Interleukin-1α treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1α)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter × 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1α-treatment, GAG loss (DMMB assay), biosynthetic activity ([³⁵SO₄]-sulfate and [³H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.     

Heat induced capsid disassembly and DNA release of bacteriophage λ

Successive structural changes of bacteriophage λ upon heating were characterized with quantitative experimental methods. In the commonly used Tris-Mg buffer, differential scanning calorimetry measurements first established that the protein capsid of λ phage melts at 87 °C and its genomic DNA melts at 91 °C. Interestingly, prior to the capsid melting, λDNA was found to escape out of the capsid and subject to DNase digestion above ~68 °C, as concluded from light scattering, UV absorption, and electron microscopy studies. Further investigations indicated distinct temperature-dependent behaviors of the three phage proteins. Around 68 °C, disruption of the tail first occurs and leads to the escape of λ DNA; above the capsid melting temperature of 87 °C, the auxiliary protein gpD of the phage head remains soluble in solution and resists centrifugal sedimentation, whereas the major capsid protein gpE is easily precipitated and likely exists as aggregates.     

Microparticulate P2X7 and GSDM-D mediated regulation of functional IL-1β release

Purinergic Signalling - The pro-inflammatory cytokine IL-1β is a secreted protein that is cleaved by caspase-1 during inflammasome activation upon recognition of internal and external insults...     

Macrophage-induced expression and release of matrix metalloproteinase 1 and 3 by human preadipocytes is mediated by IL-1β via activation of MAPK signaling

Obesity is associated with a chronic low‐grade inflammation and increased macrophage infiltration in adipose tissue. Matrix metalloproteinases (MMPs) are involved in adipose tissue remodeling and inflammatory responses in obesity. This study investigated whether macrophage‐derived factors modulate expression and secretion of MMP1 and MMP3 in human preadipocytes. The potential mediators and signaling pathways were also explored. MMP1 and MMP3 were primarily expressed and secreted by preadipocytes and dramatically reduced post‐differentiation. Preadipocytes were incubated with RPMI 1640 medium (control) or THP‐1 macrophage‐conditioned (MC) medium (25% and 100%) for 24 h. MC medium markedly increased mRNA levels of MMP1 (up to 122‐fold) and MMP3 (up to 59‐fold), as well as protein release of MMP1 (up to 378‐fold) and MMP3 (up to 10‐fold) in a dose‐dependent manner. Treatment with IL‐1β or TNFα, the major products of macrophages, also induced MMP1 and MMP3 secretion by preadipocytes. Neutralizing IL‐1β abolished the induction of MMP1 and MMP3 in preadipocytes by MC medium while the effects of TNFα neutralization were modest. Furthermore, MC medium or IL‐1β led to the phosphorylation of p38, ERK and JNK MAPKs. Inhibition of p38, ERK and JNK reversed the stimulatory effects of MC or IL‐1β on MMP1 and MMP3 production. MC medium and IL‐1β also activated NF‐κB p65 whereas reduced IκBα protein expression in preadipocytes. These results suggest that macrophage accumulation in adipose tissue has a central role in stimulating MMP1 and MMP3 production by preadipocytes, and this is partially mediated by IL‐1β via activation of the MAPK and NF‐κB signaling pathways. J. Cell. Physiol. 226: 2869–2880, 2011. © 2011 Wiley‐Liss, Inc.     

Oxygen-glucose deprivation and interleukin-1α trigger the release of perlecan LG3 by cells of neurovascular unit

Two of the main stresses faced by cells at the neurovascular unit (NVU) as an immediate result of cerebral ischemia are oxygen-glucose deprivation (OGD)/reperfusion and inflammatory stress caused by up regulation of IL-1. As a result of these stresses, perlecan, an important component of the NVU extracellular matrix, is highly proteolyzed. In this study, we describe that focal cerebral ischemia in rats results in increased generation of laminin globular domain 3 (LG3), the c-terminal bioactive fragment of perlecan. Further, in vitro study of the cells of the NVU was performed to locate the source of this increased perlecan-LG3. Neurons, astrocytes, brain endothelial cells and pericytes were exposed to OGD/reperfusion and IL-1α/β. It was observed that neurons and pericytes showed increased levels of LG3 during OGD in their culture media. During in vitro reperfusion, neurons, astrocytes and pericytes showed elevated levels of LG3, but only after exposure to brief durations of OGD. IL-1α and IL-1β treatment tended to have opposite effects on NVU cells. While IL-1α increased or had minimal to no effect on LG3 generation, high concentrations of IL-1β decreased it in most cells studied. Finally, LG3 was determined to be neuroprotective and anti-proliferative in brain endothelial cells, suggesting a possible role for the generation of LG3 in the ischemic brain.     

