Nitric oxide regulates angiotensin-I converting enzyme under static conditions but not under shear stress

Mechanical forces including pressure and shear stress play an important role in vascular homeostasis via the control of the production and release of a variety of vasoactive factors. An increase in vascular shear stress is accompanied by nitric oxide (NO) release and NO synthase activation. Previously, we have demonstrated that shear stress induces angiotensin-I converting enzyme (ACE) down-regulation in vivo and in vitro. In the present study, we determined whether NO participates in the shear stress-induced ACE suppression response. Rabbit aortic endothelial cells were evaluated using the NO synthase inhibitor L-NAME, and two NO donors, diethylamine NONOate (DEA/NO) and sodium nitroprusside (SNP). Under static conditions, incubation of endothelial cells with 1 mM L-NAME for 18 h increased ACE activity by 27% (from 1.000 ± 0.090 to 1.272 ± 0.182) while DEA/NO and SNP (0.1, 0.5 and 1 mM) caused no change in ACE activity. Interestingly, ACE activity was down-regulated similarly in the presence or absence of L-NAME (delta(0 mM) = 0.26 ± 0.055, delta(0.1 mM) = 0.21 ± 0.22, delta(1 mM) = 0.36 ± 0.13) upon 18 h shear stress activation (from static to 15 dyn/cm²). Taken together, these results indicate that NO can participate in the maintenance of basal ACE levels in the static condition but NO is not associated with the shear stress-induced inactivation of ACE.

Stability Analysis in Multicriteria Discrete Portfolio Optimization.

Almost every problem of design, planning and management in the technical and organizational systems has several conflicting goals or interests. Nowadays, multicriteria decision models represent a rapidly developing area of operation research. While solving practical optimization problems, it is necessary to take into account various kinds of uncertainty due to lack of data, inadequacy of mathematical models to real-time processes, calculation errors, etc. In practice, this uncertainty usually leads to undesirable outcomes where the solutions are very sensitive to any changes in the input parameters. An example is the investment managing. Stability analysis of multicriteria discrete optimization problems investigates how the found solutions behave in response to changes in the initial data (input parameters). This thesis is devoted to the stability analysis in the problem of selecting investment project portfolios, which are optimized by considering different types of risk and efficiency of the investment projects. The stability analysis is carried out in two approaches: qualitative and quantitative. The qualitative approach describes the behavior of solutions in conditions with small perturbations in the initial data. The stability of solutions is defined in terms of existence a neighborhood in the initial data space. Any perturbed problem from this neighborhood has stability with respect to the set of efficient solutions of the initial problem. The other approach in the stability analysis studies quantitative measures such as stability radius. This approach gives information about the limits of perturbations in the input parameters, which do not lead to changes in the set of efficient solutions. In present thesis several results were obtained including attainable bounds for the stability radii of Pareto optimal and lexicographically optimal portfolios of the investment problem with Savage's, Wald's criteria and criteria of extreme optimism. In addition, special classes of the problem when the stability radii are expressed by the formulae were indicated. Investigations were completed using different combinations of Chebyshev's, Manhattan and Hölder's metrics, which allowed monitoring input parameters perturbations differently.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies

A thorough understanding of protein structure and stability requires that we elucidate the molecular basis for the effects of both temperature and pressure on protein conformational transitions. While temperature effects are relatively well understood and the change in heat capacity upon unfolding has been reasonably well parameterized, the state of understanding of pressure effects is much less advanced. Ultimately, a quantitative parameterization of the volume changes (at the basis of pressure effects) accompanying protein conformational transitions will be required. The present report introduces a qualitative hypothesis based on available model compound data for the molecular basis of volume change upon protein unfolding and its dependence on temperature.

The powerful high pressure tool for protein conformational studies

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2º, 3º and 4º structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

Nucleotides as Antiviral Compounds. On the Feasibility of an Esterase-dependent Prodrug Strategy for 2-5A

