Structural and functional consequences of the replacement of proximal residues Cys(172) and Cys(192) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.     

Construction, electrochemically biosensing and discrimination of recombinant plasmid (pEThIL-2) on the basis of interleukine-2 DNA insert

Construction, electrochemically biosensing and discrimination of recombinant pEThIL-2 plasmid, with 5839 bp size, on the basis of interleukine-2 (IL-2) DNA insert are described. Plasmid pEThIL-2 was constructed by PCR amplification of IL-2 encoding DNA and subcloning into pET21a(+) vector using BamHI and SacI sites. The recombinant pEThIL-2 plasmid was detected with a label-free DNA hybridization biosensor using a non-inosine substituted probe. The proposed sensor was made up by immobilization of a 20-mer antisense single strand oligonucleotide (chIL-2) related to the human interleukine-2 gene on the pencil graphite electrode (PGE) as a probe and then the sensing of recombinant pEThIL-2 plasmid was conducted by anodic differential pulse voltammetry (ADPV) based on guanine oxidation signal. Selectivity of the detection was assessed with pET21a(+) non-complementary plasmid, with 5443 bp size, lacking IL-2 encoding DNA. Different factors such as electrode activation conditions and washing strategy were tested in order to eliminate the nonspecific adsorption of pET21a(+). We have found that the PGE activation for 300 s produces a condition in which desorption of nonspecifically adsorbed plasmids from the electrode surface can be achieved by 300 s washing of the electrode in 20 mM Tris–HCl buffer solution (pH 7.0) containing 20 mM NaCl. Diagnostic performance of the biosensor is described and the detection limit is found to be 10.31 pg/μL.     

Study on induction of apoptosis on HeLa and Vero cells by recombinant shiga toxin and its subunits

Verotoxin (VT) or shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae is AB5 holotoxin with potent protein synthesis inhibitor. VT can induce both apoptosis and necrosis depending on the cell type, it has been shown that VT-induced apoptosis and cytotoxicity are distinct processes, and the A subunit can be necessary for apoptosis. In other words, the precise role of each subunit in apoptosis signaling has yet to be established. In this study, induction of apoptosis has been examined by using both recombinant A and B subunits, and recombinant Stx (rStx) with different doses in HeLa and Vero cells. For this purpose, the polymyxin B extract of constructs expressing A, B and AB5 recombinant proteins was used. Therefore, amounts greater than normally reported were used to induce desire effects on cell lines. The apoptotic effect of A and B subunits appear at higher doses than that of rStx. The highest apoptotic effect was observed for rStx at low concentration, compared to A and B subunits. A or B subunits separately cannot induce the signaling pathway stimulated by holotoxin though A subunit, does induce laddering pattern similar to holotoxin. We concluded that both subunits are important in complete death signaling pathway. Since different concentration of A and B subunits and rStx was required in different assay, therefore, it could be emphasized that cell death or even apoptosis caused by either of the subunits or holotoxin depends on sensitivity or specificity of the assay and cell types used.     

Spatiotemporal Correlations between Blood-Brain Barrier Permeability and Apparent Diffusion Coefficient in a Rat Model of Ischemic Stroke

Variations in apparent diffusion coefficient of water (ADC) and blood-brain barrier (BBB) permeability after ischemia have been suggested, though the correlation between ADC alterations and BBB opening remains to be studied. We hypothesized that there are correlations between the alteration of ADC and BBB permeability. Rats were subjected to 2 h of transient middle cerebral artery occlusion and studied at 3 and 48 h of reperfusion, which are crucial times of BBB opening. BBB permeability and ADC values were measured by dynamic contrast-enhanced MRI and diffusion-weighted imaging, respectively. Temporal and spatial analyses of the evolution of BBB permeability and ADC alteration in cortical and subcortical regions were conducted along with the correlation between ADC and BBB permeability data. We found significant increases in BBB leakage and reduction in ADC values between 3 and 48 h of reperfusion. We identified three MR tissue signature models: high K_i and low ADC, high K_i and normal ADC, and normal K_i and low ADC. Over time, areas with normal K_i and low ADC transformed into areas with high K_i. We observed a pattern of lesion evolution where the extent of initial ischemic injury reflected by ADC abnormalities determines vascular integrity. Our results suggest that regions with vasogenic edema alone are not likely to develop low ADC by 48 h and may undergo recovery.     