Effect of gabaculine on metabolism and release of γ-aminobutyric acid in synaptosomes

Synaptosomes isolated from mouse brain were incubated with [¹⁴C]glutamate and [³H]-aminobutyric acid ([³H]GABA), and then [¹⁴C]GABA (newly synthesized GABA) and [³H]GABA (newly captured GABA) in the synaptosomes were analysed. (1) the [³H]GABA was rapidly degraded in the synaptosomes, (2) when the synaptosomes were treated with gabaculine (a potent inhibitor of GABA aminotransferase), the degradation of [³H]GABA was strongly inhibited, (3) the gabaculine treatment brough about a significant increase in Ca²⁺-independent release of [³H]GABA with no effect on Ca²⁺-dependent release, (4) no effects of gabaculine on degradation and release of [¹⁴C]GABA were observed. The results indicate that there are at least two pools of GABA in synaptosomes and support the possibilities that GABA taken up into a pool which is under the influence of GABA aminotransferase is released Ca²⁺-independently and that GABA synthesized in another pool which is not under the influence of GABA aminotransferase is released Ca²⁺-dependently.     

Termination of translation: interplay of mRNA,rRNAs and release factors?

Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain release factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.     

Macrophage inflammatory protein (MIP)1α and MIP1β differentially regulate release of inflammatory cytokines and generation of tumoricidal monocytes in malignancy

The C–C chemokines, macrophage inflammatory protein (MIP)1α and MIP1β are potent chemoattractants for the monocytes, which form an important component of the stroma of tumor tissue and may regulate tumor growth and associated inflammation. We examined the role of MIP1α and MIP1β in inducing the release of inflammatory cytokines and the generation of tumoricidal monocytes from the peripheral blood monocytes (PBM) of healthy women and patients with carcinoma of breast (CaBr). Interleukin-1 (IL-1) and tumor necrosis factor (TNF) α release by the PBM was markedly stimulated by MIP1α in CaBr patients, but only marginally so in healthy women. In contrast, MIP1β stimulated the release of these cytokines by the PBM of healthy women, but failed to do so in CaBr patients. MIP1α, but not MIP1β, synergized with LPS in inducing the release of IL-1 from the PBM of both healthy women and CaBr patients. Both MIP1α and MIP1β augmented respiratory bursts in PBM and generated tumoricidal PBM that killed T24 cells, MIP1α being more effective in CaBr patients and MIP1β in healthy women. IFN-γ co-stimulated and IL-4 suppressed MIP1α and β-induced cytotoxicity in PBM. The synergy of IFN-γ was more marked with MIP1α than with MIP1β. The differential effects of MIP1α and MIP1β on the PBM of healthy women and CaBr patients co-related with the levels of expression of CCR1 and CCR5 in these monocytes. The expression of CCR5 was higher than that of CCR1 in the PBM of healthy women and the PBM of the CaBr patients showed overexpression of CCR1 and downregulation of CCR5.     

Model of synthesis and release of insulin from rat islet β-cells and the effect of pretreatment with tolbutamide

A kinetic model involving synthesis of proinsulin in the rough endoplasmic reticulum, maturation through the Golgi apparatus and granules, with conversion to insulin, is proposed to account for data on the amount of insulin and of proinsulin both secreted during various time intervals and remaining in islets. Introducing three compartments for granules makes it possible to account for the measurement of both hot (pulse labeled with tritiated leucine) and cold proinsulin and insulin over a period of 21/2 hr under constant glucose. Data from islets from animals pretreated with tolbutamide are also presented and modeled. The model is then expanded so that it can be successfully applied to available data on the effects of a period of glucose deprivation on secretion of both hot and cold hormone. Parameters have essentially the same values, where they overlap, as were obtained (Landahl and Grodsky, 1982Bull. math. Biol. 44, 399–410) from insulin secretion by perfused rat pancreas stimulated by a variety of temporal patterns of glucose concentration.     

