Giant extra-hepatic thrombosed portal vein aneurysm: a case report and review of the literature.

BACKGROUND: Extrahepatic Portal vein aneurysm (EPVA) is a rare finding that may be associated with different complications, e.g. thrombosis, rupture, portal hypertension and compression of adjacent structures. It is being diagnosed more frequently with the advent of modern cross-sectional imaging. Our review of the English literature disclosed 13 cases of thrombosed EPVA. CASE PRESENTATION: A 50-years-old woman presented with acute abdominal pain but no other symptom. She had no relevant medical history. Palpation of the right upper quadrant showed tenderness. Laboratory tests were unremarkable. A computed tomography showed portal vein aneurysm measuring 88 × 65 mm with thrombosis extending to the superior mesenteric and splenic vein. The patient was treated conservatively with anticoagulation therapy. She was released after two weeks and followed on an outpatient basis. At two months, she reported decreased abdominal pain and her physical examination was normal. A computed tomography was performed showing a decreased thrombosis size and extent, measuring 80 × 55 mm. CONCLUSIONS: Although rare, surgeons should be made aware of this entity. Complications are various. Conservative therapy should be chosen in first intent in most cases. We reported the case of the second largest thrombosed extra-hepatic PVA described in the literature, treated by anticoagulation therapy with a good clinical and radiological response

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate-dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates

Loss of Hyperconjugative Effects Drives Hydride Transfer during Dihydrofolate Reductase Catalysis

Hydride transfer is widespread in nature and has an essential role in applied research. However, the mechanisms of how this transformation occurs in living organisms remain a matter of vigorous debate. Here, we examined dihydrofolate reductase (DHFR), an enzyme that catalyzes hydride from C4′ of NADPH to C6 of 7,8-dihydrofolate (H2F). Despite many investigations of the mechanism of this reaction, the contribution of polarization of the π-bond of H2F in driving hydride transfer remains unclear. H2F was stereospecifically labeled with deuterium β to the reacting center, and β-deuterium kinetic isotope effects were measured. Our experimental results combined with analysis derived from QM/MM simulations reveal that hydride transfer is triggered by polarization at the C6 of H2F. The σ Cβ–H bonds contribute to the buildup of the cationic character during the chemical transformation, and hyperconjugation influences the formation of the transition state. Our findings provide key insights into the hydride transfer mechanism of the DHFR-catalyzed reaction, which is a target for antiproliferative drugs and a paradigmatic model in mechanistic enzymology

Vascular dysfunction in children conceived by assisted reproductive technologies: underlying mechanisms and future implications.

Epidemiological studies in humans have demonstrated a relationship between pathological events during fetal development and increased cardiovascular risk later in life and have led to the so called "Fetal programming of cardiovascular disease hypothesis". The recent observation of generalised vascular dysfunction in young apparently healthy children conceived by assisted reproductive technologies (ART) provides a novel and potentially very important example of this hypothesis. This review summarises recent data in ART children demonstrating premature subclinical atherosclerosis in the systemic circulation and pulmonary vascular dysfunction predisposing to exaggerated hypoxia-induced pulmonary hypertension. These problems appear to be related to the ART procedure per se. Studies in ART mice demonstrating premature vascular aging and arterial hypertension further demonstrate the potential of ART to increase cardiovascular risk and have allowed to unravel epigenetic alterations of the eNOS gene as an underpinning mechanism. The roughly 25% shortening of the life span in ART mice challenged with a western style high-fat-diet demonstrates the potential importance of these alterations for the long-term outcome. Given the young age of the ART population, data on cardiovascular endpoints will not be available before 20 to 30 years from now. However, already now cohort studies of the ART population are needed to early detect cardiovascular alterations with the aim to prevent or at least optimally treat cardiovascular complications. Finally, a debate needs to be engaged on the future of ART and the consequences of its exponential growth for public health

Cryo-kinetics reveal dynamic effects on the chemistry of human dihydrofolate reductase

Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti-cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo-measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (kHLE /kHHE ) close to unity above 0 °C; however, the enzyme KIE was increased to 1.72±0.15 at -20 °C, indicating that the coupling of protein motions to the chemical step is minimized under optimal conditions but enhanced at non-physiological temperatures. The presented cryogenic approach provides an opportunity to probe the kinetics of mammalian DHFRs, thereby laying the foundation for characterizing their transition state structure

Cardiovascular dysfunction in children conceived by assisted reproductive technologies.