Nukleotidien ja oligonukleotidien analogeilla on merkittävä rooli virusten aiheuttamien tautien hoidossa. Tämän kaltaiset yhdisteet voivat estää spesifisesti virusten proteiineja tai aktivoida luontaista immuunijärjestelmää, jossa 2-5A:ksi kutsutut lyhyet 2´,5´-sitoutuneet oligomeerit ovat keskeisiä tekijöitä. Nukleotideihin ja oligonukleotideihin pohjautuvien lääkkeiden tehokkuus riippuu pääasiassa aihiolääkestrategiasta, jolla niiden sisäänottoa soluun tehostetaan. Tavanomaisessa aihiolääkestrategiassa negatiivisesti varautuneet fosfaattiryhmät suojataan rasvaliukoisilla biohajoavilla suojaryhmillä, jotta molekyyli läpäisee solukalvon helpommin. Solun sisällä aihiolääke muuttuu aktiiviseksi lääkeaineeksi, kun suojaryhmät irtoavat solun entsyymien, kuten esteraasien vaikutuksesta. Väitöskirjassa arvioitiin esteraasin katalysoiman aihiolääkestrategian soveltuvuutta 2-5A-trimeerille syntetisoimalla kaksi erilaista 2-5A-aihiolääkekandidaattia ja tutkimalla 2-5A:n purkautumista karboksiesteraasi-entsyymin vaikutuksesta. Suojaryhmäsuunnitelma perustui esteraasilabiileihin 2,2-disubstituoituihin asyylioksipropyyliryhmiin ja asyylioksimetyyliryhmiin, joilla suojattiin trimeerien fosfaatti- ja 3´-hydroksyyliryhmät. Tulokset osoittivat, että esteraasilabiilien suojaryhmien irtoaminen 2-5A:sta hidastui merkittävästi, kun yhdisteeseen kertyi negatiivista varausta. Lisäksi suojaryhmien hajotessa muodostui elektrofiilisiä alkyloivia aineita, jotka ovat mahdollisesti toksisia. Näistä syistä johtuen kehitettiin kuusi uudenlaista 2,2,-disubstituoitua 4-asyylitio- 3-oksobutyyliryhmää fosfodiestereiden suojaamiseksi. Suojaryhmät irtoavat sekä esteraasin katalysoimana, että lämpötilan vaikutuksesta. Tämä on hyödyllinen ominaisuus silloin, kun entsyymin affiniteetti negatiivisesti varattuun substraattiin heikkenee. Suojaryhmien hydrolyyttinen ja entsymaattinen stabiilisuus on helposti säädeltävissä, jotta suojauksen purkautumisen nopeus voidaan optimoida. Vapautuneet suojaryhmät eivät ole merkittävästi alkyloivia, sillä niiden ei havaittu alkyloivan glutationia.

Cytosine arabinoside-metabolizing enzyme genes are underexpressed in children with MLL gene-rearranged acute lymphoblastic leukemia

Infant acute lymphoblastic leukemia (IALL) is characterized by mixed lineage leukemia (MLL) gene rearrangements, unique gene expression profiles, poor prognosis, and drug resistance. One exception is cytosine arabinoside (Ara-C) to which IALL cells seem to be more sensitive. We quantified mRNA expression of Ara-C key enzymes in leukemic lymphoblasts from 64 Brazilian ALL children, 15 of them presenting MLL gene rearrangement, and correlated it with clinical and biological features. The diagnosis was based on morphological criteria and immunophenotyping using monoclonal antibodies. MLL gene rearrangements were detected by conventional cytogenetic analysis, RT-PCR and/or fluorescence in situ hybridization. The DCK and HENT1 expression levels were determined by real-time quantitative PCR using SYBR Green I. Relative quantification was made by the standard curve method. The results were analyzed by Mann-Whitney and Fisher exact tests. A P value of £0.05 was considered to be statistically significant. DCK and HENT1 expression levels were significantly lower in children with MLL gene-rearranged ALL compared to children with MLL germ line ALL (P = 0.0003 and 0.03, respectively). Our results differ from previous ones concerning HENT1 mRNA expression that observed a higher expression level in MLL gene-rearranged leukemias. In conclusion, the expression of the genes related to Ara-C metabolism was lower in MLL-positive children in the sample studied, suggesting the presence of population differences in the expression profile of these genes especially for HENT1.

