Previous Manuscript Research Articles

Functional properties and biodistribution of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) mixtures formed with nucleic acid

The clinical success of non-viral gene delivery reagents is hampered by their inefficient cellular transgene delivery, which is largely influenced by carrier properties that are currently undefined and misunderstood. In an attempt to further define and understand the requirements for a safe and efficient non-viral gene delivery reagent, research labs often engineer and evaluate many putative products with subtle physiochemical differences in order to delineate requirements for improved in vitro and in vivo success. The synthesis of many putative reagents is often time-intensive, laborious and costly. In a previous manuscript published by our lab, different amounts of poly(triethylenetetramine/cystamine bisacrylamide) (p(TETA/CBA) and its pegylated counterpart, poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) (p(TETA/CBA)-g-PEG) were mixed together to easily identify optimal reagent properties and candidates in vitro, while avoiding the synthesis of many putative candidates for study. This report uses the aforementioned facile approach to evaluate reagent properties of products that were obtained via one-pot synthesis, which improved synthetic ease. As such, synthesis time was reduced from 6days to 3days and had comparable or improved transfection and viability compared to previous works. Moreover, this synthesis resulted in higher molecular weight products than were used in the previous study and allow for lower polymer doses to be used for complexation, which is useful for systemic delivery that is used herein. The physiochemical properties of the formulations derived using these novel reagents was studied prior to investigating their in vivo biodistribution profiles in a murine colon adenocarcinoma model. Interestingly, negatively charged complexes exhibited greater passive tumor accumulation compared to positively charged complexes following their systemic administration. These studies warrant further investigation for the use of negatively charged drug and gene delivery reagents for passive tumor targeting, and they substantiate the use of polycation/PEG-polycation mixtures for facile product evaluation in order to elucidate design and formulation mandates for the clinical success of non-viral gene delivery formulations.

Generation and annihilation of point defects by doping impurities during FZ silicon crystal growth

The main purpose of this paper is to confirm the conclusion of a previous manuscript that the generation of silicon interstitials is the result of the relaxation of the lattice strain induced due to the thermal gradient. In this paper, we consider the relaxation of the lattice strain from a different point of view due to impurity doping during FZ crystal growth. Doping with nitrogen molecules annihilates both the A and D defects, which are the secondary defects of silicon interstitial and vacancy, respectively. The first half of this paper describes such peculiar behavior of nitrogen molecules in crystals doped with both a high concentration of vacancies and nitrogen molecules. The following four important values: the estimated vacancy concentrations, the deep levels at 0.44eV under the conduction band for n-type and at 0.66eV over the valence band for p-type for pure vacancies and the diffusion coefficient of the silicon interstitials DI-FZ=1.3exp(−4.5eV/kT) are determined. The last half of the paper demonstrates how impurity doping is systematically correlated with the generation and annihilation of point defects. This phenomenon occurs in accordance with Vegard's law as tested with seven kinds of impurities, which have covalent bonding radii that are smaller or larger than that of silicon. Silicon interstitials are generated by doping with impurities that have smaller covalent bonding radii than silicon to maintain the essential lattice constant of silicon at around 1300°C, and vacancies are increased above the equilibrium concentration by doping with impurities that have larger covalent bonding radii than silicon.

Addendum to “Construction of spline functions in spreadsheets to smooth experimental data” [Adv Eng Software 2008;39(5):422–429

A previous manuscript detailed how spreadsheet software can be programmed to smooth experimental data via cubic splines. This addendum corrects a few errors in the previous manuscript and provides additional necessary programming steps.

Cardiology Fellowship Education in the Era of High-density Training, Data Tracking, and Quality Measures.

Background:Educating trainees during cardiology fellowship is a process in constant evolution, with program directors regularly adapting to increasing demands and regulations as they strive to prepare graduates for practice in today’s healthcare environment.Methods and Results:In a 10-year follow-up to a previous manuscript regarding fellowship education, we reviewed the literature regarding the most topical issues facing training programs in 2010, describing our approach at The Ohio State University.Conclusion:In the midst of challenges posed by the increasing complexity of training requirements and documentation, work hour restrictions, and the new definitions of quality and safety, we propose methods of curricula revision and collaboration that may serve as an example to other medical centers.

