Cardiac hemodynamics before, during and after elective cesarean section under spinal anesthesia in low-risk women.

OBJECTIVE: The aim of this study was to describe maternal central hemodynamic parameters before and during delivery as well at the early puerperium in healthy women undergoing elective cesarean section (CS) at term. STUDY DESIGN: The noninvasive Cardiac System (NICaS, NI Medical, Petah-Tikva, Israel) is a regional impedance device that measures cardiac output (CO) and its derivatives with a good correlation with the gold standard Swan-Ganz catheter. We performed a prospective longitudinal study of healthy women with a singleton pregnancy at term. Maternal hemodynamic parameters were assessed by the NICaS at six time points: a few minutes before undergoing an elective CS, immediately after receiving spinal anesthesia, immediately after delivery of the fetus and placenta, after abdominal fascia closure, and within 24 to 36 and 48 to 72 h postpartum. RESULT: Sixty-one consenting women were recruited during the study period (January 2015 to June 2015). Baseline (pre-CS) mean arterial pressure (MAP) was 87.7±7.9 mm Hg, baseline CO was 7.5±1.7 l per min and baseline total peripheral resistance (TPR) was 994±301 dyne × s per cm5. After spinal anesthesia CO significantly increased by 13%, no significant changes were observed in MAP or TPR. Immediately after delivery, a nadir for all parameters was reached: MAP and TPR were significantly reduced by 8% and 26%, respectively (comparing to pre-CS), and CO further increased by 9% (24% comparing to pre-CS). After fascia closure, partial recoveries of all parameters were observed. Twenty-four to thirty-six hours postpartum MAP returned to pre-CS values, while CO and TPR reached -9% and +11% comparing to baseline, respectively. None of the parameters differed significantly between 24 to 36 and 48 to 72 h postpartum. CONCLUSION: Significant hemodynamic changes (reduction of TPR and increase of CO) take place at the time of delivery of fetus and placenta. Knowledge of normal hemodynamic values using a reliable noninvasive technique during various stages of pregnancy and the postpartum period is feasible, and might assist clinicians in assessing the level of patient deviation from expected cardiac performance, especially in high-risk women.

The Role of ß-Carboline Alkaloids in the Pathogenesis of Essential Tremor.

Essential tremor (ET) is one of the most prevalent neurological disorders in the world. Environmental factors have been implicated in the pathogenesis of ET. In particular, epidemiological studies have suggested that neurotoxic agents, especially ß-carboline alkaloids (ßCAs), might be generated through Maillard-type reaction. ßCAs are molecules which are members of a large group of heterocyclic amines (HCAs, the so-called products of cooking meat). ßCAs are highly tremorogenic in animals, producing a marked generalized action tremor soon after systemic administration in a wide range of laboratory animals such as mice, rats and monkeys. Administration of ßCAs remains currently the main experimental model of ET. We review the pathogenesis of ET, with a focus on the biochemistry of ßCAs, their occurrence and biological activity, their endogenous biosynthesis, their formation in food, their toxicokinetics and their neurotoxicity. We highlight open questions regarding the effects of ßCAs in humans.

Bystander killing of malignant cells via the delivery of engineered thymidine-active deoxycytidine kinase for suicide gene therapy of cancer.

Activity and specificity of chemotherapeutic agents against solid tumors can be augmented via the targeted or localized delivery of 'suicide' genes. Selective activation of specific prodrugs in cells expressing the 'suicide' gene drives their elimination by apoptosis, while also enabling the killing of adjacent bystander cells. Strong bystander effects can compensate for poor 'suicide' gene delivery, and depend on the prodrugs used and mechanisms for the acquisition of activated drug by the bystander population, such as the presence of gap junctional intercellular communications. Although a number of 'suicide' gene therapies for cancer have been developed and characterized, such as herpes simplex virus-derived thymidine kinase (HSV-tk)-based activation of ganciclovir, their limited success highlights the need for the development of more robust approaches. Limiting activation kinetics and evolution of chemoresistance are major obstacles. Here we describe 'suicide' gene therapy of cancer based on the lentivirus-mediated delivery of a thymidine-active human deoxycytidine kinase variant. This enzyme possesses substrate plasticity that enables it to activate a multitude of prodrugs, some with distinct mechanisms of action. We evaluated the magnitude and mechanisms of bystander effects induced by different prodrugs, and show that when used in combination, they can synergistically enhance the bystander effect while avoiding off-target toxicity.

[Surgical management of cutaneous malignant melanoma. Review]. / Mise au point sur la prise en charge chirurgicale du mélanome malin cutané. Revue de la littérature.

