Nucleotide pool and the proliferative process: an NMR study of the liver regeneration

The metabolic changes occurring during liver regeneration after partial hepatectomy have been followed by examination of the perchloric extracts by means of NMR spectroscopy. Proton spectra show an abrupt decrease of glycogen, glucose and nucleotides, which are essentially ribonucleotides, in the first hours after hepatectomy. Nucleotides begin to store up again in the third day after hepatectomy, while glucose and glycogen storage builds up from the second day. 31P data evidence a sharp drop of phosphomonoesters, i.e. monophosphosugars (including AMP) and phosphocholine, and phosphodiesters, i.e. GPC and GPE, soon after hepatectomy and a recovery of the initial levels approximately at the 60th hour and a further increase of PDE at later times. The NMR findings are in agreement with the biochemical knowledge of the course of liver regeneration.     

Anthracycline gels. Preparation and some physico-chemical properties

Gels have been prepared from aqueous solutions of anthracyclines by addition of salts. The gels are thixotropic and thermally reversible. They are stable for several months in the refrigerator and for long times even at room temperature. The gel-solution transition (melting) temperature depends on the concentration of the anthracycline and on the concentration and nature of the added salt. The melting has been followed by 1H-NMR. Only weak intermolecular interactions (stacking and hydrogen bonds) originate the drug network, within which the solvent is entrapped. 1H-NMR and polarimetric data suggest a stacked helical arrangement of the anthracycline molecules. The gelation process is cooperative.     

Interactions of ubiquinones with membrane models

The effect of the insertion of coenzyme Q10 and some of its shorter chain homologues in membrane models (Reverse Micelles, Small Unilamellar Vesicles and Liposomes) has been studied by NMR and IR spectroscopies. By 1H-NMR we have found that the stretched conformation of the isoprenoid side-chain observed in solution is maintained in membrane models. Interaction between the quinonoid moiety of the Q's and the phosphatidic groups of the phospholipids has been evidenced by 31P-NMR. A large effect of this interaction on the water microdynamics and distribution around the charged groups of the phospholipids has been observed by measurements of 1H and 2H relaxation times and by infrared spectra. The 13C-NMR spectra of the backbone of the acyl chains of phospholipids does not seem to be influenced to a noticeable extent by the presence of the Q's.     

Changes in the Ceramide Composition of Rat Forebrain Gangliosides with Age

Five major gangliosides (GM1, GD1a, GD1b, GT1b, and GQ1b) were extracted and isolated by normal-phase HPLC from the forebrain of Sprague-Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base moiety. At all ages, the C18:1 and C20:1 long-chain base species predominated, whereas the C18:0 and C20:0 ones represented 1-3% of the total. The C18:1 long-chain base species, predominant at 3 days (91-96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GD1a, GM1, GT1b, GD1b, and GQ1b, respectively. The content of the C20:1 long-chain base species, low at birth (4-9%), increased with age in all gangliosides and reached, at 2 years, 27-55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long-chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GT1b; and (d) a low, constant level for GD1b and GQ1b. All the ganglioside species containing the C20:1 long-chain base showed a constant increase during development, the increase being more marked in the first 30 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of human to mouse xenografts by umbilical cord blood

Utilizing human umbilical cord blood, it has been possible to create in irradiated animals a human to mouse xenograft. To facilitate hematopoietic reconstitution, SJL/J mice, which are functionally low in natural killer (NK) cells, were treated with anti-Asialo GM1 antibodies (anti-NK) and irradiation prior to injection of cord blood mononuclear cells. In contrast, SJL/J mice with the "beige" (bg/bg) mutation, which confers a functional NK cell deficiency, required only irradiation for successful transplantation. Human cells, detected by means of DNA probes, were demonstrated in the lungs and lymph nodes of irradiated animals up to 6 months after injection of the human cord blood cells.     

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.     

Action of α-l-fucoside fromOctopus vulgaris hepatopancreas on phospholipid vesicles containing the fucosylated ganglioside FucGM1

The behaviour of a highly purified -l-fucosidase (E.C. 3.2.1.51) extracted from octopus hepatopancreas was studied with phospholipid vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS) containing the fucosylated ganglioside FucGM1, a potential natural substrate of the enzyme. The substrate recognition and hydrolysis take place only with PS/FucGM1 mixtures via an association process of the enzyme with the vesicles at acidic pH; the enzyme rapidly and stably binds to PS vesicles but not to PC vesicles. The data suggest that only the PS-associated enzyme is able to hydrolyse FucGM1 embedded in the same bilayer. The enzyme association with FucGM1/PS vesicles is a prerequisite for ganglioside hydrolysis but is followed by irreversible enzyme inactivation.     

MicroRNA-486-3p Regulates γ-Globin Expression in Human Erythroid Cells by Directly Modulating BCL11A

MicroRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNAs target. The functional relevance of microRNAs has been proven in normal and malignant hematopoiesis. While analyzing miRNAs expression profile in unilineage serum-free liquid suspension unilineage cultures of peripheral blood CD34⁺ hematopoietic progenitor cells (HPCs) through the erythroid, megakaryocytic, granulocytic and monocytic pathways, we identified miR-486-3p as mainly expressed within the erythroid lineage. We showed that miR-486-3p regulates BCL11A expression by binding to the extra-long isoform of BCL11A 3′UTR. Overexpression of miR-486-3p in erythroid cells resulted in reduced BCL11A protein levels, associated to increased expression of γ-globin gene, whereas inhibition of physiological miR-486-3p levels increased BCL11A and, consequently, reduced γ-globin expression. Thus, miR-486-3p regulating BCL11A expression might contributes to fetal hemoglobin (HbF) modulation and arise the question as to what extent this miRNA might contribute to different HbF levels observed among β-thalassemia patients. Erythroid cells, differentiated from PB CD34⁺ cells of a small cohort of patients affected by major or intermedia β-thalassemia, showed miR-486-3p levels significantly higher than those observed in normal counterpart. Importantly, in these patients, miR-486-3p expression correlates with increased HbF synthesis. Thus, our data indicate that miR-486-3p might contribute to different HbF levels observed among thalassemic patients and, possibly, to the clinical severity of the disease.