Erratum: Tuning aromaticity in trigonal alkaline earth metal clusters and their alkali metal salts

Page 2766. After several tests, in our previous manuscript,1 we concluded that “the 1A1′(σ) state is the ground state of cyclo-[M3]2− (M = Be, Mg, and Ca) species.” After the publication of our work, Giri et al.2 reported that the triplet state for these cyclo-[M3]2− species is more stable than the singlet one. In view of this conclusion, we revised our results and we find that the triplet state one reaches depends on the initial molecular orbitals (MOs) guess. Thus when one optimizes the triplet state starting from the (MOs) of the 1A1′(σ) state one reaches a different minimum than when the initial MOs are those of the 1A1′(π) state (see Figure 1). The former guess leads to the triplet states that were found in our previous paper1 and that are higher in energy than the 1A1′(σ) states (see Table1). On the other hand, when the triplet states are optimized starting from the MOs of the 1A1′(π) state, one reaches a more stable triplet state that, in fact, is the ground state for these species at both the B3LYP and CCSD(T) levels of theory (see Table2). Notwithstanding, note that for the cyclo-[Ca3]2− compound the singlet state remains more stable than the triplet state at the CCSD(T) level of theory. In fact, among singlet states, the σ-HOMO singlet configuration is the fundamental electronic state for cyclo-[Ca3]2− at the CCSD(T) level of theory (at variance with the cyclo-[Be3]2− and cyclo-[Mg3]2− where the π-HOMO singlet configuration is more stable). In summary, the ground stateof cyclo-[M3]2− species (M = Be and Mg) species is a triplet state and not a singlet one as we erroneously pointed out in our earlier publication.1 Molecular orbital picture of 1A1′(σ) and 1A1′(π) electronic configurations found in cyclo-[M3]2− clusters (M = Be, Mg, and Ca).

Design of Multi-located Small Luminaires Lighting with Brightness Image(<Special Issue>Residential Lighting Aiming at Energy Saving and Amenity)

Based on the subjective experiment for a living room described in the previous manuscript, relation among subjective impression of suitableness of lighting, characteristics of luminance image, and characteristics of brightness image, is examined. Finally a lighting design method to realize comfortable and energy saving lighting is proposed from the examination.

Sir James Black (1924–2010)

This special issue of BJP celebrates the life and work of Sir James Black. The issue also illustrates the great contributions Sir James made, not only to science, but to this journal and to the British Pharmacological Society. Sir James was elected to membership of the British Pharmacological Society (BPS) in 1961, at the winter meeting hosted by Professor GV Born's Department at the Royal College of Surgeons. He served on the Editorial board of BJP from 1967 to 1973 and as a BPS committee member from 1975 to 1978. He was elected as an honorary member of BPS in 1988 and was awarded that Society's highest distinction, the Wellcome Gold Medal, in 1997. The British Pharmacological Society has also honoured him by running the ongoing annual James Black Symposia Series, held first in Cambridge on 21–25 July 2002. Jim published 37 full papers and 50 proceedings abstracts in BJP between 1965 and 2007, a tribute to his huge commitment to the Society. He also published one full paper in BJCP. This issue contains an Appreciation of Sir James from Alan McGregor, friend and physician, first delivered at his funeral in St Columba's Church in Knightsbridge, London, on 29 March 2010. This is followed by tributes sent to the BPS by colleagues who had either worked with him or been touched in some way by his commitment to encourage young scientists. The Society is also privileged to include Sir James's last manuscript in this special issue. The article titled, “A Life in New Drug Research” was written with the assistance of his wife, Professor Rona MacKie, who has continued to help us with the preparation and publication of this special issue. It is a brief compilation of his memoirs that he wrote for a general audience. Sir James wrote this article after handing us a more technical manuscript about drug discovery. In a style that reflected his life, Jim spent part of the last few weeks of his life revising a manuscript – reducing his previous technical drug discovery manuscript into a more inclusive, generalist style that could be read by a wider, and younger audience. “Reduce” and “include” seem to be two of his most driving principles. We took the liberty of addressing Sir James here as ‘Jim’ in that very same spirit. Indeed, on a visit with him at a hospice where he was a patient at the end of last November, we noticed that his locker for personal items had been neatly labeled in stylish print with ‘Sir James’. However, ‘someone’ had taken a pen and crossed out ‘Sir James’ and simply scribbled ‘Jim’ above it. More anecdotes about his humility and other qualities are included in the section on personal tributes mentioned above. As an aside, the more technical manuscript will also be published by BJP as the lead article in an upcoming issue on drug discovery. This is followed by a selection of scientific papers published in BJP that characterize three of the major phases of his career, the discovery of beta-blockers, the discovery of H2-blockers and his fascination with receptor theory as the bedrock of pharmacology and drug discovery. These are interspersed with short pithy commentaries that he wrote on these and other papers for the 50th Anniversary issue of BJP in 1997 and which bring out his humour as well as his skill with words. We hope in some way this special issue inspires scientists to follow the ideals that drove Sir James in his scientific pursuits. Most of all, we hope, as Jim would have wanted, that young people gain the confidence to pursue their dreams and not have the ‘me too’ or herd mentality that Sir James found so antithetical to scientific discovery. JCM and RAB have declared no potential conflicts.

