Serum insulin-like growth factor-1 levels in females and males in different cervical vertebral maturation stages

OBJECTIVE: The aim of this cross sectional study was to assess serum insulin-like growth factor-1 (IGF-1) levels in female and male subjects at various cervical vertebral maturation (CVM) stages. MATERIAL AND METHODS: The study sample consisted of 60 subjects, 30 females and 30 males, in the age range of 8-23 years. For all subjects, serum IGF-1 level was estimated from blood samples by means of chemiluminescence immunoassay (CLIA). CVM was assessed on lateral cephalograms using the method described by Baccetti. Serum IGF-1 level and cervical staging data of 30 female subjects were included and taken from records of a previous study. Data were analyzed by Kruska-Wallis and Mann Whitney test. Bonferroni correction was carried out and alpha value was set at 0.003. RESULTS: Peak value of serum IGF-1 was observed in cervical stages CS3 in females and CS4 in males. Differences between males and females were observed in mean values of IGF-1 at stages CS3, 4 and 5. The highest mean IGF-1 levels in males was observed in CS4 followed by CS5 and third highest in CS3; whereas in females the highest mean IGF-1 levelswas observed in CS3 followed by CS4 and third highest in CS5. Trends of IGF-1 in relation to the cervical stages also differed between males and females. The greatest mean serum IGF-1 value for both sexes was comparable, for females (397 ng/ml) values were slightly higher than in males (394.8 ng/ml). CONCLUSIONS: Males and females showed differences in IGF-1 trends and levels at different cervical stages. .

OBJETIVO: o objetivo do presente estudo transversal foi avaliar os níveis do fator de crescimento semelhante à insulina-1 (IGF-1 sérico) em pacientes de ambos os sexos e em diferentes estágios de maturação das vértebras cervicais (MVC). MÉTODOS: a amostra consistiu de 60 pacientes, sendo 30 do sexo masculino e 30 do sexo feminino, com idades entre 8 e 23 anos. Amostras de sangue foram colhidas de todos os pacientes, cujos níveis de IGF-1 sérico foram avaliados por meio do método de imunoensaio quimioluminescente (CLIA). O estágio de MVC foi avaliado por meio de radiografias cefalométricas de perfil por meio do método descrito por Baccetti. O nível de IGF-1 sérico e o estágio de maturação das vertebras cervicais de 30 pacientes do sexo feminino foram avaliados e os dados retirados dos registros de um estudo prévio. Os dados foram submetidos aos testes de Kruskal-Wallis e de Mann-Whitney. A correção de Bonferroni foi calculada e o valor de alfa foi de 0,003. RESULTADOS: o valor de pico do IGF-1 sérico foi encontrado no estágio CS3, para mulheres, e CS4, para homens. Foram encontradas diferenças entre as médias dos valores de IGF-1 entre homens e mulheres nos estágios CS3, 4 e 5. O valor médio mais alto para os níveis de IGF-1 nos homens foi observado no estágio CS4, seguido do estágio CS5 e CS3. Nas mulheres, o valor médio mais alto foi observado em CS3, seguido do estágio CS4 e CS5. Diferenças também foram encontradas quanto à curva do IGF-1, em relação ao estágio de maturação das vértebras cervicais nos pacientes de ambos os sexos. O valor médio de IGF-1 sérico mais alto foi comparado. As pacientes do sexo feminino apresentaram valores ligeiramente mais altos (397ng/ml) em comparação aos pacientes do sexo masculino (394.8ng/ml). CONCLUSÕES: homens e mulheres apresentam valores de IGF-1 diferentes em estágios de maturação das vértebras cervicais diferentes. .

Effects of sevoflurane on tight junction protein expression and PKC-alpha translocation after pulmonary ischemia-reperfusion injury

