Simultaneous liver resection and double cardiac valve replacement. A case report.

INTRODUCTION: We present a rare case in which both a double cardiac valve replacement was performed as well as a hepatic resection. PRESENTATION OF CASE: We report the case of a 36 year old patient who presented with intra abdominal bleeding thought to have been caused by a liver haemangioma she also had severe autoimmune cardiac valve disease. She underwent a simultaneous right hepatectomy with cardiac valve replacement. DISCUSSION: Management of this challenging case is discussed. CONCLUSION: We advocate the possibility of performing combined operations where both valve replacement and hepatic resection is required.

Selective Interarterial Radiation Therapy (SIRT) in Colorectal Liver Metastases: How Do We Monitor Response?

Radioembolisation is a way of providing targeted radiotherapy to colorectal liver metastases. Results are encouraging but there is still no standard method of assessing the response to treatment. This paper aims to review the current experience assessing response following radioembolisation. A literature review was undertaken detailing radioembolisation in the treatment of colorectal liver metastases comparing staging methods, criteria, and response. A search was performed of electronic databases from 1980 to November 2011. Information acquired included year published, patient numbers, resection status, chemotherapy regimen, criteria used to stage disease and assess response to radioembolisation, tumour markers, and overall/progression free survival. Nineteen studies were analysed including randomised controlled trials, clinical trials, meta-analyses, and case series. There is no validated modality as the method of choice when assessing response to radioembolisation. CT at 3 months following radioembolisation is the most frequently modality used to assess response to treatment. PET-CT is increasingly being used as it measures functional and radiological aspects. RECIST is the most frequently used criteria. Conclusion. A validated modality to assess response to radioembolisation is needed. We suggest PET-CT and CEA pre- and postradioembolisation at 3 months using RECIST 1.1 criteria released in 2009, which includes criteria for PET-CT, cystic changes, and necrosis.

Cell growth inhibition by sequence-specific RNA minihelices.

RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.

Operational RNA code for amino acids: species-specific aminoacylation of minihelices switched by a single nucleotide.

The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodon-independent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a species-specific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals.

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component.

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

The high-resolution structure of the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1H nuclear magnetic resonance spectroscopy. A total of 442 experimentally derived restraints including 13 dihedral angle (phi, chi 1) restraints were used. A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0.36 A for the backbone atoms and 0.96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39. Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured. The binding domain contains two short parallel alpha-helices (residues Val7 to Lys14 and Lys32 to Leu39), a3(10)-helix (residues Asp17 to Val21) and a structured loop made up of overlapping beta-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core. The loop is stabilized to a large extent by Asp34. This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24. We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites. The structured region of the binding domain, comprising 33 residues, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold. It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior. The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.

Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.

Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.

Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo rôle of gelatinases is discussed.