True myelomeningocele with exposed placode: unusual presentation of cervical myelomeningocele

Midline cystic lesions in cervical area are quite different in structure and cord function from typical myelomeningoceles (MMCs) occurring in lower spinal levels. Previously we have published the largest series of cervical MMCs from a single center. We referred to these lesions as MMCs because they all contained nervous tissue, and we classified them into 2 subgroups—fibroneurovascular stalk and myelocystocele.1 Some authors have recognized them as syringoceles, syringomyeloceles, or limited dorsal myeloschisis.2–4 Although cervical MMCs contain neural tissue inside the sac, they differ from MMCs in lower locations in that preservation of the neural tissue is recommended. Recently we encountered a case that was markedly different from previous ones in general and closely resembled MMCs in the lower levels. This report was written to consider an exception to the rule explained in our previous manuscript and to add some surgical points to our previous recommendations. This 2-day-old female infant was referred to us for treatment of a cervical MMC with CSF leakage. The cervical lesion contained an exposed placode centrally, with CSF leakage from its thin sac (Fig. 1). Brain ultrasound revealed Chiari malformation Type II associated with hydrocephalus. Surgery was performed through the standard approach. There was a big defect of the fascia and posterior elements of the cervical spine at the C3–5 levels. The cord was protruding through the dural defect, exiting from the canal, coming to the placode, and then entering the canal distally (Fig. 2). The tips of the cerebellar tonsils were observed proximal to the dural opening. All neural tissues were preserved and pia-to-pia closure of the neural placode converted it into tubule. (DOI: 10.3171/ 2010.1.PEDS09147)

The influence of different components on interpenetrating polymer network's (IPN's) characteristics as automotive top coats

The aim of this study was to investigate the influence of different components of interpenetrating polymer networks (IPNs) on their behavior in dual curing automotive applications. Dual curing is one of possible ways to obtain fast curing, scratch resistant coatings for use in OEM and car refinish applications. Dual curing systems, upon hardening, represent interpenetrating networks (IPNs) [1]. IPN's were obtained using novel acrylate-terminated hyperbranched polyester with high functionality and compared to classical 2-pack polyurethane clear coat. In our previous manuscript [1] we investigated the influence of the acrylated hyperbranched polyester (HBP(A)) content in dual curing systems. In this article we studied the influence of the reactive diluent on dual curing compositions. To this end, we have chosen IPN with 50/50 weigth ratio of polyurethane (PU) and polyacrylic (PA) component. In order to determine more clearly the role of the HBP(A) in one IPN HBP(A) was changed with hexane diol diacrylate (HDDA), keeping the same ratio with EHA. The IPNs were characterized by dynamic-mechanical analysis (DMA), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The mechanical properties of the IPNs were also investigated. The IPN with HDDA as reactive diluent, had the best performance. IPN with HBP(A) had the highest glass transition temperature Tg and the highest crosslink density, but it did not have the highest hardness.

II. Slow Oscillations in Vaginal Blood Flow: Regulation of Vaginal Blood Flow Patterns in Rat by Central and Autonomic Mechanisms