Epidemiological studies demonstrate a relationship between pathological events during foetal development and future cardiovascular risk and the term 'foetal programming of cardiovascular disease' has been coined to describe this phenomenon. The use of assisted reproductive technologies (ARTs) is growing exponentially and 2-5% of children are now born by this procedure. Emerging evidence indicates that ART represents a novel important example of foetal programming. Assisted reproductive technology may modify the cardiovascular phenotype in two ways: (i) ART involves manipulation of the early embryo which is exquisitely sensitive to environmental insults. In line with this concern, ART alters vascular and cardiac function in children and studies in mice show that ART alters the cardiovascular phenotype by epigenetic alterations related to suboptimal culture conditions. (ii) Assisted reproductive technology markedly increases the risk of foetal insults that augment cardiovascular risk in naturally conceived individuals and are expected to have similar consequences in the ART population. Given the young age of the ART population, it will take another 20-30 years before data on cardiovascular endpoints will be available. What is clear already, however, is that ART emerges as an important cardiovascular risk factor. This insight requires us to revise notions on ART's long-term safety and to engage on a debate on its future. There is an urgent need to better understand the mechanisms underpinning ART-induced alteration of the cardiovascular phenotype, improve the procedure and its long-term safety, and, while awaiting this aim, not to abandon medicine's fundamental principle of doing no harm (to future children) and use ART parsimoniously

Electric field measurements reveal the pivotal role of cofactor-substrate interaction in dihydrofolate reductase catalysis

The contribution of ligand-ligand electrostatic interaction to transition state formation during enzyme catalysis has remained unexplored, even though electrostatic forces are known to play a major role in protein functions and have been investigated by the vibrational Stark effect (VSE). To monitor electrostatic changes along important steps during catalysis, we used a nitrile probe (T46C-CN) inserted proximal to the reaction center of three dihydrofolate reductases (DHFRs) with different biophysical properties, Escherichia coli DHFR (EcDHFR), its conformationally impaired variant (EcDHFR-S148P), and Geobacillus stearothermophilus DHFR (BsDHFR). Our combined experimental and computational approach revealed that the electric field projected by the substrate toward the probe negates those exerted by the cofactor when both are bound within the enzymes. This indicates that compared to previous models that focus exclusively on subdomain reorganization and protein-ligand contacts, ligand-ligand interactions are the key driving force to generate electrostatic environments conducive for catalysis

Heavy enzymes and the rational redesign of protein catalysts

An unsolved mystery in biology concerns the link between enzyme catalysis and protein motions. Comparison between isotopically labelled “heavy” dihydrofolate reductases and their natural abundance counterparts has suggested that the coupling of protein motions to the chemistry of the catalysed reaction is minimized for hydride transfer. In alcohol dehydrogenase, non‐natural, bulky substrates that induce additional electrostatic rearrangements of the active site enhance coupled motions. This finding may provide a novel route engineer enzymes with altered substrate specificity, because amino acid residues responsible for dynamic coupling with a given substrate present as “hot spots” for mutagenesis. The routine engineering of enzymes to catalyse reactions of choice may eventually be possible through a detailed understanding of the biophysics of enzyme catalysis from insights gained from the analysis of “heavy” enzymes

Selective Phosphonylation of 5′-Adenosine Monophosphate (5′-AMP) via Pyrophosphite [PPi(III)]

We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O52−; PPi(III)], to the phosphate group of 5′-adenosine mono phosphate (5′-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO−). In this specific case of P-transfer from PPi(III) to 5′-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO−. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5′-AMP, [P(III)P(V)-5′-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus