A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes.

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time.

A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids.

Recognition of a Key Anchor Residue by a Conserved Hydrophobic Pocket Ensures Subunit Interface Integrity in DNA Clamps.

Sliding clamp proteins encircle duplex DNA and are involved in processive DNA replication and the DNA damage response. Clamp proteins are ring-shaped oligomers (dimers or trimers) and are loaded onto DNA by an ATP-dependent clamp loader complex that ruptures the interface between two adjacent subunits. Here we measured the solution dynamics of the human clamp protein, proliferating cell nuclear antigen, by monitoring the change in the fluorescence of a site-specifically labeled.

Werner Syndrome Helicase Is Required for the Survival of Cancer Cells with Microsatellite Instability.

Werner syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss-of-function mutations in WRN led to premature aging and predisposition to cancer. We evaluated synthetic lethal (SL) interactions between WRN and another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines.

Preoperative Platelet-Lymphocyte Ratio Augments CA 19-9 as a Predictor of Malignancy in Chronic Calcific Pancreatitis.

Introduction: Differentiating inflammatory from malignant head mass in the background of chronic calcific pancreatitis (CCP) is difficult, and there is no investigation which can reliably solve this dilemma. An accurate diagnosis is crucial as the treatment is different for the two cases and a failure to identify malignancy before surgery can be disastrous. We aimed to assess the accuracy of platelet-lymphocyte ratio (PLR) and to compare it with CA 19-9 in determining the nature of pancreatic head mass (PHM).

Bile duct thrombi in hepatocellular carcinoma: is aggressive surgery worthwhile?

Introduction: Obstructive jaundice as a result of bile duct tumour thrombus (BDTT) is an unusual clinical entity and an uncommon presenting feature of hepatocellular carcinoma (HCC). This study evaluates the outcome of hepatectomy for HCC with obstructive jaundice as a result of BDTT in non-cirrhotic livers.

Methods: Between 1997 and 2012, out of 426 patients with HCC in non-cirrhotic livers, 39 patients with BDTT (Group I n = 39), who underwent a hepatectomy, were analysed and compared with the non-BDTT group (Group II n = 387).

Synbiotics in Surgery for Chronic Pancreatitis: Are They Truly Effective? A Single-blind Prospective Randomized Control Trial.

Background: Postoperative infectious complications in patients undergoing pancreatic surgery are a significant cause for morbidity and mortality. Although synbiotics have beneficial effects on human health, their clinical value in surgical patients remains unclear given a paucity of applicable clinical studies.

Aim: To determine the impact of perioperative synbiotic therapy on postoperative infectious complications, morbidity and mortality in patients undergoing pancreatic surgery for chronic pancreatitis.

Impact of perioperative enteral synbiotics in hepatic and pancreatic surgery: design and rationale of a single blind placebo controlled prospective randomised control trial.

Background: Prebiotics and probiotics influence all pathogenic mechanisms of bacterial translocation. Used in combination, they are called synbiotics. Postoperative infective complications in patients undergoing hepatic and pancreatic surgery lead to a significant prolongation of hospital stay and increased costs.

Implications of the presence of an aberrant right hepatic artery in patients undergoing pancreaticoduodenectomy.

Aim: To analyze the differences in outcomes and the clinical impact following pancreatoduodenectomy (PD) in patients with and without aberrant right hepatic artery (aRHA).

Methods: All patients undergoing PD between January 2008 and December 2012 were divided into two groups, one with aRHA and the other without. These groups were compared to identify differences in the intraoperative variables, the oncological clearance and the postoperative morbidity, mortality and hospital stay.

RecG and UvsW catalyse robust DNA rewinding critical for stalled DNA replication fork rescue.

Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG.

Interaction of T4 UvsW helicase and single-stranded DNA binding protein gp32 through its carboxy-terminal acidic tail.

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA (ssDNA) binding protein gp32 is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved.

Stepwise assembly of the human replicative polymerase holoenzyme.

In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme.

Hemosuccus Pancreaticus: 15-Year Experience from a Tertiary Care GI Bleed Centre.

Background. Hemosuccus pancreaticus (HP) is a very rare and obscure cause of upper gastrointestinal bleeding. Due to its rarity, the diagnostic and therapeutic strategy for the management of this potentially life threatening problem remains undefined.

How a holoenzyme for DNA replication is formed.

Loading of the phage T4 sliding clamp gp45 by the gp44/62 clamp loader onto DNA to form the holoenzyme and their disassembly pathways were investigated using FRET-based single-molecule and ensemble kinetic studies. gp44/62-mediated assembly of gp45 onto the DNA involves a rate-limiting conformational rearrangement of the gp45-gp44/62-DNA complex. Single-molecule measurements revealed the intermediates in gp45 loading and their interconversion, suggesting that the assembly is not concerted but is broken down into many small kinetic steps.

Direct observation of stalled fork restart via fork regression in the T4 replication system.

The restart of a stalled replication fork is a major challenge for DNA replication. Depending on the nature of the damage, different repair processes might be triggered; one is template switching, which is a bypass of a leading-strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching.

The human lagging strand DNA polymerase δ holoenzyme is distributive.

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro.

Analysis of the DNA translocation and unwinding activities of T4 phage helicases.

Helicases are an important class of enzymes involved in DNA and RNA metabolism that couple the energy of ATP hydrolysis to unwind duplex DNA and RNA structures. Understanding the mechanism of helicase action is vital due to their involvement in various biological processes such as DNA replication, repair and recombination. Furthermore, the duplex DNA unwinding property of this class of enzymes is closely related to their single-stranded DNA translocation.

Single-molecule studies of DNA replisome function.

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication.

Approaches to the simultaneous inactivation of metallo- and serine-beta-lactamases.

A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-beta-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-beta-lactamase. With respect to interactions with the classes A, C, and D serine beta-lactamases, as judged by their correspondingly low K(m) values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes.

Processive and unidirectional translocation of monomeric UvsW helicase on single-stranded DNA.

UvsW protein from bacteriophage T4 controls the transition from origin-dependent to origin-independent initiation of replication through the unwinding of R-loops bound to the T4 origins of replication. UvsW has also been implicated through genetic and biochemical experiments to play a role in DNA repair processes such as replication fork regression and Holliday junction branch migration. UvsW is capable of unwinding a wide variety of substrates, many of which contain only duplex DNA without single-stranded regions.

Beta-ketophosphonates as beta-lactamase inhibitors: Intramolecular cooperativity between the hydrophobic subsites of a class D beta-lactamase.

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme.

Synthesis and evaluation of ketophosph(on)ates as beta-lactamase inhibitors.

A series of amidoketophosph(on)ates of general structure PhCH2OCONHCH(R)COCHR'(CH2)n(O)P(O2-)(O)R'' (R = H, CH3; R' = H, CH3; n = 0, 1; R'' = H, CH3, Et, Ph) have been prepared as a potential source of beta-lactamase inhibitors. The phosphonates (n = 0) were obtained by means of the Arbuzov reaction while most of the phosphates were achieved from reaction of phosph(or/on)ic acids with the appropriate diazoketone PhCH2OCONHCH(R)COCR'N2. The electrophilicity of the carbonyl group in the resulting phosph(on)ates was assessed by the degree of hydration in aqueous solution, determined from NMR spectra.