Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry

Small heat shock proteins (sHSPs) and the related alpha-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95-156 and 228-252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Xanthine mimetics as potent dipeptidyl peptidase IV inhibitors

A series of xanthine mimetics containing 5,5 and 5,6 heterocycle fused imidazoles were synthesized as dipeptidyl peptidase IV inhibitors. Compound 7 is potent (h-DPPIV K_i = 2 nM) and exhibits excellent selectivity and no species specificity against rat and human enzymes. The X-ray structure confirms that the binding mode of 7 to rat DPPIV is similar to the parent xanthines.     

Water-soluble formulation of Coenzyme Q10 inhibits Bax-induced destabilization of mitochondria in mammalian cells

Oxidative stress leads to mitochondrial dysfunction, which triggers the opening of the permeability transition pores (PTP) and the release of pro-apoptotic factors causing apoptotic cell death. In a limited number of cell systems, anti-oxidants and free-radical scavengers have been shown to block this response. We have previously reported that coenzyme Q₁₀ (CoQ₁₀), an electron carrier in the mitochondrial respiratory chain, is involved in the reactive oxygen species (ROS) removal and prevention of oxidative stress-induced apoptosis in neuronal cells. However, the mechanism of this protection has not been fully elucidated. In the present study we investigated the effects of CoQ₁₀ on the mitochondrial events characteristic to apoptosis, especially on the function of pro-apoptotic protein Bax. Our results demonstrated that following a brief exposure of two human cell lines (fibroblasts and HEK293 cells) to H₂O₂ the intracellular levels of ROS and the association of Bax with the mitochondria significantly increased and the cells underwent apoptosis. Both of these events, as well as the release of cytochrome c from the mitochondria, were blocked by a 24 h pre-treatment with CoQ₁₀. It is therefore believed that CoQ₁₀ prevented the collapse of the mitochondrial membrane potential in response to the H₂O₂ treatment. Recombinant Bax protein alone caused the ROS generation and release of cytochrome c from isolated mitochondria and, again, CoQ₁₀ inhibited these Bax-induced mitochondrial dysfunctions.     

Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF at 48 g dry cell wt l⁻¹ during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as ~64 g dry cell wt l⁻¹, 223 mg hG-CSF g⁻¹ dry cell wt and 775 mg hG-CSF l⁻¹ h⁻¹, respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose. Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show valuable decrease in activity in purified form.     

Multiplex standardized RT-PCR for expression analysis of many genes in small samples

Standardized RT-PCR (StaRT-PCR) enables numerical quantification as well as intra- and inter-laboratory comparison of gene expression. Multiplex StaRT-PCR, using two rounds of amplification, was conducted on Stratagene Universal Reference RNA. In the first round, cDNA, competitive template (CT) mix, and primers for up to 96 genes were amplified for varying numbers of cycles. Next, products from round one were diluted, combined with primers for one gene, and amplified for an additional 35 cycles. No additional cDNA or CT mix was added. Expression values obtained by uniplex and multiplex StaRT-PCRs were highly correlated (R=0.993, p<0.001). Products from round one could be diluted as much as 100,000-fold and still be quantified following round two amplification. Thus, using multiplex StaRT-PCR, 96 genes were measured in the same amount of cDNA typically used to measure one gene with uniplex StaRT-PCR. Multiplex StaRT-PCR was also used to measure 18 genes in the fine needle biopsy of a primary lung carcinoma.     

Potential role of dietary omega-3 essential fatty acids on some oxidant/antioxidant parameters in rats' corpus striatum

