Hemoglobina Stanleyville II [alfa78(EF7)Asn → Lys]. Primer caso descrito en España / Hemoglobin Stanleyville II [alpha78(EF7)Asn → Lys]. First case described in Spain

FUNDAMENTO Y OBJETIVO: Las hemoglobinopatías estructurales son el resultado de mutaciones enlos genes de globina que determinan una alteración cualitativa en la expresión de dichos genes.En la mayoría de ellas la alteración estructural no condiciona ningún cambio significativo,por lo que cursan de forma silente o asintomática. En este trabajo presentamos un nuevo casode hemoglobina (Hb) Stanleyville II.PACIENTES Y MÉTODO: El probando es una mujer de 72 años, raza blanca y origen canario. En laanalítica presentaba Hb de 14,3 g/dl, hematocrito del 44,4%, volumen corpuscular medio de85,8 fl, Hb corpuscular media de 27,7 pg y concentración de Hb corpuscular media de 32,2g/l; el índice de anisocitosis era del 15,1%, reticulocitos del 1,2%, HbA2 del 3,1% y HbF del1,6%. En la electroforesis en acetato de celulosa a pH alcalino y en el isoelectroenfoque se separóuna Hb anormal a la altura de la HbS. En agar citrato a pH ácido la Hb anormal no se separabade la HbA. Por cromatografía líquida de alta resolución de fase reversa se eluyó una cadenaalfa anormal más precoz que la alfa normal.RESULTADOS: En el análisis molecular, que se completó con la secuenciación de los productos deamplificación por reacción en cadena de la polimerasa de los genes alfa1 y alfa2, se demostró lamutación AAC → AAA en el codón 78 del segundo exón del gen alfa2 en estado heterocigoto, quedetermina un cambio de asparagina por lisina.CONCLUSIONES: La sustitución de un aminoácido con carga neutra, como la asparagina, por otrocon carga muy positiva, como la lisina, en el segmento EF, que corresponde a la superficie externade la estructura terciaria de la cadena de globina, determina un cambio neto en la cargade la cadena. Esto permite su fácil diferenciación por métodos electroforéticos y cromatográficos.Sin embargo, como la localización no es fundamental para la estabilidad, solubilidad y afinidadpor el oxígeno del tetrámero, cursa de forma silente o asintomática. La Hb Stanleyville IIse había descrito hasta ahora en familias de raza negra del Congo, Uganda, Zaire, EE.UU., Alsaciay Brasil. Este caso representa el primero descrito en España

BACKGROUND AND OBJECTIVE: Structural hemoglobinopathies are the result of mutations in the genesof globin, which determine a qualitative alteration in the expression of these genes. Mostalterations do not originate any significant change, and correspond to silent or asymptomaticforms. This study proves a new case of hemoglobin (Hb) Stanleyville II.PATIENTS AND METHOD: The propositus was a 72 years old Caucasian woman, from the Canary Islands.Her hematological data were: Hb 14.3 g/dl; hematocrit 44.4%; mean corpuscular volume85.8 fl; mean corpuscular hemoglobin 27.7 pg; red cell distribution width 15.1%; reticulocytes1.2%; HbA2 3.1% and HbF 1.6%. Electrophoretic studies in cellulose acetate electrophoresisat alkaline pH = 8.6 and isoelectrofocusing showed an anomalous Hb similar to HbS. The anomalousHb did not appear in agar citrate electrophoresis (pH 6.0). The analysis by reverse phasehigh perfomance liquid chromatography for globin chains showed an alphaX anomalous after alphaA.RESULTS: Molecular analysis by sequentiation of the polymerase chain reaction products genesalpha1 and alpha2 showed the mutation AAC → AAA at CD78 of second gene alpha2 in heterozygote state,which leads the change of asparagine to lysine.CONCLUSIONS: The substitution of an amino acid with neutral charge like asparagine for anotherone with positive charge like lysine in the segment EF, which corresponds to the external surfaceof the tertiary structure of the chain of globin, determines the change of charge in the chain.This allows an easy differentiation by electrophoretic and chromatographic methods. Nevertheless,owing to its position in the chain, which is not critique for the stability, solubility and affinityfor the oxygen allows for silent or asymptomatic forms. The Hb Stanleyville II had beendescribed before in black families of the Congo, Uganda, USA, Alsace and Brazil. This case representsthe first case described in Spain

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration.

