Toll-like signaling and the cytokine IL-6 regulate histone deacetylase dependent neuronal survival

Histone deacetylase (HDAC) proteins have a role in promoting neuronal survival in vitro, but the mechanism underlying this function has not been identified. Here we provide evidence that components of the neuronal microenvironment, including non-neuronal cells and defined culture media, can mitigate midbrain neuronal cell death induced by HDAC inhibitor treatment. Using microarrays we further identified gene expression changes taking place in non-neuronal cells as a result of HDAC inhibition. This analysis demonstrated that HDAC inhibitor treatment results in the down-regulation of immunity related signaling factors, in particular the Toll-like receptors (TLR). TLR signaling is active in cultured midbrain cells, yet blocking TLR receptors is not sufficient to cause neuronal cell death. In contrast, selective activation of this pathway using TLR ligands can modestly block the effects of HDAC inhibition. Furthermore, we observed that the negative effects of HDAC inhibitor treatment on neuronal survival could be more substantially blocked by the cytokine Interleukin-6 (IL-6), which is a major downstream target of TLR signaling. These data suggest that HDACs function to promote neuronal survival by activating a TLR and IL-6 dependent pathway.     

Analysis of the RNA recognition motifs of human neuronal ELAV-like proteins in binding to a cytokine mRNA

Human neuronal Elav-like proteins contain three RNP-type RNA recognition motifs (RRMs). Previous reports demonstrated that a single RRM of the proteins is not sufficient to bind to the uridine-rich stretch in the 3' untranslated region of mRNAs and that the bi-RRM peptide consisting of the first two RRMs is necessary for the binding. The present study was designed to examine the potential contributions of the first two RRMs when binding to a cytokine mRNA. Deletions of the internal or terminal amino acid residues of the first RRM (RRM1) of the HuC/ple21 ELAV-like protein completely abolished RNA binding. However, removal of any region of the second RRM (RRM2) except for the eight amino acid residues, which correspond to the potent fourth beta-sheet structure of RRM2, did not affect RNA binding. Conjugation of the eight amino acid residues to RRM1 enhanced the RNA binding as well as the entire RRM2, indicating that the octapeptide of RRM2 can be compensated for by the binding function of RRM2. The present study also showed that the substitutions of glutamic acid at 42 for aspartic acid and leucine at 44 for phenylalanine in the first potent alpha-helix structure of RRM1, as were seen in another ELAV-like protein Hel-N1, markedly affected the RNA binding.     

Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies

Therapeutic antibodies have captured substantial attention due to the relatively high rate at which these products reach marketing approval, and the subsequent commercial success they frequently achieve. In the 2000s, a total of 20 antibodies (18 full-length IgG and 2 Fab) were approved by the Food and Drug Administration (FDA) or European Medicines Agency (EMA). In the 2010s to date, an additional 3 antibodies (denosumab, belimumab, ipilimumab) have been approved and one antibody-drug conjugate (brentuximab vedotin) is undergoing regulatory review and may be approved in the US by August 30, 2011. However, a less heralded group of antibody-based therapeutics comprising proteins or peptides fused with an Fc is following the success of classical antibodies.     

Relationship between clinical classification of hyperthyroidism for three grades of severity and level of triiodothyronine (T3) and thyroxine (T4) concentrations in serum]

The aim of the study was to assess the relationship between the clinical classification of hyperthyroidism based on the 3-degree score system and T3 and T4 serum concentration. 161 patients with Graves disease or toxic goiter were studied. By comparing the number of scores separating the 3 subgroups in relation to the severity of disease with T3 and T4 serum concentration of tyreotoxic patients we found a very high statistically significant correlation. We also found the marked (by +50%) statistically significant increase in the serum T3 concentration related to the degree of hyperthyroidism severity.     

Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu-dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu-dopamine complex mediates the uptake of (64)CuSO(4) into the Raúl Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m M) (63%, p < 0.001) and nomifensine (2 microM) (77%, p < 0.001). Copper sulfate (1 m M) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO(4) and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO(4), dopamine, or CuSO(4) and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO(4), dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO(4) increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu-dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome.     

Overexpression of glutathione peroxidase increases the resistance of neuronal cells to Abeta-mediated neurotoxicity

Senile plaques are neuropathological manifestations in Alzheimer's disease (AD) and are composed mainly of extracellular deposits of amyloid beta-peptide (Abeta). Various data suggest that the accumulation of Abeta may contribute to neuronal degeneration and that Abeta neurotoxicity could be mediated by oxygen free radicals. Removal of free radicals by antioxidant scavengers or enzymes was found to protect neuronal cells in culture from Abeta toxicity. However, the nature of the free radicals involved is still unclear. In this study, we investigated whether the neuronal overexpression of glutathione peroxidase (GPx), the major hydrogen peroxide (H2O2)-de-grading enzyme in neurons, could increase their survival in a cellular model of Abeta-induced neurotoxicity. We infected pheochromocytoma (PC12) cells and rat embryonic cultured cortical neurons with an adenoviral vector encoding GPx (Ad-GPx) prior to exposure to toxic concentrations of Abeta(25-35) or (1-40). Both PC12 and cortical Ad-GPx-infected cells were significantly more resistant to Abeta-induced injury. These data strengthen the hypothesis of a role of H2O2 in the mechanism of Abeta toxicity and highlight the potential of Ad-GPx to reduce Abeta-induced damage to neurons. These findings may have applications in gene therapy for AD.     

Relationship between the severity of experimental diabetes and altered lung phospholipid metabolism

Glucose intolerance was induced in rats by iv infusion of streptozotocin (STZ) in doses of 30, 40, 50, and 100 mg/kg. Serum glucose concentrations were elevated versus controls and weight gains were reduced in a dose-dependent fashion up to 50 mg/kg. Urine outputs and blood urea nitrogen (BUN) values were higher than control values in the animals treated with 40 and 50 mg/kg and serum albumin concentrations were decreased after infusion with 50 mg STZ/kg. Lung phosphatidylcholine (PC) concentrations and dry-to-wet weight ratios were unchanged by STZ treatment, while lung protein and disaturated phosphatidylcholine (DSPC) concentrations were depressed in the 50-mg/kg group. Animals surviving treatment with 100 mg/kg demonstrated increased fasting blood glucose levels, BUN values, and 48-hr urine outputs, and decreased lung protein levels. However, these alterations were less than those found in the 50-mg/kg animals. Pulmonary concentrations of PC, DSPC, and lung dry-to-wet weight ratios were unchanged. It was found advantageous to express the results relative to fasting blood glucose levels. This demonstrated that urine output and BUN values increased and weight gain decreased with rising glucose concentrations, but serum albumin decreased only in moderate and severe hyperglycemia. Fasting glucose concentrations greater than 400 mg/dl were associated with reduced lung DSPC and protein levels, while pulmonary PC and dry-to-wet weight ratios demonstrated no change with increasing hyperglycemia.     

Influence of industrial vibration on the level of antibodies against regulatory proteins of the nervous tissue

We studied the concentration of antibodies (ABs) to the regulatory proteins of the nervous tissue revealed in the blood serum of healthy men working for a long period of time and of the patients suffering from the vibration-induced disease (VID). A characteristic feature of autoimmune response development in workers who are exposed to vibration for an extensive period of time is the decline in ABs against virtually all examined antigens (AGs), which indicates an overall reduction of immune reactivity in relation to nervous tissue proteins. At the same time, we discovered characteristic changes in certain AB concentrations, both in the spectrum and in the intensity, depending on the severity of a pathologic process. Changes in the AB concentration in long-term workers who do not exhibit signs of any health conditions induced by vibration may indicate nascent changes in the specialized structure of nervous tissue; for VID patients, the presence of changes in the levels of ABs may reflect continuous neuro-destructive processes in nervous tissue.     

Relationship between hair elements and severity of atrioventricular block in horses

The aim of this study was to investigate the relationships between the mean concentrations of trace elements and the severity of the seconddegree atrioventricular (AV) block in the mane hair of horses. Electrocardiographs of horses were continually recorded for 6 h using a holter cardiac monitor to determine dropped ventricular beats (DVBs) which can be used as an indicator of the severity of the AV block. Mane hair Ca, Cu, Mg, and Zn concentrations were measured by the particle-induced X-ray emission method. The Zn/Cu ratio and Ca concentration in mane hair were significantly and positively correlated with the hourly DVBs in horse with a second-degree AV block (p<0.01, r2=0.485; p<0.05, r2=0.351, respectively). Proposed diagnostic cutoff points for hair Ca concentration and Zn/Cu ratio based on receiver operating characteristics curves analysis in detecting second-degree AV block were set at 1536 μg/g and 26.0, respectively. Those results with horse hair suggest that the evaluation of the Ca, Cu, Mg, and Zn status in mane hair by this method is strongly related to the severity of second-degree AV block and might predict the susceptibility of an individual much before the development of the symptom.     

Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis.

Hybrid selected translation was used to map the genome of herpesvirus saimiri, a lymphotropic and oncogenic herpesvirus. RNA extracted from virus-infected cells was hybridized to cloned genomic fragments, and the hybrid selected mRNAs were translated in vitro in a rabbit reticulocyte lysate. Forty-five virus-induced polypeptides were identified and correlated to their coding regions on the herpesvirus saimiri genome. Inhibition of the replication of viral DNA with phosphonoacetic acid showed that 22 of these polypeptides belong to the early group of herpesvirus saimiri gene products.     

