Influence of melatonin and serotonin on the number of rat pineal “synaptic” ribbons and spherules in vitro

Summary Previous studies have shown that the synaptic ribbons (SR) and spherules (SS) of the mammalian pineal gland may respond differently under physiological and various experimental conditions. The aim of the present study was to gain insight into the mechanisms that may be responsible for the numerical changes of these organelles during a 24-h cycle. As the possibility exists that the structures are influenced by substances synthesized within the pinealocyte, rat pineal glands were cultured with and without added melatonin or serotonin, using an experimental protocol such that the addition of melatonin and serotonin mimicks the circadian changes of the respective substances within the pineal. The tissue was processed for electron microscopy and the numbers of SR and SS were counted in a unit area of pineal tissue. The results obtained indicate that melatonin added to the incubation medium increases the number of SR in the first half of the night; serotonin decreases SR numbers in the morning. SS numbers, by contrast, decrease following melatonin administration in the afternoon, and increase in the morning following serotonin administration. It thus appears that the numbers of SR and SS are influenced by melatonin and serotonin and that the two structures are regulated by differential, but nevertheless biochemically closely related mechanisms.Financial support of the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Neuroendokrinologie, Vo 135/8-4), the Polish Academy of Sciences (Research Program 10.4.04.6), and the Freunde der Universität Mainz e.V. is gratefully acknowledged.On leave from Department of Anatomy, University Medical School, Pécs, Hungary     

NanoAOX—a novel nanoparticle and antioxidant mixture enhances the growth of plants in vitro and in vivo

In Vitro Cellular & Developmental Biology - Plant - The global food crisis is an issue affecting 1 billion people worldwide—it is critical that a solution be developed to provide the...     

Effect of ethanol on the response of the rat urinary bladder to in vitro ischemia: Protective effect of α-lipoic acid

Purpose: Ethanol exposure has been used to demonstrate the increase of oxidative stress to a variety of tissues. We studied the effect of ethanol on the response of isolated strips of rat bladder to In vitro hypoxia in the absence of glucose (In vitro ischemia). Secondly, we determined if -lipoic acid (LA) could alter the response to ethanol + In vitro ischemia.Methods: Sixty-four rats were used for the these experiments. Each rat was anesthetized and its urinary bladder excised. The bladder body was cut into two longitudinal strips and each strip mounted in individual baths filled with oxygenated Tyrodes solution containing glucose at 37 C. Ethanol (0.3%, 1%, or 3%) was placed in the first six baths (two strips at each concentration). The last two baths did not receive ethanol. Each strip was incubated for 1 h and then stimulated with field stimulation at 2, 8, and 32 Hz. Each strip was stimulated with 10 M carbachol, washed three times with fresh oxygenated buffer and ethanol re-added to their respective baths. Each strip was then stimulated with 120 mM KCl and washed three times as before. Strips were then subjected to 1 h In vitro ischemia (incubation in the absence of glucose with Tyrodes equilibrated with nitrogen instead of oxygen). During the ischemic period, each strip was stimulated for 5 s every 10 min by 32 Hz FS to simulate hyperreflexia. At the end of the hour, the tissues were incubated for an additional hour in the presence of oxygen + glucose and subjected to a second series of stimulations as before. At all times, ethanol was maintained in baths 1–6. In set 2, 1% ethanol was added to the first six baths. LA was added to every other bath, and the experiments performed as mentioned earlier.Results: (a) Ethanol at 0.3% or 1% had no effect on the contractile responses prior to exposure to In vitro ischemia; 3% was inhibitory. (b) In vitro ischemia mediated a significant decrease in the contractile responses to all forms of stimulation except for carbachol. (c) Ethanol mediated a dose-response enhancement of the contractile dysfunctions caused by In vitro ischemia. (d) LA completely reversed the effects of ethanol on contractile responses following In vitro ischemia except for carbachol.Conclusions: The results demonstrate that direct exposure to ethanol significantly enhanced contractile dysfunctions mediated by In vitro ischemia followed by re-oxygenation and that the presence of LA significantly inhibits this effect of ethanol. (Mol Cell Biochem 271: 133–138, 2005)This material is based upon work supported in part by the Office of Research and Development, Department of Veterans Affairs, and NIH RO-1-DK067114     

