Mireya Fernández

High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteoporotic donors

There are disparate observations on central and peripheral effects of leptin, but several studies consistently support its role as a link between fat and bone. Bone marrow stroma contains mesenchymal stem cells (MSCs), which differentiate into osteoblasts and adipocytes, among others. In this study we assessed the expression of leptin receptors protein in MSCs from control and osteoporotic postmenopausal donors and their change during osteogenic and adipogenic differentiation. Also, we assessed the effects of leptin on osteogenic and adipogenic differentiation of these cells. We demonstrated high affinity leptin binding (KD = 0.36 ± 0.02 nM) in both types of cells. Binding was very low under basal, but increased significantly (2–3 times) through osteogenic and adipogenic differentiation. Osteoporotic MSCs showed lower leptin binding capacity than control cells at an early osteogenic and adipogenic differentiation time, which could restrict cell sensitivity to the protective action of leptin. In this regard, we observed that leptin significantly inhibited adipocyte differentiation in control but not in osteoporotic MSCs, while it exerted a low stimulatory effect on calcium deposition (10%–20%) in both types of MSCs cells. In summary, we report the presence of high affinity leptin receptors on control and osteoporotic MSCs, which were modified distinctly by osteogenic and adipogenic stimulation and a direct and distinct effect of leptin on both type of cells.

Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women.

Osteoblasts, the cells responsible for bone formation, derive from mesenchymal stem cells (MSCs) in bone marrow. To acquire a new cell phenotype, uncommitted MSCs must undergo several proliferation and differentiation changes. Although, it is known that extracellular signal‐regulated protein kinases (ERKs) mitogen‐activated protein (MAP) kinase pathway signaling is involved in the proliferation and differentiation processes, the role of ERKs in osteogenic differentiation it is controversial, at present. In addition, the function that ERK could play in MSCs derived from osteoporotic patients it is not well documented. In this study, we analyze whether previously observed differences in the dynamic response of MSCs from normal and osteoporotic postmenopausal women can be explained by changes in the activation of this signal transduction pathway. Levels of ERK phosphorylation and their correlation with osteogenic differentiation were evaluated in cultures of MSCs derived from osteoporotic postmenopausal women and “healthy” controls. The results show that, under basal conditions, MSCs derived from osteoporotic donors show a level of ERK phosphorylation 2.5 times higher than MSCs derived from control donors. The addition of the osteogenic stimulus only slightly increases the p‐ERK level in cells derived from osteoporotic donors, and is higher in cells derived from control women. Important differences in the ability of PD98059 to inhibit phosphorylation of ERK in both types of cells were also observed, as well as the effect that this inhibition produced on calcium deposition. We conclude that the MAP kinase pathway signaling is differentially activated in MSCs derived from osteoporotic postmenopausal women. The high p‐ERK levels in MSC derived from osteoporotic donors could determine the unresponsiveness of these cells to the osteogenic differentiation stimulus.

Cryopreservation of rainbow trout (Oncorhynchus mykiss) spermatozoa using programmable freezing

Abstract In an attempt to establish a protocol for the cryopreservation of the spermatozoa of the rainbow trout ( Oncorhynchus mykiss ), we studied the effect of various cryoprotective agents (CA) in spermatozoa motility and viability, before, during, and after freezing. Freezing was performed by using a controlled rate freezing system, which allows the accurate setting of different cooling rates, as well as a proper recording of intra-sample temperatures throughout the procedure. Results obtained indicate that before the initiation of freezing, spermatozoa motility is affected more by the length of time of exposure to CA than by the chemical nature of the agents. Exposure periods longer than 10 min affected motility irreversibly, which seems to be related to the high osmolarity of the extender solutions. To study the changes in spermatozoa motility and viability during and after cryopreservation, cells in the cryoprotective solution (glycerol, DMSO or DMSO-sucrose) were processed in a programmable biological freezer at slow (1 °C and 10 °C min −1 ) or rapid (30 °C min −1 ) cooling rates. Results obtained indicated that both during (−60 °C) and after completion of the freezing program (−80 °C, followed by storage under liquid nitrogen), motility and viability was well preserved only in the cells in DMSO-sucrose when subjected to a rapid cooling rate. Under these conditions, approximately 63% of spermatozoa were alive and showed progressive motility. Together, the average fertilization potential of cryopreserved semen was 58% (47% to 85%) compared with that of fresh semen. The procedure described here provides consistency and precision, and permits processing, freezing and storage of trout spermatozoa in less than 15 min.

Urease of Spirulina maxima

Abstract Urease (EC 3.5.1.5) was purified from Spirulina maxima by ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200. The enzyme had maximum activity at pH 8.7, a K m for urea of 0.12 mM and a MW of ca 232 000. A MW of 38 000 was determined for the subunits. The enzyme was inactivated by p -hydroxymercuribenzoate.

Adhesive Interactions in the Hematopoietic System: Regulation by Cytokines

Five years ago, in collaboration with the late Medhi Tavassoli (1) we wrote a minireview on the adhesive interactions taking place in bone marrow and their functional significance (2). In that review the emphasis was placed on the homing mechanisms of progenitor cells to the marrow stroma. Although, at that time information was available on other adhesive processes occurring at the progenitor-stroma interphase, the data were mainly descriptive. Recent research has not only expanded the knowledge about the adhesive mechanisms operating in bone marrow, but has resulted in a wider understanding of their role in steady-state and diseased hematopoiesis. Moreover, new data have illustrated the way in which the expression and/or function of several cytoadhesive molecules or their respective ligands is modulated by cytokines. The latter will be briefly reviewed in this article.

