1,721,034 research outputs found
hASC and DFAT, multipotent stem cells for tissue engineering: comparison of their differentiation potential in vitro
No full textAdipose tissue (AT) contains adipocytes, preadipocytes, fibroblasts, endothelial cells and mesenchymal stem cells (MSC) with a surface’s antigenic profile similar to MSC derived from bone marrow. DFAT (dedifferentiated fat cells), cells derived by dedifferentiation to mature adipocytes is another cell population with characteristics of stem cell isolated from adipose tissue. The aim of this research is to compare the characteristics of stemness, proliferation and differentiation of two cell populations obtained from the human subcutaneous AT to evaluate their potentiality in studies of tissue engineering and regenerative medicine. Stem cell population of vascular fraction of AT (hASC) and cells obtained from mature adipocytes cultured in a "ceiling" and dedifferentiated (DFAT) were analized for identification of surface markers. Cells were seeded in monolayer in presence of growth medium to evaluate the proliferative capacity. Cell populations were always cultured with osteogenic, adipogenic and condrogenic factors and the differentiation capacity was assessed by hystochemical techniques and molecular biology. HASC and DFAT were positive for MSC markers as CD13, CD73, CD90 and CD105 and negative for CD14, CD34 and CD45. Clonogenic test in methylcellulose exclude the acquisition of a pathological phenotype by DFAT for p10 next steps. The proliferative capacity expressed by doubling time obtained in vitro was similar in the populations. DFAT and hASC induced with specific factors into the culture medium had the ability to differentiate in three mesodermal lines. The DFAT were able to accumulate intracellular lipids after 7 days of adipogenic differentiation and gene expression of LPL and Adiponectin was much greater compared to that of hASC. The gene expression of ALP and Runx2 was greater at 14 Days in HASC respect to DFAT, where increases after 3 weeks, while at 21 days, the levels of protein expression are similar in both populations. Both cell populations were also able to differentiate in chondrocytes, showing a positive staining with Alcian blue and gene expression of Sox9 and Aggrecan. In conclusion, both stem cell populations derived from human adult AT have an high differentiation capacity and could be used for regenerative medicine. The results also suggests that DFAT are stem cells more committed to the adipose lineage and may be preferred as cell model in the study on the adipose tissue in vitro and in vivo
PREVALENZA DEI PROCESSI METABOLICI NEL TRASPORTO DELLA RIBOFLAVINA IN ENTEROCITI ISOLATI DI RATTO
No full textIn isolated rat enterocytes, both normal (normoenergized) and rotenone-deenergized, riboflavin intracellular metabolic processes, operating in association with membrane specific transport mechanism, were investigated. The time course contents of unlabeled (endogenous) and labeled (exogenous) riboflavin compounds (riboflavin, RF; flavin mononucleotide, FMN; and flavin-adenine dinucleotide, FAD) were determined by a HPLC analytical procedure before and after incubation with tritium labeled riboflavin. In normoenergized enterocytes total labeled riboflavin content, which is the sum of riboflavin membrane transported and intracellular metabolized, steadily increased to a plateau up to 20 min incubation; FMN content reached a plateau after 3-20 min, while FAD and free RF increased constantly. The phosphorylated forms prevailed over the free RF form, being about 60% of total flavins. In deenergized enterocytes total riboflavin content was significantly lower than in normoenergized enterocytes and reached a plateau after only 3 min incubation; FMN and FAD contents were significantly lower than in normoenergized enterocytes and free RF represented the prevailing form (about 70% of total RF content). In both normoenergized and deenergized enterocytes the contents of unlabeled total RF, FMN and FAD significantly decreased after 20 min incubation, whereas free RF significantly increased only in normoenergized enterocytes
Aquaporin-6 expression in rat gastrointestinal tract
No full textAquaporins (AQPs) are a family of channel proteins belonging to the broad super family of the Major Intrinsic Protein (MIP) transmembrane channels, that facilitate the osmotic movement of water and/or small solutes across biological membranes. Thirteen distinct aquaporins have been identified in mammals and they have been functionally subdivided in orthodox aquaporins (AQP1, 2, 4, and 5), aquaglyceroporins (AQP3, 7, 9 and 10) and unorthodox aquaporins (AQP6, 8, 11 and 12) on the basis of their permeability to water, uncharged or charged solutes. Differently from other AQPs, AQP6 has a preeminent intracellular localization and exhibits an extremely low water permeability; its H+ or Hg2+ activation is accompanied by an increased conductance to inorganic anions (NO3−> I−> Br−> Cl−). For these characteristics it has been suggested that AQP6 works more as an anion-selective channel than as a water channel. Some AQPs, have been identified in the gastrointestinal tract suggesting their involvement in fluid and small solutes movements. AQP6 have been previously identified in renal duct type A intercalated cells and in some extra renal tissues. Here, using RT-PCR, in situ hybridization and immunohistochemical techniques, we demonstrate AQP6 expression also in the gastrointestinal tract.
