Probing 13C chemical shielding tensors in cryptolepine and two bromo-substituted analogs for antiplasmodial activity

Density functional theory calculations were applied to investigate ¹³C chemical shielding tensors in cryptolepine and its bromo-substituted analogs, 2-bromocryptolepine and 2,7-dibromocryptolepine. The fact that bromo-substituted cryptolepine shows higher antiplasmodial activity than cryptolepine raises the question of whether this effect can be related to the electronic properties around carbon atoms. The results show that changes to the principal components of the shielding tensors upon substitution are significant. In particular, σ ₃₃ is the most affected tensor for carbons in the substituted ring, which could be related to the increased antiplasmodial activity of bromosubstituted cryptolepine. The analyses were also focused on atomic charges and dipole moment.     

RasGAP-derived fragment N increases the resistance of beta cells towards apoptosis in NOD mice and delays the progression from mild to overt diabetes

The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-Akt-dependent manner. Fragment N protects various cell types, including insulin-secreting cells, against different types of stresses. Whether fragment N exerts a protective role during the development of type 1 diabetes is however not known. Non-obese diabetic (NOD) mice represent a well-known model for spontaneous development of type 1 diabetes that shares similarities with the diseases encountered in humans. To assess the role of fragment N in type 1 diabetes development, a transgene encoding fragment N under the control of the rat insulin promoter (RIP) was back-crossed into the NOD background creating the NOD-RIPN strain. Despite a mosaic expression of fragment N in the beta cell population of NOD-RIPN mice, islets isolated from these mice were more resistant to apoptosis than control NOD islets. Islet lymphocytic infiltration and occurrence of a mild increase in glycemia developed with the same kinetics in both strains. However, the period of time separating the mild increase in glycemia and overt diabetes was significantly longer in NOD-RIPN mice compared to the control NOD mice. There was also a significant decrease in the number of apoptotic beta cells in situ at 16 weeks of age in the NOD-RIPN mice. Fragment N exerts therefore a protective effect on beta cells within the pro-diabetogenic NOD background and this prevents a fast progression from mild to overt diabetes.     

GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.     

Vitamin E and telmisartan attenuates doxorubicin induced cardiac injury in rat through down regulation of inflammatory response

ABSTRACT: BACKGROUND: The importance of doxorubicin (Dox), as a potent antitumor antibiotic, is limited by the development of life-threatening cardiomyopathy. It has been shown that free radicals are involved in acute doxorubicin-induced toxicity. The aim of this study was to determine the protective effect of vitamin E and telmisartan in acute doxorubicin induced cardiotoxicity. METHODS: Thirty two male Sprague - Dawly rats were involved in this study and were randomly separated into 4 groups, eight rats in each group, one group received normal saline I.P as control and second group received doxorubicin 20 mg/kg I.P, the other two groups also received doxorubicin 20 mg/kg I.P as single dose after seven cumulative doses (for seven days) of vitamin E (100 mg/kg) and telmisartan (1 mg/kg) respectively. Immunofluorescent staining for monocytes infiltration and analyses of plasma by (ELISAs) for MCP-1and troponin I. Western immunoblotting assay for ICAM-1, while left ventricular function was analyzed by microcatheter, also estimated the level of oxidative stress parameters (MDA and Catalase) and cardiac enzymes activities (CK-MB and LDH) before starting drugs treatment and after treatment period by 48 hours. RESULTS: The immunofluorescent staining showed that administration of vitamin E and telmisartan are attenuated of mononuclear cell infiltration; (p < 0.05 vs. Dox group), also reduced the level of chemokines MCP-1 and ICAM-1 expression compared with Dox group only, and there is marked reduction of myocardial troponin-I levels with improved LV function in vitamin E and telmisartan treated group. Doxorubicin treatment increased MDA, LDH, CK-MB levels significantly (P < 0.01), and were counteracted by administration of vitamin E and telmisartan, but did not significantly affect serum catalase activity. CONCLUSIONS: Antioxidant effect (Vitamin E and telmisartan) have been shown to decrease doxorubicininduced cardiotoxicity.     

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.     

Plc1p is required for proper chromatin structure and activity of the kinetochore in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by facilitating recruitment of the RSC complex

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p’s involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessment of yield loss of wheat cultivars caused by Heterodera filipjevi under field conditions

Among the cereal cyst nematodes, Heterodera filipjevi is the dominant species of Cereal cyst nematodes (CCNs) in most cereal growing areas of Iran. To evaluate the impact of H. filipjevi on wheat cultivars, a field trial was performed in two infested fields in Isfahan province, Iran 2014 and 2015. The trials were conducted in a factorial experiment based on the complete randomized block design. The treatments consisted of three winter wheat cultivars (Back‐cross Rowshan, Pishtaz and Parsi) which were planted with and without applying nematicide aldicarb Each cultivar was planted in 6 m² (2 × 3 m) plots which were replicated five times. The nematode reproduction factor was calculated after determining the initial and final population of H. filipjevi in each plot before sowing the seeds and after harvesting. The grain yields and growth parameters were recorded and the variables of 2 years experiment evaluated by linear regression analysis. Cultivar × nematicide treatment combinations in the first and second years showed that H. filipjevi significantly affected grain yield and growth parameters in all three cultivars. The results revealed significant reduction of grain yield by 24.8%, 24.8% and 20.4% in Back‐cross Rowshan, Pishtaz and Parsi cultivars, respectively. The nematode reproduction factor ranged from 0.32 to 1.76 in plots plus and minus nematicide application, respectively. The analysis showed linear inverse relationships between the initial population (Pi) and the yields of wheat cultivars in check plots without aldicarb application.     

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni–NTA (Ni²⁺-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.     

Acute Toxicity and Gastroprotection Studies with a Newly Synthesized Steroid

Background

Synthetic steroids, such as 9α-bromobeclomethasonedipropionate, have shown gastroprotective activity. For example, the potent glucocorticoid steroid, beclomethasone dipropionate, has been used for treatment of bowel ulcerations. The purpose of the present study was to evaluate the effect of a synthetic steroid, (20S)-22-acetoxymethyl-6β-methoxy-3α,5-dihydro-3′H-cyclopropa[3α,5]-5α-pregnane (AMDCP), on ethanol-induced gastric mucosa injuries in rats.

Methodology/Principal Finding

Rats were divided into 8 groups. The negative control and ethanol control groups were administered Tween 20 (10%v/v) orally. The reference control group, 20 mg/kg omeprazole (10% Tween 20, 5 mL/kg), was administrated orally. The experimental groups received 1, 5, 10, 15 or 20 mg/kg of the AMDCP compound (10% Tween 20, 5 mL/kg). After 60 min, Tween 20 and absolute ethanol was given orally (5 mL/kg) to the negative control group and to the rest of the groups, and the rats were sacrificed an hour later. The acidity of gastric content, gastric wall mucus and areas of mucosal lesions were assessed. In addition, histology and immunohistochemistry of the gastric wall were assessed. Prostaglandin E2 (PGE₂) and malondialdehyde (MDA) content were also measured. The ethanol control group exhibited severe mucosal lesion compared with the experimental groups with fewer mucosal lesions along with a reduction of edema and leukocyte infiltration. Immunohistochemical staining of Hsp70 and Bax proteins showed over-expression and under-expression, respectively, in the experimental groups. The experimental groups also exhibited high levels of PGE₂ as well as a reduced amount of MDA. AMDCP decreased the acidity and lipid peroxidation and increased the levels of antioxidant enzymes.

Conclusion/Significance

The current investigation evaluated the gastroprotective effects of AMDCP on ethanol-induced gastric mucosal lesions in rats. This study also suggests that AMDCP might be useful as a gastroprotective agent.