Peptides with Dual Antimicrobial-Anticancer Activity Derived from the N-terminal Region of H. pylori Ribosomal Protein L1 (RpL1)

International Journal of Peptide Research and Therapeutics - Whereas the traditional approaches of cancer therapy including radiotherapy, chemotherapy, and immunotherapy have failed to properly...     

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.     

The survivin molecule as a double-edged sword in cellular physiologic and pathologic conditions and its role as a potential biomarker and therapeutic target in cancer

Survivin is a member of the family of apoptosis inhibitory proteins with increased expression level in most cancerous tissues. Evidence shows that survivin plays regulatory roles in proliferation or survival of normal adult cells, principally vascular endothelial cells, T lymphocytes, primitive hematopoietic cells, and polymorphonuclear neutrophils. Survivin antiapoptotic role is, directly and indirectly, related to caspase proteins and shows its role in cell division through the chromosomal passenger complex. Survivin contains many genetic polymorphisms that the role of some variations has been proven in several cancers. The −31G/C polymorphism is one of the most important survivin mutations which is located in the promoter region on a CDE/CHR motif. This polymorphism can upregulate the survivin messenger RNA. In addition, its allele C can increase the risk of cancers in 1.27-fold than allele G. Considering the fundamental role of survivin in different cancers, this protein could be considered as a new therapeutic target in cancer treatment. For this purpose, various strategies have been designed including the prevention of survivin expression through inhibition of mRNA translation using antagonistic molecules, inhibition of survivin gene function through small inhibitory molecules, gene therapy, and immunotherapy. In this study, we describe the structure, played roles in physiological and pathological states and genetic polymorphisms of survivin. Finally, the role of survivin as a potential target in cancer therapy given challenges ahead has been discussed.     

Lipoprotein lipase inhibits hepatitis C virus (HCV) infection by blocking virus cell entry

A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.     

Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.     

Multi-Pronged Interactions Underlie Inhibition of α-Synuclein Aggregation by β-Synuclein

The intrinsically disordered protein β-synuclein is known to inhibit the aggregation of its intrinsically disordered homolog, α-synuclein, which is implicated in Parkinson's disease. While β-synuclein itself does not form fibrils at the cytoplasmic pH?7.4, alteration of pH and other environmental perturbations are known to induce its fibrilization. However, the sequence and structural determinants of β-synuclein inhibition and self-aggregation are not well understood. We have utilized a series of domain-swapped chimeras of α-synuclein and β-synuclein to probe the relative contributions of the N-terminal, C-terminal, and the central non-amyloid-β component domains to the inhibition of α-synuclein aggregation. Changes in the rates of α-synuclein fibril formation in the presence of the chimeras indicate that the non-amyloid-β component domain is the primary determinant of self-association leading to fibril formation, while the N- and C-terminal domains play critical roles in the fibril inhibition process. Our data provide evidence that all three domains of β-synuclein together contribute to providing effective inhibition, and support a model of transient, multi-pronged interactions between IDP chains in both processes. Inclusion of such multi-site inhibitory interactions spread over the length of synuclein chains may be critical for the development of therapeutics that are designed to mimic the inhibitory effects of β-synuclein.     

The PI3K/Akt signaling axis in Alzheimer’s disease: a valuable target to stimulate or suppress?

Among the long list of age-related complications, Alzheimer’s disease (AD) has the most dreadful impact on the quality of life due to its devastating effects on memory and cognitive abilities. Although a plausible correlation between the phosphatidylinositol 3-kinase (PI3K) signaling and different processes involved in neurodegeneration has been evidenced, few articles reviewed the task. The current review aims to unravel the mechanisms by which the PI3K pathway plays pro-survival roles in normal conditions, and also to discuss the original data obtained from international research laboratories on this topic. Responses to questions on how alterations of the PI3K/Akt signaling pathway affect Tau phosphorylation and the amyloid cascade are given. In addition, we provide a general overview of the association between oxidative stress, neuroinflammation, alterations of insulin signaling, and altered autophagy with aberrant activation of this axis in the AD brain. The last section provides a special focus on the therapeutic possibility of the PI3K/Akt/mTOR modulators, either categorized as chemicals or herbals, in AD. In conclusion, determining the correct timing for the administration of the drugs seems to be one of the most important factors in the success of these agents. Also, the role of the PI3K/Akt signaling axis in the progression or repression of AD widely depends on the context of the cells; generally speaking, while PI3K/Akt activation in neurons and neural stem cells is favorable, its activation in microglia cells may be harmful.     

Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were–544.00 and–469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.     

Twelve year experience of laparoscopic gastric plication in morbid obesity: development of the technique and patient outcomes

ABSTRACT: BACKGROUND: Laparoscopic Gastric Plication (LGP) is a new restrictive bariatric surgery, previously introduced by the author. The aim of this study is to explain the modifications and to present the 12-year experience, regarding early and long term results, complications and cost. METHODS: We used LGP for morbid obesity during the past 12 years. Anterior plication (10 cases), one-row bilateral plication while right gastroepiploic artery included (42 cases), and excluded from the plication (104 cases) and two-row plication (644 cases). The gastric greater curvature was plicated using 2/0 prolen from fundus at the level of diaphragm preserving the His angle to just proximal to the pylorus. The anatomic and functional volume of stomach was 50cc and 25cc respectively in two-row method. Ordered postop visits also included evaluation of weight loss, complications, change of diet and control of exercise. RESULTS: LGP was performed in 800 cases (mean age: 27.5, range: 12 to 65 years, nine under 18). Female to male ratio was 81% to 19% and average BMI was 42.1 (35-59). The mean excess weight loss (EWL) was 70% (40% to 100%) after 24 months and 55% (28% to 100%) after 5 years following surgery. 134 cases (16.7%) did not completed long term follow-up. The average time of follow up was 5 years (1 month to 12 years). 5.5% and 31% of cases complained from weight regain respectively during 4 and 12 years after LGP. The mean time of operation was 72 (49--152) minutes and average hospitalization time was 72 hours (24 hours to 45 days). The cost of operation was 2000 $ less than gastric banding or sleeve and 2500 $ less than gastric bypass. Eight patients out of 800 cases (1%) required reoperation due to complications like: micro perforation, obstruction and vomiting following adhesion of His angle. Other complications included hepatitis pneumonia, self-limiting intra-abdominal bleeding and hypocalcaemia. CONCLUSION: The percentage of EWL in this technique is comparable to other restrictive methods. The technique is safe with 1.6% complication (1% reoperated), and 31% regain during 12 years. The cost of operation is less than the other methods.