Effects of PRELI in Oxidative-Stressed HepG2 Cells.

Protein of relevant evolutionary and lymphoid interest (PRELI) is known for preventing apoptosis by mediating intramitochondrial transport of phosphatidic acid. However, the role of PRELI remains unclear. This study has demonstrated functions of PRELI through PRELI-knockdown in hepatocellular carcinoma (HepG2) cells exposed to oxidative stress by hydrogen peroxide. Results show that PRELI has three functions in HepG2 cells with regard to oxidative stress. First, PRELI affects expressional regulation of SOD-1 and caspase-3 genes in HepG2 cells. PRELI knockdown HepG2 cells have shown up-regulation of caspase-3 and down-regulation of SOD-1. Second, PRELI suppresses mitochondrial apoptosis in HepG2 cells. Fluorescence intensity related to mitochondrial apoptosis in PRELI-knockdown HepG2 cells increased more than two-fold compared to normal HepG2 cells. Third, PRELI suppresses senescence of HepG2 cells with oxidative stress. PRELI knockdown HepG2 cells showed higher levels of senescence than normal HepG2 cells. These results suggest that PRELI is a crucial protein in the suppression of apoptosis in HepG2 cells in response to oxidative stress.

DISTINCTIVE LOCALIZATION OF GROUP 3 LATE EMBRYOGENESIS ABUNDANT SYNTHESIZING CELLS DURING BRINE SHRIMP DEVELOPMENT.

Despite numerous studies on late embryogenesis abundant (LEA) proteins, their functions, roles, and localizations during developmental stages in arthropods remain unknown. LEA proteins protect crucial proteins against osmotic stress during the development and growth of various organisms. Thus, in this study, fluorescence in situ hybridization was used to determine the crucial regions protected against osmotic stress as well as the distinctive localization of group 3 (G3) LEA(+) cells during brine shrimp development. Several cell types were found to synthesize G3 LEA RNA, including neurons, muscular cells, APH-1(+) cells, and renal cells. The G3 LEA(+) neuronal cell bodies outside of the mushroom body projected their axonal bundles to the central body, but those inside the mushroom body projected their axonal bundles toward the deutocerebrum without innervating the central body. The cell bodies inside the mushroom body received axons of the G3 LEA(+) sensory cells at the medial ventral cup of the nauplius eye. Several glands were found to synthesize G3 LEA RNA during the nauplius stages of brine shrimp, including the sinus, antennal I and II, salt, and three ectodermal glands. This study provides the first demonstration of the formation of G3 LEA(+) sinus glands at the emergence stages of brine shrimp. These results suggest that G3 LEA protein is synthesized in several cell types. In particular, specific glands play crucial roles during the emergence and nauplius stages of brine shrimp.

Developing new methods to answer old and new questions in neurodegenerative diseases: 21(st) Workshop of the HUPO Brain Proteome Project (HBPP) 23-24 January 2014, Honolulu, Hawaii.

The HUPO Brain Proteome Project (HUPO BPP) held its 21(st) workshop in Honolulu, Hawaii. During the 23-24 January 2014 the island became the center of the open workshop of the scientific community.

Advanced glycation end-products and receptor for advanced glycation end-products expression in patients with idiopathic pulmonary fibrosis and NSIP.

Advanced glycation end products (AGEs) are associated with the pathogenesis of various diseases. AGEs induce excess accumulation of extracellular matrix and expression of profibrotic cytokines. In addition, studies on receptor for advanced glycation end products (RAGE) have shown that the ligand-RAGE interaction activates several intracellular signaling cascades associated with several fibrotic diseases. We investigated the expression of AGEs and RAGE in samples from patients with idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP). Lung tissues and plasma samples from patients with IPF (n=10), NSIP (n=10), and control subjects (n=10) were obtained. Expression of AGEs and RAGE was determined by immunofluorescence assay of lung tissue. Circulating AGEs were measured by Western blot and enzyme-linked immunosorbent assay. Lungs with IPF showed strong expression for both AGEs and RAGE compared to that in NSIP and controls. However, no difference in AGE or RAGE expression was observed in lungs with NSIP compared to that in the controls. Levels of circulating AGEs also increased significantly in lungs of patients with IPF compared to those with NSIP and normal control. Increased AGE-RAGE interaction may play an important role in the pathogenesis of IPF.

Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases.

Silkworm 30K protein inhibits ecdysone-induced apoptosis by blocking the binding of ultraspiracle to ecdysone receptor-B1 in cultured Bm5 cells.

This study investigates the mechanism through which increased 30K protein inhibits ecdysone-induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20-hydroxyecdysone (20E) after transfection with the pIZT/V5-His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5-His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti-30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5-His-transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor-B1 (EcR-B1). Anti-30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5-His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR-B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR-B1-containing complexes from Bm5 cells transfected with control pIZT/V5-His vector and treated with 20E showed that EcR-B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5-His-transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR-B1 through formation of a 30K/EcR-B1 complex, resulting in inhibition of 20E-induced Bm5 cell apoptosis.