Nowadays managing a cutaneous malignant melanoma can concern different kind of physicians: dermatologists, general or plastic surgeons The primary surgical procedure is a major step of the treatment. Biopsy must be total to properly determine the thickness of the tumor in case of malignancy. Wide local excision of the scar is often necessary to decrease the local and general recurrence rates. Wide local excision must be performed conforming to its own surgical rules. Managing tumor located on the face or limb extremities is a matter of plastic surgery. Sentinel node biopsy has succeeded to elective lymph node dissection. This procedure allows research of lymphatic spreading of the disease. Practice of sentinel node biopsy must be achieved in a protocolar way. Topography of the lesion can modified achievement and results of this procedure. Prognosis benefit of sentinel biopsy is now clear. Elective lymph node dissection is only performed in case of invaded sentinel node or clinically invaded lymph nodes. Local or locoregional recurrences mainly respond to surgical treatment using wide excision. However, alternative solutions are being evaluated (isolated limb perfusion).

Analysis of burns caused by pre-filled gas canisters used for lamps or portable camping stoves.

The use of pre-filled valveless gas canisters for lamps or camping stoves has caused a number of serious burn incidents. We performed a retrospective analysis of all of the patients who were victims of such incidents admitted to the Marseille Burn Centre between January 1990 and March 2004. There were a total of 21 patients burned in such conditions. Adult males made up the majority of the victims of this sort. Lesions were often extensive (60% of the patients were burned over more than 10% of their body surface) and systematically deep. In order of frequency, burn locations were: the lower limbs, the upper limbs, the hands and the face. The incidents principally occurred during replacement of the canister near an open flame. The marketing of a canister with a valve in order to avoid gas leaks did not cause the old canisters to be taken off the market. On the contrary, European Safety Standard EN417, updated in October 2003, validated the use of these valveless canisters. The severity of the lesions caused and the existence of safe equivalent products requires the passage of a law that forbids valveless canisters.

[Surgical treatment of lymphatic malformations]. / Traitement chirurgical des malformations lymphatiques.

Lymphatic malformations remain a therapeutic challenge. Many treatments by the past led to poor success. The wide variety of clinical presentations makes it difficult to outline specific management programs. Often, diagnosis or complication circumstances as infection, bleeding, airway obstruction, or handicap force acting. Thus, a specific follow-up joined to a multidisciplinary analysis are the key of well-planned surgery. Through our experience and a review of literature, we will describe principles of lymphatic malformations surgery and specific technics for specific locations.

Potentiating AZT activation: structures of wild-type and mutant human thymidylate kinase suggest reasons for the mutants' improved kinetics with the HIV prodrug metabolite AZTMP.

The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate (AZTMP), the partially activated AZT metabolite, by human thymidylate kinase (TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures of different TMPK nucleotide complexes indicate that steric hindrance by the azido group of AZTMP prevents formation of the catalytically active closed conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP 20-fold faster than the wild-type enzyme. The structural basis of the increased activity is assigned to stabilization of the closed P-loop conformation.

C-C chemokine-encoding DNA vaccines enhance breakdown of tolerance to their gene products and treat ongoing adjuvant arthritis.

Depending on the method of immunization, a single administration of CFA may result in the development of a local inflammatory process or chronic polyadjuvant-induced arthritis (AA). We administered naked DNA vaccines encoding MIP-1 alpha, MCP-1, MIP-1 beta, and RANTES to Lewis rats and confirmed that each of these vaccines induced immunological memory to the corresponding gene product. Upon induction of disease, this memory effectively inhibited the development of the autoimmune condition. Self-specific Ab's developed in DNA-vaccinated animals were neutralizing in vitro and could adoptively transfer the beneficial effect of each vaccine. Repeated administration of the constructs encoding MCP-1, MIP-1 alpha, or RANTES inhibited the development and progression of AA, even when each vaccine was administered only after the onset of disease. This suggests a highly effective way by which the immune system could be re-educated to generate protective immunity against its own harmful activities.

Acoustic wave scattering from a composed shell reinforced by a single rib: characteristics of peripheral waves

The steady-state axisymmetrical problem of a plane acoustic wave scattering from a composed shell is considered. The shell has a cylindrical part and two hemispherical endcaps. The rib is a ring of rectangular cross-section that divides the shell into two equal parts. The motion of the shell and the rib is described by the equations of elasticity theory, and the liquid is described by the Helmholtz equation. The solution is obtained numerically by a coupled finite element/boundary element model. Two peripheral waves are generated in the shell: the membrane S0 wave and the bending type water-borne A wave. The form function, acoustic spectrogram and dispersion curves of the phase velocities are presented, and the effect of the rib on the peripheral waves is discussed.

Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.