The effect of PGP on β-hexosaminidase and histamine secretion in rat peritoneal mast cells in vitro

The investigation of the effect of peptide prolyl-glycyl-proline (PGP) on β-hexosaminidase and histamine secretion by mast cells in primary culture has shown that incubation of mast cells with PGP (6 × 10⁻⁵ M) before their activation by synacten significantly decreased the amount of secreted histamine and β-hexosaminidase in comparison with the action of synacten only. The peptide in investigated concentration had no influence on the level of spontaneous secretion. Incubation of cells with PGP did not prevent their activation by compound 48/80. Therefore, PGP can have a direct effect on isolated rat mast cells in vitro and diminish their secretory activity under activation by synacten.     

Soluble extracellular domains of human SIRPα and CD47 expressed in Escherichia coli enhances the phagocytosis of leukemia cells by macrophages in vitro

Signal regulatory protein (SIRP) α, a transmembrane protein belonging to the immunoglobulin superfamily, is a receptor for CD47. The interaction between SIRPα and CD47 plays an important role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Blocking antibodies against CD47 have been shown to promote phagocytosis of LSCs by macrophages. Here, we consider an alternative way to interrupt the interaction between CD47 and SIRPα. We expressed the extracellular domains of the human SIRPα (hSIRP(ext)) and the human CD47 (hCD47(ext)) in Escherichia coli as Trx fusion proteins, and purified them by using affinity chromatography. We show that the purified fusion protein Trx-SIRP(ext) could interact in vitro with Trx-hCD47(ext). Moreover, Trx-SIRP(ext) could effectively bind to Jurkat T-ALL cells, which expressed CD47 at a high level. CD47(ext), on the other hand, bound to human macrophages. In vitro phagocytosis assay showed that these fusion proteins could enhance the phagocytosis of Jurkat cells by macrophage, with Trx-hSIRP(ext) showed a higher efficiency than Trx-CD47(ext). These results indicated that the soluble Trx-hSIRP(ext) and Trx-CD47(ext) polypeptides could be alternative molecules to interrupt CD47-SIRPα interaction between leukemia cells and macrophages, and might be potentially useful for the targeted therapy of leukemia.     

Coupling primary and stem cell–derived cardiomyocytes in an in vitro model of cardiac cell therapy

The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (μtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell–derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell–cell junction that degrade force transmission between cells. Moreover, we developed a computational model of μtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell–cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes.     

Wnt/β-catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro

Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, β-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/β-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3β inhibitor, of Wnt/β-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.     

A model of the role of natural killer cells in immune surveillance — I

Summary The theory of immune surveillance of Thomas and Burnet stated in part that antigenic differences between neoplastic and normal cells provide the stimulus for their destruction by cells of the immune system. Burnet pointed to the T lymphocyte as the cell which mediated this surveillance. The existence of some form of surveillance in cases of no T lymphocyte functioning presents the possibility that surveillance, if present at all, is mediated by non T cells.Cells identified as naturally cytotoxic killer (NK) cells appear to have properties required of a surveillance effector population. This paper utilizes properties of NK cells and the effects of interferon on this population to construct a mathematical model of the characteristics that an NK cell surveillance would have. A two level theory of immune surveillance is proposed.This research has been supported in part by the National Science Foundation under grant #NSF-Eng. 7904852     

A model of the role of natural killer cells in immune surveillance — II

The functioning of natural killer (NK) cells as immune surveillance effector cells against tumors is explored. In part I (J. Math. Biol. 12, 363-373 (1981], it was predicted that susceptible tumors would be eliminated if they have parameter lambda 0 value negative. They would not be eliminated if lambda 0 greater than 0. As the lambda 0 less than 0 result was local, one expected either that tumors of all sizes with lambda 0 less than 0 will be eliminated (global stability) or that tumor population will go to zero if in a domain of attraction of the critical point which is not all of the positive orthant. In this paper, the second is shown to be true. The general results are illustrated by a specific model.     

