Targeting the Warburg Effect in cancer; relationships for 2-arylpyridazinones as inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3).

High-throughput screening of a small-molecule library identified a 5-triazolo-2-arylpyridazinone as a novel inhibitor of the important glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3). Such inhibitors are of interest due to PFKFB3's control of the important glycolytic pathway used by cancer cells to generate ATP. A series of analogues was synthesized to study structure-activity relationships key to enzyme inhibition. Changes to the triazolo or pyridazinone rings were not favoured, but limited-size substitutions on the aryl ring provided modest increases in potency against the enzyme. Selected analogues and literature-described inhibitors were evaluated for their ability to suppress the glycolytic pathway, as detected by a decrease in lactate production, but none of these compounds demonstrated such suppression at non-cytotoxic concentrations.

Quantitation of hepatitis A virus and enterovirus levels in the lagoon canals and Lido beach of Venice, Italy, using real-time RT-PCR.

In order to assess the microbial water quality of the lagoon canals of Venice, Italy and nearby beach on Lido island, a study was conducted using real-time RT-PCR to enumerate levels of hepatitis A virus (HAV) and enteroviruses in these marine waters over a 3-year period from 2003 to 2005. A total of 17 sites (9 lagoon canal and 8 beach sites) were assayed. For the canals of the Venice Lagoon, 78% were positive for both HAV and enteroviruses, with levels ranging from 75 to 730 and 3 to 1,614 genome copies/L, respectively. At Lido beach, HAV was never detected, but enteroviruses were detected in all Lido beach samples at levels ranging from 2 to 71 genome copies/L. There was a statistically significant correlation between thermotolerant coliform densities and HAV levels (p=0.0002), but the relationship between thermotolerant coliform densities and enterovirus levels was not significant (p>0.05). Despite the fact that enteroviruses were detected at low levels in the surfzone at Lido beach, the risk for enteroviral infection (calculated using the beta-Poisson model) for recreational exposure from swimming, was in the range of 1.9 x 10(-3) - 6.1 x 10(-2), yielding a disease risk at or below the level (5% for gastroenteritis) deemed acceptable by European Guide standards.

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Detection and quantification of hepatitis A virus in seawater via real-time RT-PCR.

A real-time RT-PCR method utilizing SYBR Green chemistry was developed to detect and enumerate hepatitis A virus (HAV) in ocean water. Ocean water samples were taken at the Tijuana River mouth (Tijuana, Mexico) and Imperial Beach pier (1.4 km north of the Tijuana River mouth in San Diego, California) following four separate rain events. A total of eight samples were collected, one from each location, each consisting of 4 l of ocean water. Using conventional RT-PCR and primers based on the conserved sequence at the VP3-VP1 genes of HAV, a 247 bp cDNA was amplified from six out of eight rain event water samples. HAV cDNA (confirmed by sequence analysis) was cloned into a TOPO vector (Invitrogen, Carlsbad, CA), and four primer sets were designed for application in SYBR Green real-time RT-PCR. The water samples were shown to contain inhibitors that affected real-time RT-PCR amplifications, however diluting the cDNA solution enabled successful amplification. Using real-time RT-PCR, HAV could be detected in all eight samples. Depending on the rain event, the viral load in these samples varied from 90 to 3523 copies of HAV/L of ocean water near the mouth of the Tijuana River, and 347 to 2656 copies/l near the Imperial Beach pier. The sensitivity, quantitative ability and the high throughput nature of SYBR Green real-time RT-PCR will be useful in monitoring HAV contamination in seawater.

Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors.

Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.

Tat peptide-mediated cellular delivery: back to basics.

Peptides are emerging as attractive drug delivery tools. The HIV Tat-derived peptide is a small basic peptide that has been successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. The 'transduction domain' or region conveying the cell penetrating properties appears to be confined to a small (9 amino acids) stretch of basic amino acids, with the sequence RKKRRQRRR [S. Ruben, A. Perkins, R. Purcell, K. Joung, R. Sia, R. Burghoff, W.A. Haseltine, C.A. Rosen, Structural and functional characterization of human immunodeficiency virus tat protein, J. Virol. 63 (1989) 1-8; S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 664-668; E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (1997) 16010-16017; S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836-5840.]. The mechanism by which the Tat peptide adheres to, and crosses, the plasma membrane of cells is currently a topic of heated discussion in the literature, with varied findings being reported. This review aims to bring together some of those findings. Peptide interactions at the cell surface, and possible mechanisms of entry, will be discussed together with the effects of modifying the basic sequence and attaching a cargo.