The function of microRNA-34a in osteoarthritis.

AIM: To discuss the effects and mechanism of microRNA-34a in cell apoptosis induced by osteoarthritis. METHODS: Collection of the normal and osteoarthritis synovial tissues and measurements of the miRNA-34a and TGIF2 gene expression. In the cell experiment, the cells were divided into Control, Blank and miRNA inhibitor group. The cell proliferation and apoptosis of the different groups were measured by MTT and flow cytometry and the TGIF2 protein expression in the different groups was evaluated by WB assay. The correlation between TGIF2 and miRNA-34a was analyzed by Double luciferase experiment. RESULTS: Compared with normal synovial tissues, the miRNA-34a gene expression was significantly up-regulated and TGIF2 gene expression was significantly suppressed in osteoarthritis synovial tissues (p < 0.001, respectively). The cell proliferation was significantly depressed and the cell apoptosis rate was significantly increased in miRNA inhibitor group compared with the Control group (p < 0.001, respectively). Using the WB assay it was shown that the TGIF2 protein expression of miRNA inhibitor group was significantly suppressed compared with that of Control group (p < 0.01). By Double luciferase assay, TGIF2 gene was one target gene of miRNA-34a. CONCLUSION: miRNA-34a could induce osteoarthritis synovial cell apoptosis via regulation of TGIF2 in vitro (Fig. 6, Ref. 29).

miRNA-31 over-expression improve synovial cells apoptosis induced by RA.

OBJECTIVE: The aim of this study was to evaluate the effects and mechanism of miRNA-31 in synovial cells apoptosis induced by RA. METHODS: The miRNA-31 gene expressions were extracted from synovial tissues of normal and RA patients by RT-PCR and H et E staining. The synovial cells of RA patients were isolated and randomly divided into Control, Blank and miRNA groups. The cell apoptosis of difference groups were measured by flow cytometry; the TNF-α and IL-1ß concentrations of difference groups were measured by Elisa assay; TLR4 and NF-κB proteins expressions were measured by WB assay and the correlation between TLR4 and miRNA-31 were evaluated by double luciferase target experiment. RESULTS: The miRNA-31 gene expression was significantly suppressed in RA tissues (p<0.001); Compared with control group, the cell apoptosis rate of miRNA group was significantly suppressed (p<0.001); TNF-α and IL-1ß concentrations were significantly down-regulation in culture fluid (p<0.001, respectively) and TLR4 and NF-κB proteins expressions were significantly depressed (p<0.001, respectively) in miRNA group. By double luciferase target experiment, the TLR4 was a target gene of miRNA-31. CONCLUSION: miRNA-31 is a key role in synovial cells apoptosis induced by RA (Fig. 7, Ref. 23).

A novel compact heat exchanger using gap flow mechanism.

A novel, compact gap-flow heat exchanger (GFHE) using heat-transfer fluid (HTF) was developed in this paper. The detail design of the GFHE coaxial structure which forms the annular gap passage for HTF is presented. Computational fluid dynamics simulations were introduced into the design to determine the impacts of the gap width and the HTF flow rate on the GFHE performance. A comparative study on the GFHE heating rate, with the gap widths ranged from 0.1 to 1.0 mm and the HTF flow rates ranged from 100 to 500 ml/min, was carried out. Results show that a narrower gap passage and a higher HTF flow rate can yield a higher average heating rate in GFHE. However, considering the compromise between the GFHE heating rate and the HTF pressure drop along the gap, a 0.4 mm gap width is preferred. A testing loop was also set up to experimentally evaluate the GFHE capability. The testing results show that, by using 0.4 mm gap width and 500 ml/min HTF flow rate, the maximum heating rate in the working chamber of the as-made GFHE can reach 18 °C/min, and the average temperature change rates in the heating and cooling processes of the thermal cycle test were recorded as 6.5 and 5.4 °C/min, respectively. These temperature change rates can well satisfy the standard of IEC 60068-2-14:2009 and show that the GFHE developed in this work has sufficient heat exchange capacity and can be used as an ideal compact heat exchanger in small volume desktop thermal fatigue test apparatus.

Overexpression of AT14A confers tolerance to drought stress-induced oxidative damage in suspension cultured cells of Arabidopsis thaliana.