Let-7e enhances the radiosensitivity of colorectal cancer cells by directly targeting insulin-like growth factor 1 receptor

Abnormal expression of various microRNAs (miRNAs), as regulators of biological signaling pathways, has a strong association with cancer resistance to chemotherapy and radiotherapy. The let-7 family of miRNAs as tumor suppressors have shown to be downregulated in different types of human malignancies including colorectal cancer (CRC). However, the biological function of let-7 members in the processes of resistance to radiation in CRC has not yet been completely elucidated. Insulin-like growth factor 1 receptor (IGF-1R) signaling pathway is amplified in CRC and leads to its progression, development, and also radiation resistance. So, it seems like an attractive target for anticancer therapy. In this study, by using bioinformatics analysis, it has been revealed that IGF-1R is a direct target of the let-7e member. Consistent with this, we identified that increased levels of let-7e in CRC cells reduced IGF-1R protein level and subsequently its downstream signaling pathways, which resulted in the G1 cell cycle arrest and a significant reduction in the proliferation, survival and also resistance to radiation of CRC cells. Altogether, these results suggested that let-7e by targeting the IGF-1R signaling pathway might serve as therapeutics in anticancer therapy.     

Cloning,Expression, and Assessment of Cytotoxic Effects of A-NGR Fusion Protein

Targeted drug delivery is an attractive field in cancer studies. In this study, a novel fusion protein consisting of Shiga toxin A subunit and NGR peptide has been constructed. The cytotoxic Shiga toxin A subunit has the ability to kill cancer cells while NGR is a well-known peptide that targets the whole molecule to cancer cells. Two forms of this novel fusion protein, one without linker (A-NGR) and one with linker (A-GGGGS-NGR) were studied. 3D structure prediction of the two forms carried out by I-TASSER and their validation and analysis were performed by ProSA web and RAMPAGE. Results showed that A-NGR is a better model than the one with linker. A-NGR was constructed by PCR method and cloned in pBAD/gIII A vector. Then, it was successfully expressed in Escherichia coli by induction with arabinose and subsequently purified by affinity chromatography under denaturing condition. Ultimately, the cytotoxic effect of the purified protein was evaluated on U937 cancer cells and MRC-5 normal cells by MTT assay. Conclusively, the fusion protein was successfully cloned and expressed and evaluated for its cytotoxic effects. The IC50 value of A-NGR fusion protein for U937 cell was about 26.86 µg/ml while no cytotoxic effect was observed on MRC-5 cells. Therefore, considering the promising cytotoxic effects of the fusion protein, further in vitro evaluations of this fusion protein on different cell lines are underway.     

Study of defense-related gene expression in grapevine infested by <Emphasis Type="Italic">Colomerus vitis</Emphasis> (Acari: Eriophyidae)

As the main source of lipids and proteins in honey bees, pollen is a major nutrient provider involved in development and health and has been studied for tolerance stimulation against pathogens and parasites. In the case of Varroa destructor Anderson & Trueman (Acari, Mesostigmata: Varroidae) parasitization, the lack of a complete laboratory system to rear both the bee larva and the acarian parasite limited the studies concerning larval nutrition effects on the bee tolerance and resistance against varroatosis. Due to the development of this complete rearing protocol, we managed to feed young honey bee larvae with pollen supplemented solutions and to study the effect on their later development under parasitism conditions. In our experimental conditions, pollen influences neither the deformity rate, nor the survival of bees both parasitized and unparasitized. However, pollen extract supplementation seems to significantly impact the weight of the spinning bee larvae without having an effect on the physiological weight loss during pupation, so the differences found at the larval stage remain the same as at emergence. Varroa has a deleterious effect on bee pupae and led to a steady increase of the physiological weight loss experienced during metamorphosis. Interestingly, this ponderal loss associated with Varroa parasitization seems to be reduced in the polyfloral pollen supplementation condition. Altogether, this work is to our knowledge the first to study in laboratory conditions the impact of larval nutrition on the tolerance to parasitism. A diverse pollen diet may be beneficial to the bees’ tolerance against V. destructor parasitism.