Reduced plasma levels of angiotensin-(1-7) and renin activity in preeclamptic patients are associated with the angiotensin I- converting enzyme deletion/deletion genotype

The relationship between preeclampsia and the renin-angiotensin system (RAS) is poorly understood. Angiotensin I-converting enzyme (ACE) is a key RAS component and plays an important role in blood pressure homeostasis by generating angiotensin II (Ang II) and inactivating the vasodilator angiotensin-(1-7) (Ang-(1-7)). ACE (I/D) polymorphism is characterized by the insertion (I) or deletion (D) of a 287-bp fragment, leading to changes in ACE activity. In the present study, ACE (I/D) polymorphism was correlated with plasma Ang-(1-7) levels and several RAS components in both preeclamptic (N = 20) and normotensive pregnant women (N = 20). The percentage of the ACE DD genotype (60%) in the preeclamptic group was higher than that for the control group (35%); however, this percentage was not statistically significant (Fisher exact test = 2.86, d.f. = 2, P = 0.260). The highest plasma ACE activity was observed in the ACE DD preeclamptic women (58.1 ± 5.06 vs 27.6 ± 3.25 nmol Hip-His Leu-1 min-1 mL-1 in DD control patients; P = 0.0005). Plasma renin activity was markedly reduced in preeclampsia (0.81 ± 0.2 vs 3.43 ± 0.8 ng Ang I mL plasma-1 h-1 in DD normotensive patients; P = 0.0012). A reduced plasma level of Ang-(1-7) was also observed in preeclamptic women (15.6 ± 1.3 vs 22.7 ± 2.5 pg/mL in the DD control group; P = 0.0146). In contrast, plasma Ang II levels were unchanged in preeclamptic patients. The selective changes in the RAS described in the present study suggest that the ACE DD genotype may be used as a marker for susceptibility to preeclampsia.

Stability and properties of cellulose-sodium hydroxide solution systems

Since cellulose is a linear macromolecule it can be used as a material for regenerated cellulose fiber products e.g. in textile fibers or film manufacturing. Cellulose is not thermoformable, thus the manufacturing of these regenerated fibers is mainly possible through dissolution processes preceding the regeneration process. However, the dissolution of cellulose in common solvents is hindered due to inter- and intra-molecular hydrogen bonds in the cellulose chains, and relatively high crystallinity. Interestingly at subzero temperatures relatively dilute sodium hydroxide solutions can be used to dissolve cellulose to a certain extent. The objective of this work was to investigate the possible factors that govern the solubility of cellulose in aqueous NaOH and the solution stability. Cellulose-NaOH solutions have the tendency to form a gel over time and at elevated temperature, which creates challenges for further processing. The main target of this work was to achieve high solubility of cellulose in aqueous NaOH without excessively compromising the solution stability. In the literature survey an overview of the cellulose dissolution is given and possible factors contributing to the solubility and solution properties of cellulose in aqueous NaOH are reviewed. Furthermore, the concept of solution rheology is discussed. In the experimental part the focus was on the characterization of the used materials and properties of the prepared solutions mainly concentrating on cellulose solubility and solution stability.

Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals

We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 ± 5.0 vs 16.1 ± 6.0% in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes.

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-β1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (~260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg·kg-1·day-1 (D0.01, N = 14), 1 mg·kg-1·day-1 (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-β1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P ≤ 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-β1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.

Synergistic effect of mycophenolate mofetil and angiotensin-converting enzyme inhibitor in patients with chronic allograft nephropathy

Experimental data and few clinical non-randomized studies have shown that inhibition of the renin-angiotensin system by angiotensin-converting enzyme (ACE) associated or not with the use of mycophenolate mofetil (MMF) could delay or even halt the progression of chronic allograft nephropathy (CAN). In this retrospective historical study, we investigated whether ACE inhibition (ACEI) associated or not with the use of MMF has the same effect in humans as in experimental studies and what factors are associated with a clinical response. A total of 160 transplant patients with biopsy-proven CAN were enrolled. Eighty-one of them were on ACE therapy (G1) and 80 on ACEI_free therapy (G2). Patients were further stratified for the use of MMF. G1 patients showed a marked decrease in proteinuria and stabilized serum creatinine with time. Five-year graft survival after CAN diagnosis was more frequent in G1 (86.9 vs 67.7%; P < 0.05). In patients on ACEI-free therapy, the use of MMF was associated with better graft survival. The use of ACEI therapy protected 79% of the patients against graft loss (OR = 0.079, 95%CI = 0.015-0.426; P = 0.003). ACEI and MMF or the use of MMF alone after CAN diagnosis conferred protection against graft loss. This finding is well correlated with experimental studies in which ACEI and MMF interrupt the progression of chronic allograft dysfunction and injury. The use of ACEI alone or in combination with MMF significantly reduced proteinuria and stabilized serum creatinine, consequently improving renal allograft survival.