Drought stress can affect interaction between plant cell plasma membrane and cell wall. Arabidopsis AT14A, an integrin-like protein, mediates the cell wall-plasma membrane-cytoskeleton continuum (WMC continuum). To gain further insight into the function of AT14A, the role of AT14A in response to drought stress simulated by polyethylene glycol (PEG-6000) in Arabidopsis suspension cultures was investigated. The expression of this gene was induced by PEG-6000 resulting from reverse transcription-PCR, which was further confirmed by the expression data from publically available microarray datasets. Compared to the wild-type cells, overexpression of AT14A (AT14A-OE) in Arabidopsis cultures exhibited a greater ability to adapt to water deficit, as evidenced by higher biomass accumulation and cell survival rate. Furthermore, AT14A-OE cells showed a higher tolerance to PEG-induced oxidative damage, as reflected by less H2O2 content, lipid peroxidation (malondialdehyde (MDA) content), and ion leakage, which was further verified by maintaining high levels of activities of antioxidant defense enzymes such as ascorbate peroxidase and guaiacol peroxidase and soluble protein. Taken together, our results suggest that overexpression of AT14A improves drought stress tolerance and that AT14A is involved in suppressing oxidative damage under drought stress in part via regulation of antioxidant enzyme activities.

Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays.

Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin beta1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall-plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall-plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays.

Sustained elevation of Epstein-Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma.

We have monitored Epstein-Barr virus (EBV) IgA antibody levels of 39 nasopharyngeal carcinoma (NPC) cases for up to 15 years before clinical onset of NPC, and assessed preclinical serologic status of another 68 cases. Our results identify a serologic window preceding diagnosis when antibody levels are raised and sustained. This window can persist for as long as 10 years, with a mean duration estimated to as 37+/-28 months. Ninety-seven of these 107 NPC cases exhibited such a window. Cases that did not may reflect individual antibody response to EBV. Serologic screening at enrollment identified those cases who had already entered the window and became clinically manifested earlier (median=28 months) than those who entered the window after enrollment (median=90 months). The former account for 19 of 21 cases diagnosed within 2 years of screening. Nasopharyngeal carcinoma risk levels among seropositive subjects were also highest during this period. Both prediction rates and risk levels declined thereafter; cases detected at later times were composed of increasing proportions of individuals who entered the serological window after screening. Our findings establish EBV antibody as an early marker of NPC and suggest that repeated screening to monitor cases as they enter this window has considerable predictive value, with practical consequences for cancer treatment.

Correlations between cadmium and mineral nutrients in absorption and accumulation in various genotypes of rice under cadmium stress.

The absorption and accumulation of Cd2+, Fe3+, Zn2+, Mn2+, Cu2+ and Mg2+ in the roots and leaves of 20 rice cultivars (Oryza sativa L.) with different genotypes under cadmium (Cd) stress were investigated with pot experiments. The results showed that there existed significant differences among the rice cultivars in the contents of six mineral elements in both roots and leaves at both heading and ripening periods. The statistical analysis showed that, for their contents in roots, significant and positive correlations between Cd2+ and Fe3+, Cd2+ and Zn2+, Cd2+ and Mn2+, Cd2+ and Cu2+ existed, but no significant correlation between Cd2+ and Mg2+, at the two periods. In the leaves, Cd also showed significant and positive correlations with Fe3+, Zn2+ and Cu2+ at the both periods, but a significant and negative correlation with Mn2+ and no significant correlation with Mg2+ at heading, a significant and positive correlation with Mg2+ and no significant correlation with Mn2+ at ripening. These results suggested that there were cooperative absorption between Cd2+ and Fe3+, Mn2+, Cu2+, Mn2+ in rice plants. Genotypic differences in Cd uptake and translocation among the rice cultivars suggested that paddy field of some rice cultivars may be irrigated with partially treated sewage water.

HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia.

Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.

Congenital diaphragmatic hernia misdiagnosed as pneumothorax in a newborn.