Regulation of intracellular calcium release and PP1α in a mechanism for 4-hydroxytamoxifen-induced cytotoxicity

Treatment with tamoxifen, or its metabolite 4-hydroxytamoxifen (4OHT), has cytostatic and cytotoxic effects on breast cancer cells in vivo and in culture. Although the effectiveness of 4OHT as an anti-breast cancer agent is due to its action as an estrogen receptor-alpha (ERα) antagonist, evidences show that 4OHT is also cytotoxic for ERα-negative breast cancer cells and can be effective therapy against tumors that lack estrogen receptors. These findings underscore 4OHT signaling complexities and belie the most basic understandings of 4OHT action and resistance. Here, we have investigated the effects of 4OHT on Ca²⁺ homeostasis and cell death in breast cancer cells in culture. Measurement of Ca²⁺ signaling in breast cancer cells showed that 4OHT treatment altered Ca²⁺ homeostasis and was cytotoxic for both an ERα+ and an ERα- cell line, MCF-7 and MDA-MB-231, respectively. Further investigation lead us to the novel discovery that 4OHT-induced increase of ATP-dependent Ca²⁺ release from the endoplasmic reticulum correlated with 4OHT-induced upregulation of protein phosphatase 1α (PP1α) and the inositol 1,4,5-trisphosphate receptor (IP3R). Blocking 4OHT-induced PP1α upregulation by siRNA strategy reduced the effects of 4OHT on both Ca²⁺ signaling and cytotoxicity. Results from these investigations strongly suggest a role for PP1α upregulation in a mechanism for 4OHT-induced changes to Ca²⁺ signaling that ultimately contribute to the cytotoxic effects of 4OHT. Aliccia Bollig, Liping Xu- contributed equally to this work     

β-adrenergic insulin release and adrenergic innervation of mouse pancreatic islets

Summary An investigation of the stereospecificity of -adrenergic insulin release, its relation to -adrenergic blockade and the adrenergic innervation of the pancreatic islets was performed in the mouse. It was observed that in vivo -adrenergic stimulation of insulin release by isopropylnoradrenaline was stereospecific for the L-stereoisomer and selectively blocked by the L-isomer of the -adrenergic antagonist L-propranolol. D-propranolol had no effect. Pretreatment of mice with a dose of D-isopropylnoradrenaline devoid of insulin releasing activity, slightly increased the subsequent insulin response to a halfmaximal dose of L-isopropylnoradrenaline. Basal insulin secretion was blocked by L-propranolol (-adrenergic blockade) and increased by phentolamine (-adrenergic blockade). A -blocked insulin response to L-isopropyl-noradrenaline could be overcome by -adrenergic blockade depending on the dose of the -agonist, suggesting a close association between the adrenergic receptors.The adrenergic innervation of the islet cells was studied by electron microscopic autoradiography after injection of ³H-L-noradrenaline. It was observed that labelled adrenergic nerve terminals were associated with both A₁(D-), A₂ and B-cells. The nerves were mainly distributed in the periphery of the islets either as single axons or as bundles. The majority of the terminals were associated with A₂-cells, the most frequent cell type in the islet periphery. However, in all islets examined terminals were found close to B-cells. Adrenergic terminals often caused indentations in the contour of an islet cell and were separated from the islet cell membrane only by a narrow intercellular space, about 20 nm in width.It is concluded that the islet cells of the mouse are equipped with the morphological substrate for direct adrenergic regulation. Further it is suggested that the B-cell is supplied with L-stereospecific -adrenergic receptors and that the - and -adrenergic receptors are at least partially interrelated.     

Effects of thiazole analogs of vitamin B1 on neuromuscular transmission and α-latrotoxin-induced transmitter release in skeletal muscles

Thiazole analogs of vitamin B₁ 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)thiazole chloride (DMHT) and 3-decyloxycarbonylmethyl-4-methylthiazole chloride (DMT) suppress quantum transmitter release from nerve terminals in the frog skeletal muscle. Intraperitoneal administration of these compounds to mice suppresses behavioral motor activity, diminishes motor coordination, and suppresses the corazol-induced seizures. Application of DMHT reduces the -latrotoxin-induced massive transmitter release from nerve terminals in the frog skeletal muscle and suppresses latrotoxin-induced seizures in mice. In model experiments, DMHT blocks Ca²⁺ entry through the ion channels formed by -latrotoxin in a bilayer lipid membrane. It has been suggested that the effectiveness of DMHT and DMT is determined by the presence of a thiazole cycle in their molecules that, among all endogenous biologically active compounds, is possessed only by vitamin B₁ and its metabolites.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 368–374, September–December, 1995.     