A new method for assessing female sexual arousal through changes in slow oscillatory patterns in vaginal blood flow was first described in the previous manuscript [1]. This method was translational and discriminated between normal healthy volunteers and women with female sexual arousal disorder. These studies addressed the influence of autonomic and central nervous systems on slow vaginal blood flow oscillations in rats. Vaginal blood flow oscillations were measured in urethane-anesthetized rodents using laser Doppler flowmetry. Acquired data were filtered for frequency analysis range of 0.013-2.5 Hz. Data were assessed for changes in a high frequency range (HF = 0.6-2.5 Hz), and low frequency range (LF = 0.013-0.6 Hz). The basal HF oscillatory component of vaginal blood flow was primarily vagally mediated, although could be modulated pharmacologically with p-chloroamphetamine in the absence of vagal innervation. The LF component could be modulated by antagonists of noradrenergic receptors but did not appear to be dependent upon tonic activation of sympathetic circuitry. The non-selective dopamine receptor agonist apomorphine induced changes in vaginal blood flow oscillations consistent with sexual arousal during metestrus in the presence of the peripheral antagonist domperidone but not in the presence of the centrally acting antagonist haloperidol. Electrical stimulation of the paraventricular nucleus (PVN) of the hypothalamus induced an anti-arousal response in vaginal blood flow oscillations. These data demonstrated that manipulation of the central nervous system alone (via centrally acting apomorphine or electrical stimulation of the PVN) could produce either a pro-arousal or an anti-arousal response in vaginal blood flow oscillations. Alterations in the LF/HF ratio measured from vaginal laser Doppler flowmetry were independently regulated from vasculature in the trunk, the tongue, and electrocardiogram-derived heart rate variability, and were independent of overall vasocongestion of the vagina as measured by mean blood flow. These data indicated that slow oscillations in vaginal blood flow from rodents may be utilized as an animal model of female sexual arousal. Changes in these oscillations are driven by the central nervous system and modulated by the autonomic nervous system.

Assessment of a Large Subsurface Controlled Drainage and Irrigation System: II. Corn Performance

Abstract Controlled subsurface drainage irrigation systems have been designed to promote agronomic production by optimizing water availability. In a previous manuscript we described the design of a 40 ha controlled subsurface drainage irrigation system, the soil resource and indicated the soil water properties. In this manuscript we describe the performance of corn (Zea mays L.) using a controlled subsurface drainage/irrigation system, with a focus on nutrient uptake at black layer formation. In a subsequent manuscript we will describe nutrient concentrations from tile drain effluents and note their potential impact on surface water resources. Crop yields using the controlled subsurface drainage/irrigation system substantially increased grain yields in 2008, 2011 and 2012 relative to previous corn production prior to the installation of the controlled subsurface drainage/irrigation system. Nutrient uptake (N, P, K, Ca, Mg, S, Fe, Mn, B, Cu, and Zn) was partitioned into leaf blades (blades), leaf sheaths (sheaths), culm (stem), tassel, ear leaves, shank, cob and grain. Nutrient concentrations in plant parts were estimated using plant tissue analysis, plant populations and dry matter production and expressed on a field basis (kg ha−1). The nutrient uptake by plant part at black layer showed that N, P, and S were more than 50% vested with grain. The remaining nutrients were primarily associated with the non-grain plant parts, especially K, Ca and B.

Retraction

RETRACTIONRetractionN. E. Brooks, M. D. Schuenke, and R. S. HikidaN. E. Brooks, M. D. Schuenke, and R. S. HikidaPublished Online:01 Dec 2009https://doi.org/10.1152/japplphysiol.00131.2009-retr.2009Retracted articleMoreSectionsPDF (63 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Brooks NE, Schuenke MD, Hikida RS. Aging attenuates muscle responsiveness to creatine supplementation, but not overload, in rat plantaris muscles. J Appl Physiol (First published May 14, 2009). doi: 10.1152/japplphysiol.00131.2009—It is with sincere regret that Brooks et al. report the retraction of this manuscript. Some data were inaccurately duplicated control animal data from a previous manuscript. In the first manuscript, data were reported exactly as they were collected for CSA and myonuclear domain size. In the latter manuscript, statistical outliers were removed, resulting in data disagreement between the manuscripts. We inadvertently neglected to mention the outlier removal. Upon being questioned on this issue by APS, we returned the outlying data to the manuscript. At that point, some of our conclusions were no longer supported. In addition, reporting of a subset of data (young and old controls) for body weight, muscle weight, muscle-to-body weight ratio, and fiber type distribution in the Journal of Applied Physiology was not fully acknowledged. At the request of APS, we are dutifully retracting the paper.We apologize to the American Physiological Society for the inconvenience. We hold the Journal of Applied Physiology in high regard and assure you these errors were not intentional or of deceptive intent. We further apologize to our colleagues for any difficulties caused by the publication of this paper.This article has no references to display. Previous Back to Top Next FiguresReferencesRelatedInformationRelated articlesRetraction 01 Dec 2009Journal of Applied Physiology More from this issue > Volume 107Issue 6December 2009Pages 1989-1989 Copyright & PermissionsCopyright © 2009 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00131.2009-retr.2009PubMed19443749History Published online 1 December 2009 Published in print 1 December 2009 Metrics Downloaded 60 times

Synthesis and characterization of fluorinated magnetic core–shell nanoparticles forinhibition of insulin amyloid fibril formation