Pulmonary dysfunction caused by ischemia-reperfusion injury is the leading cause of mortality in lung transplantation. We aimed to investigate the effects of sevoflurane pretreatment on lung permeability, tight junction protein occludin and zona occludens 1 (ZO-1) expression, and translocation of protein kinase C (PKC)-alpha after ischemia-reperfusion. A lung ischemia-reperfusion injury model was established in 96 male Wistar rats following the modified Eppinger method. The rats were divided into four groups with 24 rats in each group: a control (group C), an ischemia-reperfusion group (IR group), a sevoflurane control group (sev-C group), and a sevoflurane ischemia-reperfusion group (sev-IR group). There were three time points in each group: ischemic occlusion for 45 min, reperfusion for 60 min and reperfusion for 120 min; and there were six rats per time point. For the 120-min reperfusion group, six extra rats underwent bronchoalveolar lavage. Mean arterial pressure (MAP) and pulse oxygen saturation (SpO2) were recorded at each time point. The wet/dry weight ratio and lung permeability index (LPI) were measured. Quantitative RT-PCR and Western blot were used to measure pulmonary occludin and ZO-1, and Western blot was used to measure cytosolic and membranous PKC-alpha in the lung. Lung permeability was significantly increased after ischemia-reperfusion. Sevoflurane pretreatment promoted pulmonary expression of occludin and ZO-1 after reperfusion and inhibited the translocation of PKC-alpha. In conclusion, sevoflurane pretreatment alleviated lung permeability by upregulating occludin and ZO-1 after ischemia-reperfusion. Sevoflurane pretreatment inhibited the translocation and activation of PKC-alpha, which also contributed to the lung-protective effect of sevoflurane.

Biological activity and redistribution of nucleolar proteins of two different cell lines treated with cis-dichloro-1,2-propylenediamine-N,N,N',N'-tetraacetato ruthenium (III) (RAP).

The interaction of a newly synthesized antitumor complex cis-dichloro-1,2-propylenediamine-N,N,N',N'-tetraacetato ruthenium (III) (RAP) with DNA was investigated in vitro through a number of techniques including comet assay, immunoprecipitation, and immunolocalization of certain nucleolar proteins (the upstream binding factor (UBF) and fibrillarin) involved in DNA transcription, rRNA processing, and ribosomal assembly. The results showed that RAP binds to the DNA of two cell lines (H4 and Hs-683) causing a delay in cell proliferation rate leading to a number of cellular modifications. These modifications include DNA-damage assessed by the single cell gel electrophoresis method (comet assay) and variation in the expression of nucleolar proteins; UBF was more abundant in RAP treated cells, this was explained by the high affinity of this protein to DNA modified by RAP. On the other hand, fibrillarin was found in less quantities in RAP treated cells which was explained by a de-regulation of the ribosomal machinery caused by RAP.

Synergistic Effect of Interleukin-6 and Hyaluronic Acid on Cell Migration and ERK Activation in Human Keratinocytes

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-kappaB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-kappaB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.

Autophagic failure promotes the exocytosis and intercellular transfer of alpha-synuclein

The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of alpha-synuclein aggregates and Lewy bodies, often found in PD and other alpha-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of alpha-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in alpha-synuclein-expressing cells would increase the secretion of alpha-synuclein, subsequently affecting the alpha-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of alpha-synuclein. In a mixed culture of alpha-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular alpha-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of alpha-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated alpha-synuclein exocytosis, thereby promoting alpha-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.

Rhamnogalacturonan II is a Toll-like receptor 4 agonist that inhibits tumor growth by activating dendritic cell-mediated CD8+ T cells

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.

Kurarinone promotes TRAIL-induced apoptosis by inhibiting NF-kappaB-dependent cFLIP expression in HeLa cells

This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspase-dependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the death-inducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IkappaB degradation and nuclear translocation of NF-kappaB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-kappaB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-kappaB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.

2-Deoxy-D-glucose regulates dedifferentiation through beta-catenin pathway in rabbit articular chondrocytes

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.

Endothelin 1 protects HN33 cells from serum deprivation-induced neuronal apoptosis through Ca2+-PKCalpha-ERK pathway

Endothelins (ETs), which were originally found to be potent vasoactive transmitters, were known to be implicated in nervous system, but the mode of mechanism remains unclear. ETs (ET-1, ET-2, and ET-3) were added to HN33 (mouse hippocampal neuron chi neuroblastoma) cells. Among the three types of ET, only ET-1 increased the intracellular calcium levels in a PLC dependent manner with the induction of ERK 1/2 activation. As the result of ET-1 exposure, the survival rate of HN33 cells and the PKCalpha translocation into the plasma membrane were increased. We suggest that ET-1 participated in the neuroprotective effect involving the calcium-PKCalpha-ERK1/2 pathway.