Binding and release kinetics of inhibitors of Q−A oxidation in thylakoid membranes

Oxygen flash yield patterns of dark adapted thylakoid membranes as measured with a Joliot-type O₂-electrode indicate that inhibitors that block the oxidation of the reduced primary quinone Q^?_A of Photosystem II vary greatly in the rate of binding to and release from the inhibitor / Q_B binding environment. The ‘classical’ Photosystem-II herbicides like diuron and atrazine exhibit slow binding and release kinetics, whereas, for example, phenolic inhibitors, o-phenanthroline and synthetic quinones are exchanging quite rapidly with Q_B (about once per second or faster at inhibitor concentrations causing about 50% inhibition of O₂ evolution). No general relationship between the efficiency of the inhibitor and the exchange rate is observed; it depends mainly on the type of inhibitor. Based on the classical Kok model, equations are derived in order to calculate oxygen yields evolved by thylakoids in single-turnover flashes as a function of the rate constants of inhibitor binding to and release from the inhibitor / Q_B binding environment in the presence of an oxidized or semireduced Q_A · Q_B or Q_A · inhibitor complex. Fitting of theoretical and experimental values yields that o-phenanthroline binds much faster to an oxidized than to a semireduced Q_A · Q_B complex. This fits very well with the hypothesis that the Q^?_B affinity to the site is much higher than that of Q_B. In the case of i-dinoseb, however, inhibitor / quinone exchange seems to occur mainly in the semiquinone state. Possibilities to explain this result are discussed.     

Comparison of release of endogenous dopamine and γ-aminobutyric acid from rat caudate synaptosomes

Release of endogenous dopamine (DA) and -aminobutyric acid (GABA) from superfused rat caudate synaptosomes was monitored with liquid chromatography with electrochemical detection. Dopamine was analyzed by oxidative detection following alumina extraction while GABA was analyzed with reductive detection following pre-column derivatization with trinitrobenzenesulfonic acid and extraction. Both spontaneous and K⁺-stimulated (40 mM) release were examined as well as the effect of several possible neuromodulatory agents (DA, GABA, muscimol, ascorbic acid, acetylcholine). The content of GABA in the sample and the amount released by K⁺ were approximately fifty times those of DA although the relative amounts released by repetitive K⁺ stimulations were similar. Muscimol and DA significantly attenuated both the spontaneous and stimulated release of GABA while ascorbate and acetylcholine had no effect. Acetylcholine significantly increased both the stimulated and spontaneous release of DA while the other agents had no effect. Dopamine showed an absolute dependence on calcium for stimulated release while GABA exhibited a significant calcium-independent release. These results indicate that profound differences exist in the factors which modulate the release of endogenous DA and GABA.     

Effect of oxygen–glucose deprivation on degranulation and histamine release of mast cells

The purpose of this study was to investigate the effect of oxygen–glucose deprivation (OGD) on degranulation and histamine release of mast cells. Cultured mast cells were exposed to OGD for 1, 2, 4, 8, or 16 h. At 2 h of OGD exposure, the degranulation percentage of mast cells had increased and subsequently showed a progressive further increase, associated with a similar change in lactate dehydrogenase release. Histamine release increased significantly from 1 h of OGD exposure. These results indicate that OGD induces mast cells to degranulate, possibly via a cytotoxic response. This in vitro ischemic model of mast cells might clarify their roles in the pathological processes induced by cerebral ischemia. This project was supported by the National Natural Science Foundation of China (nos. 30371638, 30472013) and the National Basic Research of China (no. 2003CB515400), and partly by the Zhejiang Provincial Natural Science Foundation of China (R303779) and the Zhejiang Provincial Scientific Research Foundations (2004C34002).     

The effect of β-endorphin on arginine-induced growth hormone and prolactin release

β-endorphin administration via constant infusion inhibited the release of growth hormone (GH) and augmented the release of prolactin (PRL) induced by arginine in normal female subjects. Although β-endorphin infusion also induced hyperglycemia, the increment in plasma glucose was insufficient to account for the observed suppression of arginine-initiated GH release. These studies demonstrate that β-endorphin influences, in opposed directions, the secretion of PRL and GH in women.     

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.