Maghemite (γ-Fe2O3) magneticnanoparticles of 15.0 ± 2.1 nm are formed by nucleation followed by controlled growth ofmaghemite thin films on gelatin-iron oxide nuclei. Uniform magneticγ-Fe2O3/poly (2,2,3,3,4,4,4-heptafluorobutylacrylate) (γ-Fe2O3/PHFBA) core–shell nanoparticles are prepared by emulsion polymerization of the fluorinated monomer2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles.The kinetics of the insulin fibrillation process in the absence and in the presence of theγ-Fe2O3/PHFBA core–shell nanoparticles are elucidated. A significant direct slow transition fromα-helixto β-sheets during insulin fibril formation is observed in the presence of theγ-Fe2O3/PHFBA nanoparticles. This is in contradiction to our previous manuscript, which illustrated that theγ-Fe2O3 core nanoparticles do not affect the kinetics of the formation of the insulin fibrils, and toother previous publications that describe acceleration of the fibrillation process by usingvarious types of nanoparticles. These core–shell nanoparticles may therefore be also usefulfor the inhibition of conformational changes of other amyloidogenic proteins that lead toneurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, mad cow andprion diseases.

A028 Il-10 restores the vascular hyporeactivity induced by lipopolysaccharide in tissue-engineered blood vessel models

Sepsis is an archetypal condition with molecular links between inflammation and coagulation. Both events can be orchestrated by the interaction between circulating and vascular cells that under activation release microparticles. Recently, we reported that increased circulating microparticles in septic patients play a pivotal role in ex vivo vascular function suggesting that they are protective against vascular hyporeactivity by maintaining a tonic pressor response (Mostefai et al. Am J Resp Crit Care Med 2008). Besides, we further demonstrated these effects of septic microparticles in human tissue-engineered blood vessel models by comparing the contractile response to histamine of two vascular constructs from human vascular smooth muscle cells and fibroblasts. The first model is composed of a media solely (TEVM), and the second contained a media and an adventitia (TEVMA). Indeed, we showed that septic microparticles increased contraction induced by histamine, in vascular constructs from TEVM but not in TEVMA. This effect was linked to their capacity to decrease vasodilators components, from inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), and increase mRNA expression of IL-10 in TEVM The present study was designed to address the question whether IL-10 is the mediator implicated in the protective effect of septic microparticles against vascular hyporeactivity observed in sepsis using the two vascular constructs, TEVM and TEVMA. For this purpose, the effect of IL-10 was investigated in the two constructs in presence or absence of lipopolysaccharide (LPS). IL-10 treatment did not modify the contractile response in both TEVM and TEVMA compared to non-treated vessels. LPS treatment induced vascular hyporeactivity in TEVM but it did not modify the contractile response in TEVMA compared to non-treated vessels. Interestingly, IL-10 restores contraction in LPS-treated TEVM towards control vessels. The mechanisms by which IL-10 enhanced the response are under investigation in relation with the COX vasoconstrictor metabolite, thromboxane A2, as reported in our previous manuscript (Mostefai et al. 2008). These data demonstrate that IL-10 restores the vascular hyporeactivity induced by LPS in tissue-engineered blood vessel models.

Comparison of real-time PCR and nested PCR for the detection of Y chromosome sequences in the peripheral blood mononuclear cells of patients with systemic sclerosis

Real-time PCR (RT-PCR) is a sensitive technique that has been recently applied to the determination and quantification of fetomaternal microchimerism.1–3 In a previous manuscript, we detected the presence of Y...

Evaluation of the effect of chronic hydrodynamical stresses on cultures of suspensed CHO‐6E6 cells

The effect of hydrodynamic forces on animal cell cultures, while extensively studied, still lacks significant, fundamental understanding. A previous manuscript reported on the acute exposure of CHO cells to hydrodynamic forces in a second generation convergent-divergent microfluidic device (Mollet et al., 2007). In this study, the use of this device is extended in a proof of concept system in which suspended animal cells, grown in a typical bioreactor, are subjected to chronic exposure of moderately high levels of hydrodynamic forces by way of a continuous recycle loop between the bioreactor and the microfluidic device. A strain of CHO cells (CHO-6E6) was grown in a batch culture under controlled pH, temperature, and dissolved oxygen conditions. At mid exponential stage of growth in the bioreactor the recycle flow was initiated. The cells either stopped growing or started dying at EDR values that were significantly lower (one to two orders of magnitude) than those previously reported to kill cells from a single, acute exposure. These observations allow further refinement in the design of bioprocess equipment since it provides a more accurate threshold, above which one does not want to subject animal cells to continuous exposure to specific levels of hydrodynamic forces.