Cytosolic accumulation of gammaH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal thatgammagammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic beta-cells through suppression of NF-kappa B activation

We demonstrated previously that Coptidis rhizoma extract (CRE) prevented S-nitroso-N-acetylpenicillamine-induced apoptotic cell death via the inhibition of mitochondrial membrane potential disruption and cytochrome c release in RINm5F (RIN) rat insulinoma cells. In this study, the preventive effects of CRE against cytokine-induced beta-cell death was assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of beta-cell destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with IL-1beta and IFN-gamma resulted in a reduction of cell viability. CRE completely protected IL-1beta and IFN-gamma-mediated cell death in a concentration-dependent manner. Incubation with CRE induced a significant suppression of IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding which correlated well with reduced levels of the iNOS mRNA and protein. The molecular mechanism by which CRE inhibited iNOS gene expression appeared to involve the inhibition of NF-kappa B activation. The IL-1beta and IFN-gamma-stimulated RIN cells showed increases in NF-kappa B binding activity and p65 subunit levels in nucleus, and IkappaBalpha degradation in cytosol compared to unstimulated cells. Furthermore, the protective effects of CRE were verified via the observation of reduced NO generation and iNOS expression, and normal insulin-secretion responses to glucose in IL-1beta and IFN-gamma-treated islets.

Implication of phosphorylation of the myosin II regulatory light chain in insulin-stimulated GLUT4 translocation in 3T3-F442A adipocytes

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca2+/ calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3- F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca2+-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin- stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca2+/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.

Iron chelator inducesMIP-3alpha/CCL20 in human intestinal epithelial cells: implication for triggeringmucosal adaptive immunity

A previous report by this laboratory demonstrated that bacterial iron chelator (siderophore) triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs). Microarray-based gene expression profiling revealed that iron chelator also induces macrophage inflammatory protein 3 alpha (MIP-3alpha)/ CC chemokine-ligand 20 (CCL20). As CCL20 is chemotactic for the cells involved in host adaptive immunity, this suggests that iron chelator may stimulate IECs to have the capacity to link mucosal innate and adaptive immunity. The basal medium from iron chelator deferoxamine (DFO)-treated HT-29 monolayers was as chemotactic as recombinant human CCL20 at equivalent concentrations to attract CCR6+ cells. The increase of CCL20 protein secretion appeared to correspond to that of CCL20 mRNA levels, as determined by real-time quantitative RT-PCR. The efficacy of DFO at inducing CCL20 mRNA was also observed in human PBMCs and in THP-1 cells, but not in human umbilical vein endothelial cells. Interestingly, unlike other proinflammatory cytokines, such as TNF-alpha and IL-1beta, a time-dependent experiment revealed that DFO slowly induces CCL20, suggesting a novel mechanism of action. A pharmacologic study also revealed that multiple signaling pathways are differentially involved in CCL20 production by DFO, while some of those pathways are not involved in TNF-alpha-induced CCL20 production. Collectively, these results demonstrate that, in addition to some bacterial products known to induce host adaptive immune responses, direct chelation of host iron by infected bacteria may also contribute to the initiation of host adaptive immunity in the intestinal mucosa.

Epigallocatechin-3-gallate Suppresses Galactose-alpha1,4-galactose-beta1,4-glucose Ceramide Expression in TNF-alpha Stimulated Human Intestinal Epithelial Cells Through Inhibition of MAPKs and NF-kappa B

Intestinal epithelial cells (IECs) have been known to produce galactose-alpha1,4-galactose-beta1,4-glucose ceramide (Gb3) that play an important role in the mucosal immune response. The regulation of Gb3 is important to prevent tissue damage causing shiga like toxin. Epigallocatechin-3-gallate (EGCG) has been studied as anti-carcinogenic, anti-oxidant, anti-angiogenic, and anti-viral activities, and anti-diabetic. However, little is known between the expressions of Gb3 on IECs. The aim of this study was to examine the inhibitory effect of EGCG, a major ingredient of green tea, on Gb3 production via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappa B) in the TNF-alpha stimulated human colon epithelial cells, HT29. To investigate how Gb3 is regulated, ceramide glucosyltransferase (CGT), lactosylceramide synthase (GalT2), and Gb3 synthase (GalT6) were analyzed by RT-PCR in HT 29 cells exposed to TNF-alpha in the presence or absence of EGCG. EGCG dose-dependently manner, inhibits TNF-alpha induced Gb3 expression by blocking in both the MAPKs and NF-kappaB pathways in HT29 cells. TNF-alpha enhanced CGT, GalT2 and GalT6 mRNA levels and EGCG suppressed the level of these enzymes enhanced by TNF-alpha treatment.

Corn silk induces nitric oxide synthase in murine macrophages

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.

Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.