BACKGROUND: Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryltransfer between ATP and TMP to yield ADP and TDP. In addition to its vital role in supplying precursors for DNA synthesis, human TMPK has an important medical role participating in the activation of a number of anti-HIV prodrugs. RESULTS: Crystal structures of human TMPK in complex with TMP and ADP, TMP and the ATP analog AppNHp, TMP with ADP and the phosphoryl analog AlF(3), TDP and ADP, and the bisubstrate analog TP(5)A were determined. The conformations of the P-loop, the LID region, and the adenine-binding loop vary according to the nature of the complex. Substitution of ADP by AppNHp results in partial closure of the P-loop and the rotation of the TMP phosphate group to a catalytically unfavorable position, which rotates back in the AlF(3) complex to a position suitable for in-line attack. In the fully closed state observed in the TP(5)A and the TDP-ADP complexes, Asp15 interacts strongly with the 3'-hydroxyl group of TMP. CONCLUSIONS: The observed changes of nucleotide state and conformation and the corresponding protein structural changes are correlated with intermediates occurring along the reaction coordinate and show the sequence of events occurring during phosphate transfer. The low catalytic activity of human TMPK appears to be determined by structural changes required to achieve catalytic competence and it is suggested that a mechanism might exist to accelerate the activity.

Modifying human thymidylate kinase to potentiate azidothymidine activation.

Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP.

Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry.

The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4. 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate. The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-).

Structural basis for efficient phosphorylation of 3'-azidothymidine monophosphate by Escherichia coli thymidylate kinase.

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5'-adenosyl)-P5-(5'-thymidyl)pentaphosphate and P1-(5'-adenosyl)P5-[5'-(3'-azido-3'-deoxythymidine)] pentaphosphate have been solved to 2.0-A and 2.2-A resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3'-azido-3'-deoxythymidine 5'-monophosphate (AZT-MP) at a 200-fold reduced turnover number (kcat) in comparison to the physiological substrate dTMP, reduction of kcat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3'-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3'-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3' substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.

Long-lasting protective immunity to experimental autoimmune encephalomyelitis following vaccination with naked DNA encoding C-C chemokines.

DNA vaccination represents a novel means of expressing Ag in vivo for the generation of both humoral and cellular immune responses. The current study uses this technology to elicit protective immunity against experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as an experimental model for multiple sclerosis. RT-PCR verified by Southern blotting and sequencing of PCR products of four different C-C chemokines, macrophage-inflammatory protein-1alpha (MIP-1alpha), monocyte-chemotactic protein-1 (MCP-1), MIP-1beta, and RANTES, were performed on brain samples from EAE rats to evaluate mRNA transcription at different stages of disease. Each PCR product was then used as a construct for naked DNA vaccination. The subsequent in vivo immune response to MIP-1alpha or MCP-1 DNA vaccines prevented EAE, even if disease was induced 2 mo after administration of naked DNA vaccines. In contrast, administration of the MIP-1beta naked DNA significantly aggravated the disease. Generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE. MIP-1alpha, MCP-1, and MIP-1beta mRNA transcription in EAE brains peaked at the onset of disease and declined during its remission, whereas RANTES transcription increased in EAE brains only following recovery. Immunization of CFA without the encephalitogenic epitope did not elicit the anti-C-C chemokine regulatory response in DNA-vaccinated rats. Thus, modulation of EAE with C-C chemokine DNA vaccines is dependent on targeting chemokines that are highly transcribed at the site of inflammation at the onset of disease.

Crystal structure of yeast thymidylate kinase complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) at 2.0 A resolution: implications for catalysis and AZT activation.

The crystal structure of yeast thymidylate kinase (TmpK) complexed with the bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-thymidyl) pentaphosphate (TP5A) was determined at 2.0 A resolution. In this complex, TmpK adopts a closed conformation with a region (LID) of the protein closing upon the substrate and forming a helix. The interactions of TmpK and TP5A strongly suggest that arginine 15, which is located in the phosphate binding loop (P-loop) sequence, plays a catalytic role by interacting with an oxygen atom of the transferred phosphoryl group. Unlike other nucleoside monophosphate kinases where basic residues from the LID region participate in stabilizing the transition state, TmpK lacks such residues in the LID region. We attribute this function to Arg 15 of the P-loop. TmpK plays an important role in the phosphorylation of the AIDS prodrug AZT. The structures of TmpK with dTMP and with AZT-MP [Lavie, A., et al. (1997) Nat. Struct. Biol. 4, 601-604] implicate the movement of Arg15 in response to AZT-MP binding as an important factor in the 200-fold reduced catalytic rate with AZT-MP. TmpK from Escherichia coli lacks this arginine in its P-loop while having basic residues in the LID region. This suggested that, if such a P-loop movement were to occur in the E. coli TmpK upon AZT-MP binding, it should not have such a detrimental effect on catalysis. This hypothesis was tested, and as postulated, E. coli TmpK phosphorylates AZT-MP only 2.5 times slower than dTMP.