Increase of 30K protein in identified motoneurons by hemolymph results in inhibition of programmed cell death in silkworm, Bombyx mori (Lepidoptera, Bombycidae).

This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph.

Comparative evaluation of hypoxic-ischemic brain injury by flow cytometric analysis of mitochondrial membrane potential with JC-1 in neonatal rats.

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic-ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V(+)/PI(+) necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V(-)/PI(-) live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

Nardostachys jatamansi extract protects against cytokine-induced beta-cell damage and streptozotocin-induced diabetes.

AIM: To investigate the anti-diabetogenic mechanism of Nardostachys jatamansi extract (NJE). METHODS: Mice were injected with streptozotocin via a tail vein to induce diabetes. Rat insulinoma RINm5F cells and isolated rat islets were treated with interleukin-1beta and interferon-gamma to induce cytotoxicity. RESULTS: Treatment of mice with streptozotocin resulted in hyperglycemia and hypoinsulinemia, which was confirmed by immunohistochemical staining of the islets. The diabetogenic effects of streptozotocin were completely abolished when mice were pretreated with NJE. Inhibition of streptozotocin-induced hyperglycemia by NJE was mediated by suppression of nuclear factor (NF)-kappaB activation. In addition, NJE protected against cytokine-mediated cytotoxicity. Incubation of RINm5F cells and islets with NJE resulted in a significant reduction in cytokine-induced NF-kappaB activation and downstream events, inducible nitric oxide synthase expression and nitric oxide production. The protective effect of NJE was further demonstrated by the normal insulin secretion of cytokine-treated islets in response to glucose. CONCLUSION: NJE provided resistance to pancreatic beta-cell damage from cytokine or streptozotocin treatment. The beta-cell protective effect of NJE is mediated by suppressing NF-kappaB activation.

Production of nattokinase by batch and fed-batch culture of Bacillus subtilis.

Nattokinase was produced by batch and fed-batch culture of Bacillus subtilis in flask and fermentor. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on the production of nattokinase. In flask culture, the highest cell growth and nattokinase activity were obtained with 50 g/L of peptone supplementation. In this condition, nattokinase activity was 630 unit/ml at 12 h. In batch culture of B. subtilis in fermentor, the highest nattokinase activity of 3400 unit/ml was obtained at 10h with 50 g/L of peptone supplementation. From the batch kinetics data, it was shown that nattokinase production was growth-associated and culture should be harvested before stationary phase for maximum nattokinase production. In fed-batch culture of B. subtilis using pH-stat feeding strategy, cell growth (optical density monitored at 600 nm) increased to ca. 100 at 22 h, which was 2.5 times higher than that in batch culture. The highest nattokinase activity was 7100 unit/ml at 19 h, which was also 2.1 times higher than that in batch culture.

The effect of intravesical electrical stimulation on bladder function and synaptic neurotransmission in the rat spinal cord after spinal cord injury.

OBJECTIVE: To investigate the effects of intravesical electrical stimulation (IVES) on bladder function and synaptic neurotransmission in the lumbosacral spinal cord in the spinalized rat, as the clinical benefits of IVES in patients with increased residual urine or reduced bladder capacity have been reported but studies on the mechanism of IVES have mainly focused on bladder A delta afferents in central nervous system-intact rats. MATERIALS AND METHODS: In all, 30 female Sprague-Dawley rats were divided equally into three groups: normal control rats, sham-stimulated spinalized rats and IVES-treated spinalized rats. IVES was started 5 weeks after spinal cord injury (SCI) and was performed 20 min a day for 5 consecutive days. At 7 days after IVES, conscious filling cystometry was performed. Sections from the L6 and S1 spinal cord segments were examined for n-methyl-d-aspartic acid receptor 1 (NMDAR1) subunit and gamma-aminobutyric acid (GABA) immunoactivity. RESULTS: In IVES-treated spinalized rats, the number and maximal pressure of nonvoiding detrusor contractions were significantly less than in sham-stimulated spinalized rats. The mean maximal voiding pressure was also lower in IVES-treated than in sham-stimulated spinalized rats. IVES significantly reduced the interval between voiding contractions compared with the untreated spinalized rats. There was an overall increase in NMDAR1 immunoactivity after SCI, which was significantly lower in IVES-treated spinalized rats. Immunoactivity of GABA after SCI was significantly lower than in the control group and was significantly higher in IVES-treated spinalized rats. CONCLUSION: Our results suggest that IVES might affect voiding contractions in addition to inhibiting C-fibre activity and that IVES seems to have a more complex effect on the bladder control pathway. For synaptic neurotransmission in the spinal cord, IVES could possibly shift the balance between excitation and inhibition towards inhibition.

NF-kappaB activates transcription of the RNA-binding factor HuR, via PI3K-AKT signaling, to promote gastric tumorigenesis.

BACKGROUND & AIMS: HuR is a RNA-binding factor whose expression is commonly upregulated in some human tumor types. We explored the molecular mechanism underlying HuR elevation and its role in gastric cancer tumorigenesis. METHODS: HuR expression and subcellular localization were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Its effect on tumor growth was characterized using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and soft agar analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor kappaB (NF-kappaB) signaling. RESULTS: Compared with normal gastric tissues, HuR was expressed at higher levels in gastric tumors, particularly in advanced versus early tumors; this increase was associated with enhanced cytoplasmic translocation of HuR. HuR overexpression increased proliferation of tumor cells, activating the G(1) to S transition of the cell cycle, DNA synthesis, and anchorage-independent growth. Small interfering RNA-mediated knockdown of HuR expression reduced tumor cell proliferation and response to apoptotic stimuli. No genetic or epigenetic alterations of HuR were observed in gastric tumor cell lines or primary tumors; overexpression depended on phosphatidylinositol 3-kinase/AKT signaling and NF-kappaB activity. AKT activation increased p65/RelA binding to a putative NF-kappaB binding site in the HuR promoter, the stability of HuR target transcripts, and the cytoplasmic import of HuR. CONCLUSIONS: HuR is a direct transcription target of NF-kappaB; its activation in gastric cancer cell lines depends on phosphatidylinositol 3-kinase/AKT signaling. HuR activation by this pathway has proliferative and antiapoptotic effects on gastric cancer cells.

Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.

c-fos expression in bladder-specific spinal neurons after spinal cord injury using pseudorabies virus.

PURPOSE: c-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV). MATERIALS AND METHODS: Forty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons. RESULTS: Immunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4+/-10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/section, and 2.7+/-1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder-specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder. CONCLUSION: We confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia.

Although acidosis may be involved in neuronal death, the participation of Na(+)/H(+) exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1 day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na(+)/Ca(2+) exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.

The time is right: proteome biology of stem cells.

In stem cell biology, there is a growing need for advanced technologies that may help to unravel the molecular mechanisms of self-renewal and differentiation. Proteomics, the comprehensive analysis of proteins, is such an emerging technique. To facilitate interactions between specialists in proteomics and stem cell biology,a new initiative has been undertaken, supported by the Human Proteome Organization (HUPO) and the International Society for Stem Cell Research (ISSCR). Here we present the Proteome Biology of Stem Cells Initiative (PBSCI) and report on its goals and future activities.

Differential immunoreactivity of microglial and astrocytic marker protein in the hippocampus of the seizure resistant and sensitive gerbils.

In the present study, we compared differences in ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) immunoreactivities for microglia and astrocytes, respectively, in the hippocampus of the seizure-resistant (SR) and seizure-sensitive (SS) gerbils. The density of Iba-1 immunoreactive microglia in the hippocampal CA1 region (CA1) and dentate gyrus (DG) of the SS gerbil was higher than that in the SR gerbil, and many Iba-1 immunoreactive microglia in the SS gerbil were hypertrophied in morphology. In contrast, we could not find significant difference in the density of GFAP immunoreactive astrocytes between the SR and SS gerbils. This result indicates that Iba-1 immunoreactive microglia in CA1 and DG of the SS gerbil are activated compared to those in the SR gerbil.

Defective cholesterol traffic and neuronal differentiation in neural stem cells of Niemann-Pick type C disease improved by valproic acid, a histone deacetylase inhibitor.

Niemann-Pick type C disease (NPC) is a neurodegenerative and lipid storage disorder for which no effective treatment is known. We previously reported that neural stem cells derived from NPC1 mice showed impaired self-renewal and differentiation. We examined whether valproic acid (VPA), a histone deacetylase inhibitor, could enhance neuronal differentiation and recover defective cholesterol metabolism in neural stem cells (NSCs) from NPC1-deficient mice (NPC1(-/-)). VPA could induce neuronal differentiation and restore impaired astrocytes in NSCs from NPC1(-/-) mice. Importantly, an increasing level of cholesterol within NSCs from NPC1(-/-) mice could be reduced by VPA. Moreover, essential neurotrophic genes (TrkB, BDNF, MnSoD, and NeuroD) were up-regulated through the repression of the REST/NRSF and HDAC complex by the VPA treatment. Up-regulated neurotrophic genes were able to enhance neural differentiation and cholesterol homeostasis in neural stem cells from NPC1(-/-) mice. In this study, we suggested that, along with cholesterol homeostasis, impaired neuronal differentiation and abnormal morphology of astrocytes could be rescued by the inhibition of HDAC and REST/NRSF activity induced by VPA treatment.

Miniaturized asymmetrical flow field-flow fractionation: application to biological vesicles.

Asymmetrical flow field-flow fractionation (AFlFFF) has been carried out in a miniaturized channel by reducing the channel dimensions. Performance of the miniaturized AFlFFF (mAFlFFF) channel was evaluated with standard proteins and polystyrene latex spheres from nanometer to micrometer size. By reducing the channel dimension, proteins or particulate materials can be separated within a few minutes without a significant loss in resolution. The mAFlFFF channel was applied for the separation of exosomes harvested from immortalized human mesenchymal stem cell line. It shows a potential to fractionate exosome vesicles according to sizes which can be useful for proteomic studies in relation to immunotherapeutic applications.