Synthesis and characterization of two stable paramagnetic octahedral chromium(IV) complexes with dianionic tridentate SNO donor ligands and of a chromium(III) complex with a ONO donor ligand

Two novel paramagnetic octahedral chromium(IV) complexes with dianionic tridentate SNO donor ligands containing extended π-system have been synthesized while only a paramagnetic octahedral chromium(III) complex is obtained when a related dianionic tridentate ONO donor ligand is used under similar conditions. These bischelate complexes [Cr(abtsal)₂] (1) (abtsalH₂ is the Schiff base of o-aminobenzenethiol and salicylaldehyde), [Cr(4-PhTSCsal)₂] · H₂O (2) (4-PhTSCsalH₂ is the Schiff base of 4-phenylthiosemicarbazide and salicylaldehyde) and K[Cr(sap)₂] · H₂O (3) (sapH₂ is the tridentate Schiff base of salicylaldehyde and o-aminophenol) are characterized by elemental analyses, magnetic moment measurements, IR, UV-Vis and EPR spectroscopic studies. Compound 3 has been structurally characterized by X-ray crystallography. Measured room temperature (RT) magnetic moment values are 2.98 BM for 1 and 2.83 BM for 2 indicating a d² system with a triplet ground state in both the cases. On the other hand, the magnetic moment value for 3 is found to be 3.74 BM at RT and is consistent with the presence of three unpaired electrons for a d³ Cr(III) ion. The magnetic moment values rule out the large spin-orbit coupling which is substantiated by the presence of RT EPR signals. Compounds 1 and 2 exhibit very similar powder EPR spectra at RT and LNT, which show the allowed transition ΔM_s = ±1 (g = 2.004 for both 1 and 2) as well as the “forbidden” half-field transition (ΔM_s = ±2) at g = 4.105 for 1 and g = 4.318 for 2, respectively. The X-band LNT frozen glass EPR spectrum of 1 in DMF shows the presence of zero-field split rhombic symmetry character, and results in the parameters g ≅ 2.0, D = 740 G, and E = 260 G. It suggests that the intensity of ΔM_s = ±2 forbidden transition is large due to the large D value. The X-band frozen glass EPR spectrum of compound 3 in DMF is found to be very similar to that reported for trans-[Cr(py)₄F₂]⁺ in DMF-H₂O-MeOH glass. The large difference (∼700 mV) in the reduction potential for the two octahedral complexes 1 (−1.40 V) and 3 (−0.70 V) is attributed to the difference in their metal ion oxidation states.     

Striatal dopamine level contributes to hydroxyl radical generation and subsequent neurodegeneration in the striatum in 3-nitropropionic acid-induced Huntington's disease in rats

We tested the hypothesis that dopamine contributes significantly to the hydroxyl radical (OH)-induced striatal neurotoxicity caused by 3-nitropropionic acid (3-NP) in a rat model of Huntington's disease. Dopamine (10–100 μM) or 3-NP (10–1000 μM) individually caused a significant increase in the generation of hydroxyl radical (OH) in the mitochondria, which was synergistically enhanced when the lowest dose of the neurotoxin (10 μM) and dopamine (100 μM) were present together. Similarly, systemic administration of l-DOPA (100–250 mg/kg) and a low dose of 3-NP (10 mg/kg) potentiated OH generation in the striatum, and the rats exhibited significant decrease in stride length, a direct indication of neuropathology. The pathology was also evident in striatal sections subjected to NeuN immunohistochemistry. The significant changes in stride length, the production of striatal OH and neuropathological features due to administration of a toxic dose of 3-NP (20 mg/kg) were significantly attenuated by treating the rats with tyrosine hydroxylase inhibitor α-methyl-p-tyrosine prior to 3-NP administration. These results strongly implicate a major contributory role of striatal dopamine in increased generation of OH, which leads to striatal neurodegeneration and accompanied behavioral changes, in 3-NP model of Huntington's disease.     

A New Target for Amyloid Beta Toxicity Validated by Standard and High-Throughput Electrophysiology

Background

Soluble oligomers of amyloid beta (Aβ) are considered to be one of the major contributing factors to the development of Alzheimer''s disease. Most therapeutic development studies have focused on toxicity directly at the synapse.

Methodology/Principal Findings

Patch clamp studies detailed here have demonstrated that soluble Aβ can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Aβ (1–42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Aβ oligomerization, using both analysis methods.

Conclusions/Significance

The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment.     

Inflammatory stress exacerbates lipid-mediated renal injury in ApoE/CD36/SRA triple knockout mice

Both lipids and inflammation play important roles in the progression of kidney disease. This study was designed to investigate whether inflammation exacerbates lipid accumulation via LDL receptors (LDLr), thereby causing renal injury in C57BL/6J mice, apolipoprotein E (ApoE) knockout (KO) mice, and ApoE/CD36/scavenger receptor A triple KO mice. The mice were given a subcutaneous casein injection to induce inflammatory stress. After 14 wk, terminal blood samples were taken for renal function, lipid profiles, amyloid A (SAA), and IL-6 assays. Lipid accumulation in kidneys was visualized by oil red O staining. Fibrogenic molecule expression in kidneys was examined. There was a significant increase in serum SAA and IL-6 in the all casein-injected mice compared with respective controls. Casein injection reduced serum total cholesterol, LDL cholesterol, and HDL cholesterol and caused lipid accumulation in kidneys from three types of mice. The expression of LDLr and its regulatory proteins sterol-responsive element-binding protein (SREBP) 2 and SREBP cleavage-activating protein (SCAP) were upregulated in inflamed mice compared with controls. Casein injection induced renal fibrosis accompanied by increased expression of fibrogenic molecules in the triple KO mice. These data imply that inflammation exacerbates lipid accumulation in the kidney by diverting lipid from the plasma to the kidney via the SCAP-SREBP2-LDLr pathway and causing renal injury. Low blood cholesterol levels, resulting from inflammation, may be associated with high risk for chronic renal fibrosis.     

Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD). Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR) signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.     

Cytokine Network in Scrub Typhus: High Levels of Interleukin-8 Are Associated with Disease Severity and Mortality

Background

Scrub typhus, caused by Orientia tsutsugamushi, is endemic in the Asia-Pacific region. Mortality is high if untreated, and even with treatment as high as 10–20%, further knowledge of the immune response during scrub typhus is needed. The current study was aimed at comparing plasma levels of a variety of inflammatory mediators in scrub typhus patients and controls in South India in order to map the broader cytokine profile and their relation to disease severity and clinical outcome.

Methodology/Principal Findings

We examined plasma levels of several cytokines in scrub typhus patients (n = 129) compared to healthy controls (n = 31) and infectious disease controls (n = 31), both in the acute phase and after recovery, by multiplex technology and enzyme immunoassays. Scrub typhus patients were characterized by marked changes in the cytokine network during the acute phase, differing not only from healthy controls but also from infectious disease controls. While most of the inflammatory markers were raised in scrub typhus, platelet-derived mediators such as RANTES were markedly decreased, probably reflecting enhanced platelet activation. Some of the inflammatory markers, including various chemokines (e.g., interleukin-8, monocyte chemoattractant peptide-1 and macrophage inflammatory protein-1β) and downstream markers of inflammation (e.g., C-reactive protein and pentraxin-3), were also associated with disease severity and mortality during follow-up, with a particular strong association with interleukin-8.

Conclusions/Significance

Our findings suggest that scrub typhus is characterized by a certain cytokine profile that includes dysregulated levels of a wide range of mediators, and that this enhanced inflammation could contribute to disease severity and clinical outcome.     

Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999