Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Metabolism of semisynthetic single-chain GM1 derivatives in cerebellar granule cells in culture

Semisynthetic single-chain GM1 derivatives containing N-acetyl-sphingosine (LIGA4) or N-dichloroacetyl-sphingosine (LIGA20) were recently reported to exert strong protection against glutamate-induced neuronal death in primary cultures of cerebellar granule cells. Elucidation of the molecular mechanism underlying the evoked effect requires knowledge of the metabolic fate of such molecules in the same cultured cells. For this, LIGA4 and LIGA20 were made radioactive on the long chain base moiety and added to cerebellar granule cells in culture in parallel with GM1 ganglioside. The metabolic fate was then investigated. It was found that both these molecules were easily taken up by the cells and promptly metabolized in a fashion qualitatively similar to that of control GM1. The highest amount processed was attributed to the different aggregation properties of LIGAs in solution. Among metabolites, higher accumulation of the single-chain ceramide residues was found after LIGA administration. Interestingly, sphingomyelin was generated, regardless the added compound, suggesting a recycling of the free long chain base.     

Cells from human bone giant cell tumors show a [Ca2+]o-sensing.

Giant cells from a human giant cell tumor of bone, showing several osteoclast features were tested for their capability of detecting the [Ca2+]o by a receptor like [Ca2+]o sensing. We found that cultured cells responded to elevation of [Ca2+]o, obtained adding 4 mM Ca2+ to the 2 mM Ca2+ containing buffer, by a transient increase of [Ca2+]i. Proliferative cells induced to differentiate by treatment with 10(-8) M 1,25 dihydroxyvitamin D3, were upregulated in their capability of responding to elevated [Ca2+]o. In fact, in this circumstance, the peak of [Ca2+]o-induced [Ca2+]i rise was increased compared to untreated cells. This suggests that 1,25 dihydroxyvitamin D3 induces a more efficient regulation of osteoclast activity.     

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

Is macrophage migration inhibitory factor a therapeutic target in systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is the prototype of a cluster of diseases that are characterized by a loss of self tolerance and chronic inflammation in organs including skin, kidney, brain and joints. Researchers have long debated the varying contributions of the components of the immune system to the pathogenesis of SLE, but the emigration of leucocytes from the microcirculation, and the subsequent tissue inflammation mediated by these inflammatory cells, are key features of chronic inflammation seen in SLE. Macrophage migration inhibitory factor (MIF) is a broad-spectrum pro-inflammatory cytokine. We hypothesize that MIF is an important inflammatory mediator in the perpetuation of immune activation in SLE, via its effects on activation of T and B cells, and endothelial and effector cells. As MIF exerts anti-apoptotic effects, it may also play a role in promoting abnormal survival of autoreactive lymphocytes, thus perpetuating autoimmune reactivity. In addition, MIF has a unique relationship with glucocorticoids, in that MIF can override the effects of glucocorticoids and may be important in steroid resistance. By virtue of its pluripotent functions, we propose that MIF may be a critical mediator of inflammation and damage in SLE, and that targeting of MIF may offer therapeutic benefits in this disease.     

Metabolomic Fingerprint of Heart Failure with Preserved Ejection Fraction

BackgroundHeart failure (HF) with preserved ejection fraction (HFpEF) is increasingly recognized as an important clinical entity. Preclinical studies have shown differences in the pathophysiology between HFpEF and HF with reduced ejection fraction (HFrEF). Therefore, we hypothesized that a systematic metabolomic analysis would reveal a novel metabolomic fingerprint of HFpEF that will help understand its pathophysiology and assist in establishing new biomarkers for its diagnosis.ConclusionsThe metabolomics approach employed in this study identified a unique metabolomic fingerprint of HFpEF that is distinct from that of HFrEF. This metabolomic fingerprint has been utilized to identify two novel panels of metabolites that can separate HFpEF patients from both non-HF controls and HFrEF patients.

Clinical Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02052804","term_id":"NCT02052804"}}NCT02052804