Evidence for cooperative effects in human liver arginase

The reaction kinetics of human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) in terms of arginine concentration is strikingly altered by varying the pH. Lowering the pH from the optimum (9.5) toward a more physiological value (7.5) there is a transition from hyperbolic to sigmoidal kinetics. The cooperative effects are observed in the presence and absence of the product ornithine. Dimers of arginase exhibit typical Michaelis-Menten kinetics even in the presence of ornithine. Dimer-dimer interactions are suggested to explain the kinetic properties of arginase at pH 7.5.

Fluorescent serum and urinary advanced glycoxidation end-products in non- diabetic subjects.

INTRODUCTION Advanced glycoxidation end-products (AGEs) are involved in age-related conditions and diabetic complications. Diet intake contributes to their circulating concentrations. AIM To measure serum and urinary AGEs in non-diabetic volunteers and relate their concentration to body composition, blood chemistry and dietary ingestion. METHODS We studied 41 adult men (31 middle-aged adults and 10 elderly). A nutritional assessment including a dietary recall designed for detection of AGE ingestion (specifically carboxymethyl-lysine(CML)), and anthropometric measurements were performed. Also serum lipoproteins, insulin, glucose, leptin and C reactive protein (CRP). AGEs were measured in serum and urine samples using size exclusion chromatography and flow injection assay (FIA); the technical procedures were first employed in 11 heterogeneous diabetics, as positive controls for this methodology. RESULTS Serum and urinary chromatograms indicated that areas under the curve were not different in younger compared with elderly adults. AGEs did not correlate with dietary intake, body composition, nor metabolic parameters, however they correlated significantly with renal function and CRP concentration. DISCUSSION In these non-diabetic volunteers, with low CML intake, serum and urinary concentration of AGEs were not related to dietary intake. AGEs were related to renal function and CRP, but not to body composition, lipoproteins, insulin and glucose.

The increased expression of receptor activator of nuclear-κB ligand (RANKL) of multiple myeloma bone marrow stromal cells is inhibited by the bisphosphonate ibandronate

The receptor activator of nuclear factor‐kappaB ligand (RANKL) and interleukin‐1beta are osteoclast activating factors which are abnormally expressed in bone marrow stromal cells and plasma cells of multiple myeloma patients. In this work we analyzed RANKL expression in human bone marrow mesenchymal stromal cells and the effect of the bisphosphonate ibandronate on RANKL expression after IL‐1beta activation of ERK pathway. Mesenchymal stromal cells were obtained from bone marrow iliac aspirates from multiple myeloma patients at stages II/III and non‐osteoporotics control donors; these cells were maintained under long‐term culture conditions. Cells were cultured in the presence or the absence of 5 ng/ml IL‐1beta and/or 5 µM ibandronate, during selected periods. mRNA for RANKL and protein levels were assayed by RT‐PCR and Western blot, respectively. Human bone marrow stromal cell line HS‐5 was used for assessing IL 1beta‐ and ibandronate‐ERK phosphorylation responses. Multiple myeloma mesenchymal stromal cells differentiate from control cells by increased basal RANKL expression. IL‐1beta up regulated RANKL expression showed dependent on activated MEK/ERK pathway. Finally, the bisphosphonate ibandronate, that hindered activation of the MEK/ERK pathway significantly inhibited both basal and IL‐1beta dependent RANKL expression by cells. Results indicate that RANKL expression involves the MEK/ERK pathway in multiple myeloma mesenchymal stromal cells, and that early obstruction of this path, such as that achieved with ibandronate, significantly deters RANKL protein expression. J. Cell. Biochem. 111: 130–137, 2010.

Subunit interactions and immobilised dimers of human liver arginase.

Incubation of soluble human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) with p-hydroxymercuribenzoate resulted in the dissociation of the enzyme into active dimers. Addition of 2-mercaptoethanol resulted in the regeneration of the tetrameric enzyme. When arginase, bound covalently to nylon, was incubated with p-hydroxymercuribenzoate, matrix-bound dimers were obtained. Incubation of these species with 2-mercaptoethanol resulted in stable, unmodified dimers. Based on this dissociation of arginase, a model with D2-symmetry is suggested for this enzyme. The specific activity, the Km value for arginine, pH optimum and the inhibition constants for ornithine and lysine were determined for monomeric, dimeric and tetrameric forms. It is concluded that the behaviour of the active sites of the monomers is not substantially altered by the interaction of these species in the oligomeric molecule.

Production of soluble CD34 by human myeloid cells.

CD34, a glycophosphoprotein present in lymphohaematopoietic stem and progenitor cells, as well as in other cell types, exists in both transmembrane and intracytoplasmic forms. Transmembrane CD34 expression, which is high in the earliest haematopoietic precursors, decreases as cells mature. However, to our knowledge, there is no information on whether a decrease in transmembrane CD34 can also predict a release of the molecule from the cell membrane into the extracellular fluid. To investigate the above possibility, we studied conditions (incubation time, cell density and proliferative status) in human myeloid cells (lines KG‐1a, KG‐1 and cord blood‐derived cells) that may cause a decrease in surface CD34 and the generation of a soluble form of the molecule. The latter, as demonstrated by Western blot analysis, adds more complexity to the proposed structural features and functional properties of CD34 in myeloid cells.