In the stomach AQP6 mRNA is expressed in the isthmus, neck and basal region, while in the small and large intestine the transcript is distributed all along the crypta-villus axis. We detect AQP6 protein expression in mucous neck cells of the stomach, and at lower levels in chief cells and in some parietal cells, while in the small and large intestine we obtain a strong staining on the apical membrane of the surface epithelial cells with no labelling of crypt cells. Our data, together with the unique functional properties of AQP6, suggest for the first time a role for AQP6 in ions and water absorption in the gastrointestinal tract epithelium and its up regulation by feeding
Functional expression of basolateral Cl-/HCO3- exchange from rat jejunum in Xenopus laevis oocytes
Full text linkPoly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl−/HCO3− antiport was investigated by means of 36Cl− uptake. Two days after injection of 50 ng of poly(A)+ RNA, Cl− uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0·2 mM DIDS, whereas endogenous Cl− uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl− uptake was detected with mRNA size fraction of about 2–4 kb in length. The expressed Cl− uptake can occur against a Cl− concentration gradient and is unaffected by the known Cl− channel blocker anthracene-9-carboxylic acid. Cl− transport mechanism has properties similar to jejunal basolateral Cl−/HCO3− exchange with regard to Na+ dependenc
Human adipose stem cells can differentiate in an osteoblastic-like phenotype on trabecular titanium scaffolds without osteogenic medium
No full textIntroduction Pluripotent human adipose tissue-derived stem cells
(hASCs) can differentiate into multiple mesenchymal cell lineages Previous studies demonstrated that the material, the geometry and the
porosity of scaffolds affect cell binding and migration depth of cellular
ingrowth, and that hASCs can differentiate in an osteoblasticlike
phenotype when seeded on trabecular titanium scaffolds with
osteogenic medium. Aim of this study was to demonstrate that trabecular
titanium scaffolds could induce osteogenic differentiation of
hASCs also without osteogenic medium.
Materials and methods hASCs were isolated from subcutaneous
adipose tissue, seeded on monolayer and on trabecular titanium
scaffolds (Ti6Al 4 V, LimaCorporate, Villanova di San Daniele del
Friuli, Italy; average porosity, 65%, average diameter of cell pores,
640 lm) and incubated at 37 C in 5% CO(2) with osteogenic and
proliferation medium in static conditions. Cells before seeding corresponded
to reference group. After seeding on scaffolds, hASCs
were cultured for 7 days in the presence of proliferation medium
before the osteogenic medium was added (Time 0). The osteogenic
differentiation was detected with real-time PCR to analyze the
expression of ALP and RUNX-2 genes, at time 0 and after 3, 7 and
21 days from differentiation. Alkaline phosphatase activity on cell
lysates was also measured after 7 days from differentiation.
Results Monolayer: both ALP and RUNX-2 gene expression and
alkaline phosphatase activity were higher in hASCs grown in presence
of osteogenic medium.
Trabecular titanium scaffolds: in hASCs grown in proliferation
medium, ALP gene expression was higher than reference group still at
time 0, and remained 2.5 fold higher at time 3 and 7. Similar results
were found with osteogenic medium. RUNX-2 gene expression was
higher in hASCs grown in osteogenic medium, and increased also in
the presence of proliferation medium respect to reference group. At
time 7, there were no difference in alkaline phosphatase activity of
hASCs grown with proliferation and osteogenic medium.
Conclusions hASCs on trabecular titanium expressed higher amount
of ALP and RUNX-2 gene expression in proliferation medium with
respect to cells on monolayer in the same condition. The expression
of osteogenic genes similar in cells grown on scaffold with both
medium, leads to the conclusion that trabecular titanium scaffold
seems to have not only osteoconductive but also osteoinductive
properties
Effects of acute and chronic ethanol administration on thiamine metabolizing enzymes in some brain areas and in other organs of the rat.
No full textThe effect of ethanol (4.7 g/kg body wt intragastrically as a single dose or once daily for 35 days) on the levels of the thiamine metabolizing enzymes (thiamine pyrophosphokinase, TPKase; thiamine pyrophosphatase, TPPase; and monophosphatase, TMPase) was studied in different organs (liver, kidney, small intestine, heart and skeletal muscle) and nervous regions (cerebral cortex, cerebellum, medulla oblongata, pons, corpus callosum, hypothalamus and sciatic nerve) of the rat. In order to evaluate the non-specific effects of the stress of gastric gavage and of the additional caloric intake, appropriate control groups of animals were treated intragastrically with water or with a saccharose solution isoenergetic with ethanol respectively. All animals were reared on a nutritionally adequate diet supplying amounts of thiamine higher than the recommended daily requirement. Enzymatic activities were determined quantitatively by biochemical methods. Tissue TPKase levels were generally reduced by both acute and chronic ethanol administration. TPPase levels were generally reduced after acute and increased after chronic ethanol treatment. Changes in brain TMPase levels were similar to those observed for TPPase. In visceral organs and skeletal muscle TMPase activity was increased by chronic ethanol treatment as compared to acute ethanol administration. In conclusion, ethanol exerts a marked influence on the tissue levels of the thiamine metabolizing enzymes: the activity of the enzymes dephosphorylating thiamine phosphates is increased whereas the activity of the thiamine pyrophosphate synthesizing enzyme is reduced. These changes may contribute to an important extent to the disturbances in thiamine cellular uptake and metabolism observed in alcoholism
The effect of ethanol and other alcohols on morphometric parameters of rat small intestinal microvillous vesicles.
No full textIntestinal alkaline phosphatase can transphosphorylate thiamin to thiamin monophosphate during intestinal transport in the rat
No full textIntestinal alkaline phosphatase (IAP) purified from calf intestine and IAP present in the brush border membrane of rat small intestine effectively transphosphorylated thiamin (T) to thiamin monophosphate (TMP) using Na2-beta-glycerophosphate or Na2-creatine phosphate as phosphate donors at pH 8.5. TMP production in the brush border membrane was very small and corresponded to 0.001-0.01 percent of the total inorganic phosphate simultaneously released by the enzyme activity. This reaction, however, could account for TMP formation independently from that much more important due to the hydrolysis of thiamin pyrophosphate during T intestinal absorption
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