d postdosing. Vitamin A supplementation for 32 d increased body stores, although unexplained exaggerated increases were seen in some subjects. An inverse linear relation was found between estimates of body stores and serum D:H at 3 d postdosing (r = -0.75, P = 0.002); at 6 d postdosing, the correlation was weaker. CONCLUSIONS: DRD can detect changes in total body stores of vitamin A, although factors affecting serum D:H need to be elucidated. Serum D:H 3 d postdosing might be used as an early indicator of total body stores of vitamin A, although a predictive equation will need to be developed.     

Primary DNA damage but not mutagenicity correlates with ciprofloxacin concentrations in German hospital wastewaters

BACKGROUND: Auscultation of patients with mitral annular calcification on echocardiography revealed a particular constellation of findings. OBJECTIVE: To test the hypothesis that a particular auscultatory constellation provides a high degree of certainty in diagnosing the combination of mitral annular calcification and aortic sclerosis so often found in the elderly. METHODS: Two groups of patients were studied to evaluate the particular auscultatory constellation under consideration which consisted of: (1) a harsh ejection systolic murmur heard from the 2nd right interspace to the cardiac apex and usually loudest between the 3rd left interspace and the apex; (2) the murmur radiates from the apex towards the left axilla and radiates poorly or not at all from the 2nd right interspace to the neck, and (3) the 2nd heart sound at the cardiac base is normal in intensity, and no ejection clicks are present. Group 1 consisted of patients with mitral annular calcification on echocardiographic examination, and group 2 consisted of patients in whom the particular constellation of auscultatory findings was present and who were then referred for echocardiographic assessment. RESULTS: The particular auscultatory constellation under investigation allowed the diagnosis of the presence of the combination of mitral annular calcification and aortic sclerosis with substantial accuracy. CONCLUSION: The findings in this exploratory study suggest that the pathologic combination of mitral annular calcification and aortic sclerosis can be diagnosed with a reasonably high degree of certainty in elderly patients, if the particular auscultatory configuration is identified.     

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.     

A view from the family: years after a loved one has died of ovarian cancer

BACKGROUND: Macrosclerotherapy in combination with compression has proven to be safe and effective in the treatment of varicose veins. Local compression is increased by pads, according to Laplace law. Firm rolls of cotton wool are fixed over the course of the entire vein to increase local compression and to reduce complications. Additional compression is given by a combination of a class I (daytime and nighttime) and class II (daytime only) medical compression hosiery. PURPOSE: To evaluate the effectiveness and side effects of sclerocompression therapy with cotton wool rolls in combination with medical compression hosiery. METHOD: Prospective study with 100 patients (120 legs) with primary varicose veins, which are treated with polidocanol as sclerosant with the empty vein technique. Immediately after the injection, a long cotton wool roll is placed over the entire vein and fixed. Additional compression is obtained with class I and class II medical compression hosiery. The interface pressure on the skin, just under the cotton wool roll, is measured on 12 legs with the aid of an interface pressure measuring instrument (Oxford Pressure Monitor). RESULTS: Good sclerosing results are obtained in all patients. Side effects are classified as early and late. In 16 patients, minor side effects which needed no treatment are observed. In only 3 cases (2.5%), intravascular blood clots (2) and phlebitis (1) needed incision and expression. The mean interface pressure of all measuring sensors under the cotton wool roll is 84 mm/Hg (68 to 122 mm/Hg). CONCLUSION: This study proves the high effectiveness of a cotton wool roll compression right at the place of treatment. By using these long cotton wool compression rolls, the compression part of sclerocompression therapy becomes more effective and much easier to perform.     

Stable association of PYK2 and p130(Cas) in osteoclasts and their co-localization in the sealing zone

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.