Congenital diaphragmatic hernia (CDH) is usually left sided and has a large defect allowing abdominal viscera herniated into thoracic cavity. The chest films usually show air-filled stomach and/or loops of bowel in the ipsilateral hemithorax with mediastinal shift. We report a newborn with CDH, presenting as hyperlucent hemithorax, right-shifted mediastinum, apparently normal pattern of abdominal bowel gas, with the tip of nasogastric tube below the left hemidiaphragm on the radiograph. It was initially misdiagnosed as pneumothorax, and the acute respiratory distress was temporarily relieved by needle aspiration. Hyperlucent hemithorax due to intrathoracic gastric dilatation alone is an unusual presentation of CDH in neonatal period. Absence of stomach bubble in the left upper quadrant of the abdomen, in both radiography and abdominal sonography, is an important clue to make diagnosis of CDH in this misleading condition.

Bone marrow transplantation from an HLA-matched unrelated donor for treatment of Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by partial albinism and large granules in all granule-containing cells. It is also associated with recurrent pyogenic infections secondary to impaired leukocyte function. Most patients with CHS enter an accelerated phase that leads to repeated infections and bleeding complications, often resulting in death. The first accelerated phase may occur shortly after birth or several years later. There are no curative treatments, and bone marrow transplantation (BMT) is the treatment of choice. Here, we report the case of a boy with CHS. The diagnosis was made at the age of 1 month, on the basis of the characteristic clinical findings and family history. He received BMT from an HLA-matched unrelated donor. After BMT, fluorescent cytometric analysis of polymorphonuclear leukocytes showed normalized cellular granularity and a normal increase in CD11b expression on N-formylmethionyl-leucyl-phenylalanine stimulation. The accelerated phase did not develop during 27 months of follow-up. Without BMT, CHS is usually fatal before the age of 10 years. BMT from an unrelated donor may be an effective treatment option for those who lack sibling donors. In addition to the characteristic leukocytic dysfunctions, fluorescent cytometric analysis of cellular granularity and surface molecules offer useful diagnostic information.

Inhibition of translocation of nascent apolipoprotein B across the endoplasmic reticulum membrane is associated with selective inhibition of the synthesis of apolipoprotein B.

In HepG2 cells, inhibition of apolipoprotein B100 (apoB) translocation across the endoplasmic reticulum by an microsomal triglyceride transfer protein (MTP) inhibitor (CP-10447) in the presence of N-acetyl-leucinyl-norleucinal, a proteasomal inhibitor, results in accumulation of newly synthesized apoB in the translocation channel. Here we demonstrated that such accumulation led to a specific reduction of apoB synthesis. ApoB mRNA levels remained unchanged, but we observed reduced rates of elongation of nascent apoB in puromycin-synchronized cells pretreated with MTP inhibitor. This observation was consistent with a longer half-ribosome transit time for the synthesis of apoB in MTP-inhibited cells. Initiation of translation of apoB mRNA was not impaired by MTP inhibition. Overall, these findings suggest that translocation arrest of apoB in the endoplasmic reticulum channel can exert a selective and negative effect on the synthesis of apoB at the stage of elongation.

Serum amyloid A in Alzheimer's disease brain is predominantly localized to myelin sheaths and axonal membrane.

Immunohistochemical localization of the injury specific apolipoprotein, acute phase serum amyloid A (A-apoSAA), was compared in brains of patients with neuropathologically confirmed Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD); Pick's disease (Pick's), dementia with Lewy bodies (DLB), coronary artery disease (CAD), and schizophrenia. Affected regions of both AD and MS brains showed intense staining for A-apoSAA in comparison to an unaffected region and non-AD/MS brains. The major site of A-apoSAA staining in both diseases was the myelin sheaths of axons in layers V and VI of affected cortex. A-apoSAA contains a cholesterol binding site near its amino terminus and is likely to have a high affinity for cholesterol-rich myelin. These findings, along with our recent evidence that A-apoSAA can inhibit lipid synthesis in vascular smooth muscle cells suggest that A-apoSAA plays a role in the neuronal loss and white matter damage occurring in AD and MS.

The acute phase response in apolipoprotein A-1 knockout mice: apolipoprotein serum amyloid A and lipid distribution in plasma high density lipoproteins.

In plasma, the bulk of apoSAA, a positive acute phase reactant protein, is transported in high density lipoproteins (HDL), especially HDLH (apoA1-rich HDL). In this study we tested whether apoA1 deficiency would adversely affect apoSAA concentration and lipid distribution in mouse plasma lipoproteins. Acute phase response (APR) was induced in C57BL/6J (apoA1+/+) and apoA1-knockout mice (apoA1-/-) by a subcutaneous injection of silver nitrate. The APR increased cholesterol concentrations in LDL of apoA1-/- mice and apoA1+/+ mice in a like manner. In contrast to apoA1+/+ mice, concentrations of cholesterol, phospholipids and proteins in both HDLL (1.063

Catabolism of lipid-free recombinant apolipoprotein serum amyloid A by mouse macrophages in vitro results in removal of the amyloid fibril-forming amino terminus.

Serum amyloid A fibrils are formed when the normally rapid catabolism of the acute-phase reactant apolipoprotein serum amyloid A (apoSAA) is incomplete; thus amyloidosis may be viewed as a condition of dysregulated proteolysis. There is evidence that apoSAA is dissociated from plasma high-density lipoprotein (HDL) prior to fibril formation. The objective of this study was to investigate degradation of lipid-free apoSAA by tissue macrophages derived from amyloid-susceptible CBA/J mice in vitro. Peritoneal macrophages derived from untreated (normal) mice converted apoSAA (12 kDa) to a single 4 kDa C-terminal peptide while splenic macrophages converted apoSAA to 10, 7 and 4 kDa C-terminal peptides and a 4 kDa peptide that lacked the C- and N-terminal regions. Similar patterns of proteolysis occurred when peritoneal and splenic macrophages from amyloidotic CBA/J mice were used. Conditioned medium prepared from peritoneal, but not splenic macrophages, degraded apoSAA. Specific sites of cleavage indicated activity of cathepsin G- and elastase-like neutral proteases. The data indicate that lipid-free apoSAA can be degraded by secreted or cell-associated neutral proteases that are generated by macrophages to yield peptides that lack fibrillogenic potential.

Evidence for local production of acute phase response apolipoprotein serum amyloid A in Alzheimer's disease brain.

Acute phase serum amyloid A (A-apoSAA), but not constitutive apoSAA (C-apoSAA), was identified by Western blotting experiments in brain protein extracts from eight of nine patients with Alzheimer's disease (AD), one with a brain tumor and one with multiple sclerosis. A-apoSAA was not detected in six subjects with Pick's or Lewy Body disease or three other non-AD brain specimens. Apolipoprotein A-I and albumin were not found in any of the brain protein extracts. A-apoSAA mRNA was detected in AD brain by reverse transcription-polymerase chain reaction (RT-PCR). These data suggest that apoSAA is locally produced in AD brain and that investigation of the neuroinflammatory effects of this injury specific apolipoprotein is warranted.

Anti-muscarinic toxins from Dendroaspis angusticeps.

Toxins from the venom of the African green mamba, Dendroaspis angusticeps, fulfill a major need for selective ligands for some of the five genetically defined subtypes of muscarinic acetylcholine receptors (m1-m5). Two toxins have been found that are highly selective antagonists for m1 and m4 receptors (m1-toxin and m4-toxin, respectively). Two other toxins (MT1 and MT2) bind with high affinity to both m1 and m4 receptors, and are agonists. Components of the venom also modify the binding of radiolabeled antagonists to m2 receptors, but an m2-selective toxin has not yet been isolated, m1-Toxin can bind to m1 receptors at the same time as typical competitive antagonists, suggesting that this toxin binds to the N-terminal and outer loops of m1 receptor molecules, rather than within the receptor pocket where typical agonists and antagonists bind. The binding of toxins to the outer parts of receptor molecules probably accounts for their much higher specificity for individual receptor subtypes than is seen with smaller ligands. Toxins are useful for identifying, counting, localizing, activating and blocking m1 and m4 receptors with high specificity.

Amino terminal region of acute phase, but not constitutive, serum amyloid A (apoSAA) specifically binds and transports cholesterol into aortic smooth muscle and HepG2 cells.