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.

Uremia and insulin resistance: N-carbamoyl-asparagine decreases insulin-sensitive glucose uptake in rat adipocytes.

BACKGROUND: In uremia, urea-derived cyanate reacts with amino groups irreversibly forming carbamoyl amino acids (C-AA) and carbamoyl proteins. Carbamoylated molecules can affect binding and trafficking and alter metabolic pathways. The C-AA role in insulin-sensitive glucose transport has not been explored and may contribute to insulin resistance in uremia. METHODS: Insulin-stimulated glucose uptake by cultured rat adipocytes was measured using both 3-minute and 3-second assays. Adipocytes were incubated for 24 hours in medium containing 0.5 micromol/mL of 15 different C-AA. 125I-insulin binding studies were done. C-asparagine in plasma from 10 uremic patients on continuous ambulatory peritoneal dialysis (CAPD) was measured using high-performance liquid chromatography (HPLC). RESULTS: Insulin-sensitive glucose uptake was reduced 34% by N-carbamoyl-l-asparagine, (N-C-Asn), in a dose-dependent manner with a half-maximally effective concentration of 0.15 micromol/mL. Fourteen other N-carbamoyl-amino acids as well as 0.5 micromol/mL of asparagine did not affect insulin sensitive glucose uptake. N-C-Asn, l-asparagine, and the other N-carbamoyl amino acids (0.5 micromol/mL) had no effect on basal glucose uptake. These data suggest that that N-C-Asn affects the insulin sensitive glucose transporter system. 125I-insulin binding studies demonstrated that N-C-Asn did not alter insulin binding. Glucose uptake measured using a 3-second assay showed that the glucose affinity of the transporter and glucose phosphorylation were not affected. In uremic patients managed by CAPD, the mean free N-C-Asn plasma level was 1.33 micromol/mL. CONCLUSION: These data suggest that N-C-Asn concentration may contribute to the insulin resistance seen in uremia.

Design and synthesis of a series of (2R)-N(4)-hydroxy-2-(3-hydroxybenzyl)-N(1)- [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors.

A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.

Interaction mechanism between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of human serum albumin.

PURPOSE: The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. METHODS: Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. RESULTS: The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: "competitive-like" strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly "independent". CONCLUSIONS: The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.

Asparagine uptake in rat hepatocytes: resolution of a paradox and insights into substrate-dependent transporter regulation.

Extracellular asparagine has previously been shown to markedly stimulate both ornithine decarboxylase and System N-mediated glutamine transport activities in hepatocytes by a transport-dependent mechanism. However, as a weak substrate of its inferred transporter System N, the specific route of asparagine uptake has remained enigmatic. In this study, asparagine transport was studied in detail and shown to be Na+-dependent, Li+-tolerant, stereospecific, and inhibited profoundly by glutamine and histidine. Coupled with competitive inhibition by glutamine (Ki = 2.63+/-1.11 mM), the data indicated that asparagine was indeed slowly transported by System N in rat hepatocytes, albeit at rates an order of magnitude less than for glutamine. The differential substrate transport velocities were shown to be attributable to a low transporter asparagine affinity (Km = 9.3 - 17.5mM) compared to glutamine (Km approximately 1 mM). Consistent with its slow uptake, asparagine accumulated to a fivefold lesser degree than glutamine after 60 min, yet stimulated System N activity to the same extent as glutamine. The transaminase inhibitor aminooxyacetate and starvation of the donor animal each enhanced asparagine uptake twofold and augmented subsequent transporter activation. Conversely, asparagine-dependent System N stimulation was abrogated by hyperosmotic media and blunted 30%-40% by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Collectively, the data suggest that System N-mediated asparagine uptake serves an autostimulatory role, mediated by cellular swelling and in part by a PI3K-dependent signal transduction pathway.

