Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Immune enhancing effect of a growth hormone secretagogue

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.     

Synthesis of 7-benzyl-5-(piperidin-1-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c][2,7]naphthyridin-1-ylamine and its analogs as bombesin receptor subtype-3 agonists

The original structure of a high-throughput screening hit obtained from an external vendor was revised based on multiple NMR studies. The active compound was re-synthesized via a novel route and its structure and biological activity as a BRS-3 agonist were unambiguously confirmed. Multi-gram quantities of the hit were prepared for pharmacokinetic and efficacy studies. The synthetic strategy allowed for the preparation of multiple analogs for SAR exploration.     

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.     

2-Piperazinecarboxamides as potent and selective melanocortin subtype-4 receptor agonists

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. The 5- and 6-alkylated piperazine compounds exhibit low bioactivation potential as measured by covalent binding in microsome preparations.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Cadmium induces a heterogeneous and caspase-dependent apoptotic response in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The toxic metal cadmium is linked to a series of degenerative disorders in humans, in which Cd-induced programmed cell death (apoptosis) may play a role. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in a yca1Δ mutant. Cadmium-dependent apoptosis was also suppressed in a gsh1Δ mutant, indicating a requirement for glutathione. Other apoptotic markers, including sub-G₁ DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. These responses were not distributed uniformly throughout the cell population, but were restricted to a subset of cells. This apoptotic subpopulation also exhibited markedly elevated levels of intracellular reactive oxygen species (ROS). The heightened ROS levels alone were not sufficient to induce apoptosis. These findings highlight several new perspectives to the mechanism of Cd-dependent apoptosis and its phenotypic heterogeneity, while opening up future analyses to the power of the yeast model system.