The bottleneck in AZT activation.

Nucleoside-based inhibitors of reverse transcriptase were the first drugs to be used in the chemotherapy of AIDS. After entering the cell, these substances are activated to their triphosphate form by cellular kinases, after which they are potent chain terminators for the growing viral DNA. The two main factors limiting their efficacy are probably interrelated. These are the insufficient degree of reduction of viral load at the commencement of treatment and the emergence of resistant variants of the virus. The reason for the relatively poor suppression of viral replication appears to be inefficient metabolic activation. Thus, for the most extensively used drug, 3'-azido-3'-deoxythymidine (AZT), whereas phosphorylation to the monophosphate is facile, the product is a very poor substrate for the next kinase in the cascade, thymidylate kinase. Because of this, although high concentrations of the monophosphate can be reached in the cell, the achievable concentration of the active triphosphate is several orders of magnitude lower. Determination of the structure of thymidylate kinase as a complex with AZT monophosphate (AZTMP) together with studies on the kinetics of its phosphorylation have now led to a detailed understanding of the reasons for and consequences of the poor substrate properties.

A controlled double blind multicenter study of the effectiveness of 5-aminosalicylic acid in patients with Crohn's disease in remission.

We evaluated the efficacy of an oral formulation of 5-amino-salicylic acid in lowering the relapse rate after remission of Crohn's disease. Included were 59 patients who had proven Crohn's disease of at least 1 year's duration, and who had been in continuous remission for at least 6 months, while taking only 5-aminosalicylic acid or no therapy at all. Remission was defined as a Harvey Bradshaw index score (Softley-Clamp modification) of < 4. Patients were given coded mesalzaine 250 mg or placebo tablets (2 x 2 day). They were seen at 0, 1, and 2 months, and then every 2 months until the end of the study. Trial endpoints were 1 year of follow-up, or clinical relapse results. After randomization, 31 patients were included in the placebo arm, and 28 in the treatment arm. There were no significant differences between the two groups at entry. Ten patients were withdrawn from the trial because of noncompliance, loss of follow-up, or headache. There were more clinical relapses in the placebo arm (15 patients, 55%) than in the treatment arm (6 patients, 27%) (p < 0.05). Mesalazine had a significant advantage over placebo (p < 0.05) only in the subgroups of patients with ileal Crohn's disease and in those older than 30 years. We conclude that mesalazine has a moderate but significant benefit in preventing relapse in Crohn's disease in remission; this occurred only in patients with small-bowel involvement or in those older than 30 years.

Design, synthesis, and characterization of a potent xylose isomerase inhibitor, D-threonohydroxamic acid, and high-resolution X-ray crystallographic structure of the enzyme-inhibitor complex.

The binding of a potent inhibitor to the enzyme D-xylose isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of xylose isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the xylose isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray crystallographic structures of D-xylose isomerase-substrate complexes position the substrate and provide evidence for metal movement during catalysis.

The X-ray crystallographic structures of the metal-activated enzyme xylose isomerase from Streptomyces olivochromogenes with the substrates D-glucose, 3-O-methyl-D-glucose and in the absence of substrate were determined to 1.96-, 2.19-, and 1.81-A resolution and refined to R-factors of 16.6%, 15.9%, and 16.1%, respectively. Xylose isomerase catalyzes the interconversion between glucose and fructose (xylose and xylulose under physiological conditions) by utilizing two metal cofactors to promote a hydride shift; the metals are bridged by a glutamate residue. This puts xylose isomerase in the small but rapidly growing family of enzymes with a bridged bimetallic active site, in which both metals are involved in the chemical transformation. The substrate 3-O-methylglucose was chosen in order to position the glucose molecule in the observed electron density unambiguously. Of the two essential magnesium ions per active site, Mg-2 was observed to occupy two alternate positions, separated by 1.8 A, in the substrate-soaked structures. The deduced movement was not observed in the structure without substrate present and is attributed to a step following substrate binding but prior to isomerization. The substrates glucose and 3-O-methylglucose are observed in their linear extended forms and make identical interactions with the enzyme by forming ligands to Mg-1 through O2 and O4 and by forming hydrogen bonds with His53 through O5 and Lys182 through O1. Mg-2 has a water ligand that is interpreted in the crystal structure in the absence of substrate as a hydroxide ion and in the presence of substrate as a water molecule. This hydroxide ion may act as a base to deprotonate the glucose O2 and subsequently protonate the product fructose O1 concomitant with hydride transfer. Calculations of the solvent-accessible surface of possible dimers, with and without the alpha-helical C-terminal domain, suggest that the tetramer is the active form of this xylose isomerase.