The human apoSAA proteins comprise both acute phase (apoSAA1, apoSAA2) and constitutive (apoSAA4) isoforms; all are expressed in human atherosclerotic lesions as well as in liver. Recombinant acute phase apoSAA binds cholesterol with an affinity of approximately 170 nM and enhances cholesterol uptake by HepG2 cells (J. Lipid Res. 1995. 36:37-46). In the present study, we sought to define the region of acute phase apoSAA involved in cholesterol binding and to investigate the ability of constitutive apoSAA4 to bind cholesterol. Binding of [3H]cholesterol to apoSAAp was inhibited by unlabeled cholesterol (1-100 nM), but not significantly by vitamin D and estradiol. Direct binding of acute phase, but not constitutive, apoSAA to the surfaces of polystyrene microtiter wells was strongly diminished in the presence of cholesterol. The ability of apoSAAp to bind cholesterol was inhibited by antibodies to human apoSAA1 and to peptide 1-18 of apoSAA1. There was only slight inhibition of cholesterol binding by antibodies to peptide 40-63, and no inhibition by antibodies to peptides spanning regions containing amino acid residues 14-44 and 59-104. [3H]cholesterol uptake by neonatal rabbit aortic smooth muscle and HepG2 cells was enhanced by a synthetic peptide corresponding to amino acids 1-18 of hSAA1, but not by peptides corresponding to amino acids 1-18 of hSAA4. [3H]cholesterol uptake by HepG2 cells was slightly increased by a peptide corresponding to amino acids 40-63 of hSAA1. These findings suggest that apoSAA modulates the local flux of cholesterol between cells and lipoproteins during inflammation and atherosclerosis.

Polymorphism of acute-phase serum amyloid A isoforms and amyloid resistance in wild-type Mus musculus czech.

Until CE/J mice and their offspring were characterized as amyloid-resistant, all mice were thought to be amyloid-susceptible to multiple injections of azocasein or a single injection of silver nitrate following administration of amyloid enhancing factor. We now report, for the first time, that wild-type Mus musculus czech and F1 hybrids bred by crossing M. musculus czech with amyloid-susceptible CBA/J mice are also amyloid resistant. Based on the derived amino acid sequences of two serum amyloid A (SAA) cDNA clones, we describe two unusual SAA gene isoforms in M. musculus czech, one of which differs from four previously characterized acute-phase apoSAA isoforms at several amino acid residues. Our findings support the hypothesis that protection against amyloid fibril formation in wild-type M. musculus czech mice and their offspring is linked to apoSAA gene mutations (molecular motif).

Degradation of serum amyloid A in amyloid-susceptible and amyloid-resistant mouse strains.

Degradation of serum amyloid A (apoSAA) by resident peritoneal cells (RPCS) and conditioned medium (CDM), prepared with RPCS, from amyloid-susceptible CBA/J mice, amyloid-resistant CE/J mice and their amyloid-resistant CBA/J x CE/J F1 progeny was investigated in vitro. Serum amyloid A was derived from murine acute phase (AP) plasma and associated with high density lipoprotein (HDL). Degradation of apoSAA by RPCS and CDM from CBA/J mice was complete while degradation by RPCS and CDM from CE/J mice did not occur. Degradation of apoSAA by RPCS and CDM from CBA/J x CE/J F1 hybrid mice was indistinguishable from that by RPCS and CDM from the CBA/J parent. Intermediate fragments were not detected with either RPCS or CDM from CBA/J mice or CBA/J x CE/J F1 hybrid mice. Degradation of apoSAA was inhibited by phenylmethanylsulfonyl fluoride (PMSF) indicating that the enzyme, secreted into the fluid phase, was a serine esterase. Unlike apoSAA, HDL-associated apoA-1 remained intact. It was thus concluded that while selective degradation of HDL-associated apoSAA (apoSAA-HDL) by RPCS from the CBA/J and CE/J mice was significantly different, the genetic study did not support the hypothesis that there was direct linkage between impaired degradation of apoSAA-HDL in the CE/J mouse strain and protection against amyloid fibril formation. As amyloid resistance in CBA/J x CE/J F1 hybrid mice is not attributable to failure to express the amyloidogenic isoform apoSAA2, the study supports the original hypothesis that amyloid resistance may be linked to expression of apoSAAcej.