Omega-3 (omega-3) is an essential fatty acid (EFA) found in large amounts in fish oil. It contains eicosapentaenoic acid and docosahexaenoic acid (DHA). DHA is one of the building structures of membrane phospholipids of brain and necessary for continuity of neuronal functions. Evidences support the hypothesis that schizophrenia may be the result of increased reactive oxygen species mediated neuronal injury. Recent reports also suggest the protective effect of omega-3 EFA against neuropsychiatric disorders including schizophrenia. This study proposed to assess the changes in antioxidant enzyme and oxidant parameters in the corpus striatum (CS) of rats fed with omega-3 EFA diet (0.4g/kg/day) for 30 days. Eight control rats and nine rats fed with omega-3 were decapitated under ether anesthesia, and CS was removed immediately. Thiobarbituric acid-reactive substances (TBARS) and nitric oxide (NO) levels as well as total superoxide dismutase (t-SOD) and xanthine oxidase (XO) enzyme activities in the CS were measured. Rats treated with omega-3 EFA had significantly lower values of TBARS (P<0.001), NO (P<0.002) and XO (P<0.005) whereas higher values of t-SOD enzyme activity (P<0.002) than the control rats. These results indicate that omega-3 EFA rich fish oil diet reduces some oxidant parameters in CS. This may be revealed by means of reduced CS TBARS levels as an end product of lipid peroxidation of membranes in treated rats. Additionally, reduced XO activity and NO levels may support this notion. On the other hand, although the mechanism is not clear, omega-3 EFA may indirectly enhance the activity of antioxidant enzyme t-SOD. Taken together, this preliminary animal study provides strong support for a therapeutic effect of omega-3 EFA supplemented to classical neuroleptic regimen in the treatment of schizophrenic symptoms and tardive dyskinesia.     

Effects of caffeic acid phenethyl ester and alpha-tocopherol on reperfusion injury in rat brain

Oxygen-derived free radicals have been implicated in the pathogenesis of cerebral injury after ischaemia-reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. The purpose of the present study was to investigate the effects of ischaemia and subsequent reperfusion on rat brain and to investigate the effects of two free radical scavengers, CAPE and alpha-tocopherol, on this in vivo model of cerebral injury. Ischaemia was induced by bilateral occlusion of the carotid arteries for 20 min and reperfusion was achieved by releasing the occlusion to restore the circulation for 20 min. Control rats underwent a sham operation. CAPE at 10 micromol kg(-1) or alpha-tocopherol at 25 micromol kg(-1) was administered intraperitoneally before reperfusion. Reperfusion led to significant increase in the activity of xanthine oxidase and higher malondialdehyde levels in the brain. Acute administration of both CAPE and alpha-tocopherol suppressed ischaemia-reperfusion-induced cerebral lipid peroxidation and injury, but CAPE seems to offer a better therapeutic advantage over alpha-tocopherol.     

Cobalt(II) salophen-modified carbon-paste electrode for potentiometric and voltammetric determination of cysteine

A chemically modified electrode constructed by incorporating N,N(')-bis(salicylidene)-1,2-phenylenediaminocobalt(II) into carbon-paste matrix was used as a sensitive electrochemical sensor for detection of cysteine. The resulting electrode exhibits catalytic properties for the electrooxidation of cysteine and lowers the overpotential for the oxidation of this compound. The faster rate of electron transfer results in a near-Nernstian behavior of the modified electrode and makes it a suitable potentiometric and voltammetric sensor for the fast and easy determination of cysteine. A linear response in concentration range from approximately 2 microM to 0.01 M was obtained with a detection limit of 1 microM for the potentiometric detection of cysteine. The modified electrode was also used for the amperometric and differential pulse voltammetric determination of cysteine and the results were compared with those of the potentiometric method.     

Development of an oral biosensor for salivary amylase using a monodispersed silver for signal amplification

An amperometric biosensor for monitoring the level of protein amylase in human saliva is described. A novel design and the preparation of amylase antibodies and antigens, essential for the development of the biosensor, are reported. The biosensor sensing elements comprise a layer of salivary antibody (or antigen) self-assembled onto Au-electrode via covalent attachment. Molecular recognition between the immobilized antibody and the salivary amylase proteins was monitored via an electroactive indicator (e.g., K(3)Fe(CN)(6)) or a monodispersed silver layer present in solution or electrochemically deposited onto the solid electrode. This electroactive indicator was oxidized or reduced and the resulting current change provided the analytical information about the concentration of the salivary proteins. The limit of detection of 1.57 pg/ml was obtained, in comparison to detection limits of 4.95 pg/ml obtained using potassium ferrocyanide as the redox probe and 10 ng/ml obtained using enzyme-linked immunosorbent assay. Cross-reactivity was tested against cystatin antibodies and was found to be less than 2.26%.