The Safety of Fresh Frozen Plasma for the Treatment of Hereditary Angioedema

Fresh frozen plasma (FFP) has been used as a treatment option for patients with Hereditary Angioedema (HAE); however, it has been hypothesized to have the potential to worsen an acute attack of HAE. In a previous manuscript, we reviewed patient records at one institution and the medical literature which suggested that FFP is safe and effective in the treatment of HAE. Now, the aim is to present the data at two separate institutions regarding the treatment of HAE with FFP.

Erratum to: Detection of Chlamydia in the peripheral blood cells of normal donors using in vitro culture, immunofluorescence microscopy and flow cytometry techniques

Upon close examination of the representative image depicted in 'figure 2' from [1] and a figure published elsewhere [2], we realized that the 'figure 2' of this work was a lower magnification of a section of the same microscope slide used in the previous manuscript [2]. The experiments leading to the data for these two manuscripts were generated during the same time period, in the same laboratory and were compiled, annotated and stored in the same folder, since pictures are taken in bulk at our microscopy facility on a fee for service basis. The figures both describe tissue culture of Chlamydia from clinical samples on the same tissue culture substrate (J774A.1 cells) and were in a single data base of figures with different alpha numeric designations. We believe that sometime during the compilation and annotation process, the representative 'figure 2' previously shown in this work was incorrectly labeled. After careful scrutiny, we have determined that the image was in fact taken from a culture coverslip that was infected with lysates from pediatric bronchial lavage samples and not normal blood donor buffy coat (BC) samples. The original published figure was a 600× immunofluorescence image [2], part of which can be seen in the lower magnification image (400×) in this work. The authors deeply regret this error in image annotation and later image selection and therefore submit a representative image from this work to replace the earlier incorrect image depicted in 'figure 2', which is provided with this manuscript (see figure ​figure11). Figure 1 This image is provided as a replacement for the original figure 2; Chlamydiae within Peripheral Blood Transfer Infection when Cultured in vitro on J774A.1 host cell monolayers. BC from NBD peripheral blood samples were isolated, lysed and layered onto ...

Evolution of Enzymatic Activities in the Orotidine 5‘-Monophosphate Decarboxylase Suprafamily: Structural Basis for Catalytic Promiscuity in Wild-Type and Designed Mutants of 3-Keto-l-gulonate 6-Phosphate Decarboxylase

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and D-arabino-hex-3-ulose 6-phosphate synthase (HPS), members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily, catalyze reactions that involve the formation of Mg(2+)-ion stabilized 1,2-enediolate intermediates. The active sites of KGPDC and HPS share several conserved residues, including the presumed ligands for the Mg(2+) and a catalytic histidine residue that has been implicated in protonation of the intermediate in the KGPDC-catalyzed reaction. As reported in the previous manuscript, both enzymes are naturally promiscuous, with KGPDC from Escherichia coli catalyzing a low level of the HPS reaction and the HPS from Methylomonas aminofaciens catalyzing a significant level of the KGPDC reaction. Interestingly, the promiscuous HPS reaction catalyzed by KGPDC can be significantly enhanced by replacing no more than four active site residues from KGPDC reaction with residues from HPS. In this manuscript, we report structural studies of wild-type and mutant KDGPC's that provide a structural explanation for both the natural promiscuity for the HPS reaction and the enhanced HPS activity and diminished KGPDC activity catalyzed by active site mutants.

Reanalysis of the testosterone displacement from its HSA binding site by DHEA using competitive Langmuir isotherm

In a previous manuscript [C. Andre, A. Berthelot, J.F. Robert, M. Thomassin, Y.C. Guillaume, J. Pharm. Bio. Med., in press], the Mg 2+ effect on the testosterone displacement equilibrium from its human serum albumin (HSA) binding site by DHEA was investigated using a thermodynamic approach. In this paper, a novel concept based on the competitive Langmuir distribution isotherms was proposed to calculate the association constant of the HSA–hormone binding and to confirm the Mg 2+ role on the testosterone displacement equilibrium from its HSA binding site by DHEA. Thus, both the HSA–hormone binding and the displacement equilibrium processes were reanalysed. The results obtained confirmed that: – DHEA and testosterone bound on the same HSA site; – DHEA displaced well testosterone to its HSA binding site; – Mg 2+ cation led a change on both the HSA–hormone binding and the displacement equilibrium at a critical Mg 2+ concentration x c around 1 mM.