Pharmacokinetic study of a tripeptide HIV-1 protease inhibitor, KNI-174, in rats after intravenous and intraduodenal administrations.

Recently, as a new type of anti-AIDS drug, an HIV-1 protease inhibitor, KNI-174, has been synthesized; it shows a potent and selective HIV-1 protease inhibitory activity in vitro. In this study, we developed an HPLC assay system for KNI-174 in rat plasma and examined the pharmacokinetics of KNI-174 in rats using this assay method after both intravenous (i.v.) and intraduodenal (i.d.) administrations to obtain the disposition characteristics and bioavailability of this new anti-AIDS drug. This HPLC assay method is specific to KNI-174 and the standard curve was linear from 0.02 to 30 micrograms ml-1 plasma. After i.v. administration, 10.0 mg kg-1, KNI-174 disappeared from the rats' plasma in a three-exponential decay. The mean terminal elimination half-life, t1/2 lambda z, was 3.97 +/- 0.19 (S.E.) h, the total body clearance, CLtot, was 9.53 +/- 1.08 ml min-1 and the distribution volume at steady state, Vd,ss, was 7070 +/- 960 ml kg-1. In the case of the i.d. administration, 10.0 mg kg-1, the mean peak plasma concentration, Cmax, and the peak time, tmax, were 0.196 +/- 0.076 micrograms ml-1 and 0.444 +/- 0.193 h, respectively. The bioavailability of KNI-174 till infinity, BA(0-infinity), was 5.37 per cent. Because the IC50 of KNI-174 against HIV-1 in PHA-PBM was 138 ng ml-1, the time needed for maintaining the concentrations above IC50 after a single i.d. administration of KNI-174 is estimated to be 0.350 +/- 0.184 h.

Papel da geracao de oxaloacetato no exercicio fisico moderado em ratos: consequencias da suplementacao de aspartato, asparagina e carnitina / The role of the production of oxaloacetate in the moderate exercise in rats: consequence of the supplementation of aspartate, asparagine and carnitine

A importancia na geracao de oxaloacetato foi investigada atraves da determinacao da atividade da piruvato carboxilase nos musculos estriados e da suplementacao de seus precursores (aspartato e aspargina) na dieta de ratos. A atividade da piruvato carboxilase eleva-se durante o exercicio fisico e, portanto, deve fornecer mais oxaloacetato para a etapa inicial do ciclo de Krebs. A suplementacao cronica (5 semanas) de aspartato e aspargina promove aumento da resistencia ao esforco em ratos treinados em natacao durante 1 hora diaria por 5 semanas. Este efeito foi acompanhado de elevacao no numero e tamanho das mitocondrias e alteracao no metabolismo de glicose dos musculos esqueleticos (elevacao do conteudo de glicogenio e de sua sintese e diminuicao da glicolise). Esses resultados sugerem que a geracao de oxaloacetato desempenha papel fundamental na manutencao do esforco prolongado. A suplementacao de aspartato e aspargina na dieta melhora a performance nessas condicoes, porem causa lesoes na ultraestrutura muscular (mitocondrias, linha "Z" e microfiblilas)

Asparagine transport and the induction of ornithine decarboxylase in H-35 rat hepatoma cells.

A number of amino acids, most noticeably asparagine, were capable of inducing ornithine decarboxylase in H-35 rat hepatoma cells. The effective amino acids were all neutral and were substrates of the Na+-dependent transport systems A, ASC and N. Transport inhibitor studies indicated that there was an excellent correlation between the level of enzyme activity induced and the initial rate of asparagine transport into the cells by the A and the N systems. It is proposed that the activation of the Na+-dependent, pH-sensitive amino acid transport systems and the subsequent intracellular pH and ionic perturbation constitute part of the initiation signals for cell activation.