Ultrastructure of sympathetic ganglion cells and granule-containing cells in the paracervical (Frankenhäuser) ganglion of the newborn rat

Summary A study was made of the ultrastructure of the paracervical (Frankenhäuser) ganglion of the newborn rat, using immersion fixation by glutaraldehyde (2.5%) followed by OsO₄ (1%), or KMnO₄ (3%) fixation. The cells containing dense—core vesicles were divided into three groups: (1) primitive sympathetic cells, (2) cells containing some dense-core vesicles 700–1100 Å in size and structurally resembling sympathetic neurons, called principal neurons, and (3) cells containing many dense-core vesicles with a larger, darker dense core, 800–2000 Å in diameter, called granule-containing cells. Using glutaraldehyde-osmium fixation, the principal neurons were further divided into dark and light cells on the basis of electron opacity of the cytoplasmic matrix. The granule-containing cells were believed to correspond to the small, intensely fluorescent cells (SIF-cells) previously described using the formaldehyde-induced fluorescence technique. On the basis of the amount of granules, the granulecontaining cells were classified as mature or maturing SIF-cells and as more primitive SIF-cells, and developing sympathicoblasts. The development of synapses in autonomic ganglia was discussed.Grant: The Finnish Medical Foundation.     

Expression of the α-subunit of glycoprotein hormones in the pars tuberalis-specific glandular cells in rat,mouse and guinea-pig

The nature of the hormone(s) secreted by the pars tuberalis (PT) is still unknown. This pituitary lobe is mainly formed by specific glandular cells that differ in their ultrastructural features from the other adenohypophysial cell types. Data from the literature indicate the presence of thyroid-stimulating hormone immunoreactivity in the PT-specific cells of the rat and the Djungarian hamster but not of other species, including the mouse and guinea-pig. The PT also encloses variable numbers of pars distalis cells, essentially gonadotrophs that are mainly dispersed in its caudal area. We studied the expression of the glycoprotein hormone -subunit in the PT of the rat, mouse and guinea-pig by in situ hybridization and immunocytochemistry. In situ hybridization, using an oligonucleotide probe complementary to rat cDNA sequence 196–237 revealed the expression of the -subunit gene throughout the PT of the rat and the mouse; in the guinea-pig, the probe labelled no pituitary cells. Light-and electron-microscopic immunocytochemistry demonstrated -subunit immunoreactivity in the secretory granules of the PT-specific cells in the three species examined. These cells did not react with a specific antibody against the -subunit of luteinizing hormone, an antibody that labelled scattered gonadotrops. The present data suggest that hormone(s) produced by the PT-specific glandular cells are, at least partly, related to glycoprotein hormones.     

Expression of the Thomsen-Friedenreich antigen and of its putative carrier protein mucin 1 in the human placenta and in trophoblast cells in vitro

The Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope Galbeta1-3GalNAcalpha-O-) has been known for a long time as a carcinoma-associated antigen. In normal tissues the occurrence of TF antigen is restricted to a few immunologically privileged areas. Here we report on the identification of the TF epitope and its putative carrier protein mucin 1 (MUC1) in human placental tissue, on isolated trophoblast cells in vitro and on trophoblast tumour cell lines BeWo and Jeg3. Cryosections of placental and decidual tissues of the first, second and third trimester were double stained with monoclonal antibodies directed against the TF epitope (IgM) and against MUC1 (IgG). In the first trimester of pregnancy we found strong expression of TF antigen and MUC1 at the apical side of the syncytiotrophoblast directed towards the maternal blood. This expression was consistent in the second trimester of pregnancy, and to a lesser degree in the third trimester. In addition, we found positive staining for TF antigen and MUC1 on extravillous trophoblast cells in the decidua during the first and second trimester of pregnancy. Trophoblast tumour cells of the cell line BeWo, which form a syncytium in vitro, were also positive for TF antigen and MUC1, whereas Jeg3 cells, which are unable to form a syncytium, expressed only MUC1. Freshly isolated trophoblast cells from first trimester placentas showed strong staining for MUC1; however, only a few of these cells (less than 1%) were positive for TF antigen, and might consist of digested fragments of the syncytium. In summary, TF antigen and MUC1 are expressed by the syncytiotrophoblast at the feto-maternal interface and by extravillous trophoblast cells invading the decidua, whereas villous cytotrophoblast cells in situ as well as freshly isolated trophoblast cells from first trimester placentas only express MUC1 but not TF antigen.     

Proteome analysis of the left ventricle in the vitamin D₃ and nicotine-induced rat vascular calcification model

Vitamin D? and nicotine (VDN) serve as an animal model of arterial calcification. The vascular calcification induced by the VDN model is always accompanied by compensatory left ventricular (LV) hypertrophy and impaired cardiac performance. To determine the possible mechanisms that are responsible for the effects of VDN on the LV, a 2-DE based proteomics approach was used to evaluate the changes in protein expression of the left ventricle in VDN rats, to our knowledge, for the first time. We identified sixteen proteins that were markedly altered and involved in mitochondrial function, heat shock protein activity, myocyte cytoskeleton composition and enzyme activity for energy metabolism. We describe, for the first time, a novel pathway (NDPK) that is involved in LV hypertrophy and enzyme activities of three of the sixteen clinical identified proteins: lactate dehydrogenase (LDH), SOD [Mn] and GST.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

Rapamycin increases the yield and effector function of human γδ T cells stimulated in vitro

Clinical strategies to exploit Vγ2Vδ2 T cell responses for immunotherapy are confronted with short-term increases in cell levels or activity and the development of anergy that reduces the response to therapy with succeeding treatments. We are exploring strategies to increase the yield and durability of elicited Vγ2Vδ2 T cell responses. One approach focuses on the mammalian target of rapamycin (mTOR), which is important for regulating T cell metabolism and function. In Vγ2Vδ2 T cells, mTOR phosphorylates the S6K1 and eIF4EBP1 signaling intermediates after antigen stimulation. Rapamycin inhibited these phosphorylation events without impacting Akt or Erk activation, even though specific inhibition of Akt or Erk in turn reduced the activation of mTOR. The effects of rapamycin on the T cell receptor signaling pathway lead to increased proliferation of treated and antigen-exposed Vγ2Vδ2 cells. Rapamycin altered the phenotype of antigen-specific Vγ2Vδ2 cells by inducing a population shift from CD62L + CD69− to CD62L-CD69+, higher expression of CD25 or Bcl-2, lower levels of CCR5 and increased resistance to Fas-mediated cellular apoptosis. These changes were consistent with rapamycin promoting cell activation while decreasing the susceptibility to cell death that might occur by CCR5 or Fas signaling. Rapamycin treatment during antigen-stimulation of Vγ2Vδ2 T cells may be a strategy for overcoming current obstacles in tumor immunotherapy.     

Histochemical studies on the distribution of β-glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of the rat

Summary The present study compares the distribution of -glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of rat. In young cells there are indications of cyclic activity of these enzymes, i.e., in some stages there are perinuclear concentrations of the enzymes, at other times -glucuronidase and succinic dehydrogenase are uniformly distributed throughout the cytoplasm. These stages have been discussed with the identical distribution of mitochondria. However, in old spinal ganglion cells both -glucuronidase and succinic dehydrogenase become mainly concentrated in the pigment areas, suggesting thereby their possible role in the production of pigment, through the medium of the mitochondria.