Relationship between pre-partum relaxin concentrations and farrowing intervals in the pig

Gilts were treated on Day 112 of gestation with saline or a prostaglandin (PG) F-2 alpha analogue. In control gilts there was a rise in the relaxin concentration from 48 h before the onset of delivery, peaking between 12 and 28 h pre partum followed by a steep fall. The relaxin concentrations at each 1-h time interval were analysed in relation to the farrowing interval for each gilt using correlation analysis. There was a significant (at least P less than 0.05) positive correlation between the relaxin concentration and the farrowing interval at every time period from 14 to 2 h before delivery. In contrast there was little relationship between concentrations of progesterone, oestrone and oestradiol-17 beta and farrowing intervals. The gilts treated with PGF-2 alpha analogues had steroid profiles indistinguishable from those in controls but differing relaxin secretion patterns. Relaxin concentrations peaked at 1-2 h after PGF-2 alpha injection and this was followed by a second smaller increase closer to the time of delivery in 7 out of 12 gilts. The 'two-peak' gilts had significantly higher relaxin concentrations at farrowing and took significantly longer to farrow than did the 'one-peak' gilts (P less than 0.005). These results suggest that high relaxin concentrations during the last 14 h before the onset of parturition are associated with increased farrowing times, but are not associated with any increase in neonatal mortality.     

Giant vesicles as models to study the interactions between membranes and proteins

The interaction between polypeptides and membranes is a fundamental aspect of cell biochemistry. Liposomes have been used in this context as in vitro systems to study such interactions. We present here the case of giant vesicles (GVs), which, due to their size (radius larger than 10 microns), mimic more closely the situation observed in cell membranes and furthermore permit to study protein-membrane interactions by direct optical monitoring. It is shown that GVs formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by electroformation are permeable to certain low molecular weight molecules such as the nucleic acid dye YO-PRO-1 and fluorescein diphosphate whereas conventional liposomes (large or small unilamellar liposomes) are not. In addition, it is shown that non-membrane proteins, such as DNases or RNases, added to the selected GVs from the outside, are able to convert their substrate, which is strictly localized on the internal side of the membrane. This effect is only seen in GVs (also when they are removed from the original electroformation environment) and is absent in conventional liposomes. The fact that these effects are only present in GVs obtained by electroformation and not in conventional small liposomes is taken as an indication that certain physico-chemical properties of the bilayer are affected by the membrane curvature, although the mechanism underlying such differences could not be established as yet.     

4-Phenylpyridine (4PP) and MPTP: the relationship between striatal MPP+ concentrations and neurotoxicity

Because of the chemical and structural similarity between 4-phenylpyridine (4PP) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the effects of 4PP alone and in combination with MPTP on striatal dopamine (DA) concentrations were studied in mice. 4PP did not deplete striatal DA, even when given in maximally tolerated doses (five times that required for MPTP neurotoxicity). However, when 4PP was administered prior to MPTP, it provided significant protection against the DA-depleting effects of MPTP. Additional experiments showed that 4PP pretreatment reduced striatal concentrations of 1-methyl-4-phenylpyridinium ion (MPP+) - the putative toxic biotransformation product of MPTP, and that the concentration of this metabolite closely mirrored striatal DA depletion in MPTP-treated mice. In vitro studies established that 4PP probably lowers MPP+ concentrations by inhibiting the biotransformation of MPTP to MPP+. These observations could be of clinical interest in view of the lower incidence of cigarette smoking among Parkinson's disease patients, and the fact that 4PP is known to be present in cigarettes.     

Relationship between synthesis and cleavage of poliovirus-specific proteins.

Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-initiation to occur. This suggests that the poliovirus proteinase is highly unstable.     

Relationship between the self-incompatibility and cAMP level in Lilium longiflorum.

The elongation of pollen tubes in Lilium longiflorum cv. Hinomoto after self-incompatible pollination stopped halfway, but that after cross-compatible pollination (cross with cv. Georgia) did not. The elongation of pollen tubes after self-pollination was enhanced by exogenous cAMP and by pertussis toxin or cholera toxin, which activates adenylate cyclase. The level of endogenous cAMP in pistils after self-pollination was approximately one half of that after cross-pollination. Furthermore, the activity of adenylate cyclase in pistils after self-pollination was also approximately one half of that after cross-pollination. By contrast, cAMP phosphodiesterase in pistils after self-pollination was approximately 2 times as high as that after cross-pollination. A possible correlation between self-incompatibility and the low level of endogenous cAMP in lily pistils is discussed on the basis of these results.     

Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr

The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.