RESUMEN
Many people newly diagnosed with HIV are lost to follow-up before timely initiation of antiretroviral therapy (ART). A randomised controlled trial (RCT); WelTel Kenya1; demonstrated the effectiveness of the WelTel text messaging intervention to improve clinical outcomes among patients initiating ART. In preparation for WelTel Retain; an RCT that will evaluate the effect of the intervention to retain patients in care immediately following HIV diagnosis; we conducted an informative qualitative study with people living with HIV (n = 15) and healthcare providers (HCP) (n = 5) in October 2012. Study objectives included exploring the experiences of people living with HIV who have attempted to engage in HIV care; the use of cell phones in everyday life; and perceptions of communicating via text message with HCP. Participants were recruited through convenience sampling. Semi-structured; qualitative interviews were conducted and recorded; transcribed verbatim and analysed using NVivo software. Analysis was guided by the Theory of Reasoned Action and the Technology Acceptance Model. Results indicate that while individuals have many motivators for engaging in care after diagnosis; structural and individual barriers including poverty; depression and fear of stigma prevent them from doing so. All participants had access to a mobile phone; and most were comfortable communicating through text messages; or were willing to learn. Both people living with HIV and HCP felt that increased communication via the text messaging intervention has the potential to enable early identification of problems; leading to timely problem solving that may improve retention and engagement in care during the first year after diagnosis
Asunto(s)
Comunicación , Estudios de Seguimiento , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Kenia , Telemedicina/instrumentación , Telemedicina/métodos , Envío de Mensajes de Texto/estadística & datos numéricosRESUMEN
The K+-dependent ATPase activity of the H,K-ATPase was irreversibly inhibited by the carboxyl activating reagent, dicyclohexylcarbodiimide (DCCD). The inhibition was first order and displayed a concentration dependence with the K0.5 (DCCD) = 0.65 +/- 0.04 mM. KCl protected 70% of the ATPase activity from DCCD-dependent inhibition in a concentration-dependent manner with a K0.5 (K+) = 0.58 +/- 0.1 mM KCl. DCCD modification selectively inhibited the K+-dependent rather than ATP-dependent partial reactions including eosin fluorescence responses and ligand-stabilized initial tryptic cleavage patterns of the membrane-associated enzyme. DCCD modification also inhibited the binding of 86Rb+ and the fluorescent responses of the K+-competitive, fluorescent inhibitor 1-(2-methylphenyl)-4-methylamino-6-methyl-2, 3-dihydropyrrolo[3,2-c]quinoline. [14C]DCCD was incorporated into the H,K-ATPase in a time course identical to that describing the inactivation of the K+-dependent ATPase activity of the H,K-ATPase. A component of the [14C]DCCD incorporated into the H,K-ATPase was K+-sensitive where K+ reduced the [14C]DCCD incorporated into the enzyme by 1.6 nmol of [14C]DCCD/mg of protein. Membrane-associated tryptic peptides resolved from the [14C]DCCD-modified H,K-ATPase exhibited various K+ sensitivities with peptides at 23, 9.6, 8.2, 7.1, and 6.1 kDa containing 10-78%, 23-52%, 24-36%, 2%, and 3-4% K+-sensitivity, respectively. The N-terminal sequence of the K+-sensitive, approximately 23- and 9.6-kDa peptides was LVNE857, a C-terminal fragment of the ATPase alpha-subunit. The mass of the smaller peptide limited the residue assignment to the transmembrane M7/M8 domains and an intervening extracytoplasmic loop. An N-terminal sequence, SD840IM, was obtained from a 3.3-kDa, [14C]DCCD-labeled peptide resolved from a V8 digest of the partially purified alpha-subunit. This mass was sufficient to include LVNE but would exclude M8 and the intervening loop between M7 and M8. Glu857 is a unique residue present in each of the proteolytic preparations of the H,K-ATPase modified by [14C]DCCD. These data provide functional evidence of the selective inactivation of the K+-dependent partial reactions of the H,K-ATPase and show that Glu857 located at the M7 boundary in the C terminus of the pump molecule is a significant site of DCCD modification. These data are interpreted to indicate that this carboxyl residue is important for cation binding function.
Asunto(s)
ATPasa Intercambiadora de Hidrógeno-Potásio/química , Estómago/enzimología , Aminoquinolinas/metabolismo , Animales , Diciclohexilcarbodiimida/farmacología , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes/metabolismo , Fragmentos de Péptidos/química , Cloruro de Potasio/metabolismo , Conformación Proteica , Rubidio/metabolismo , Tripsina/metabolismoRESUMEN
86Rb+ binding to the H,K-ATPase was measured in the Mg(2+)-vanadate-inhibited enzyme at 4 degrees C. The concentration dependence of 86Rb+ binding in detergent-free preparations exhibited two components, one saturable with a K0.5 (Rb+) of 0.76 +/- 0.3 mM and a binding capacity of 2626 +/- 690 pmol of Rb+/mg of protein and the second nonsaturable, but linearly dependent, upon the 86Rb+ concentration. The concentration dependence of 86Rb+ binding was unaffected by digitonin treatment with a K0.5 (Rb+) of 0.63 +/- 0.09 mM and a binding capacity of 2824 +/- 152 pmol of Rb+/mg of protein, but the amplitude of the nonsaturable component was eliminated. The level of 86Rb+ binding was optimized by vanadate and decreased by ADP and ATP, suggesting that cation binding is stabilized in the E2-like conformation and antagonized in the E1 conformation. The Rb(+)-dependent stabilization of the E2 enzyme conformation was confirmed from the fluorescent quench response of the fluorescein isothiocyanate (FITC)-labeled enzyme, where 86Rb+ bound to the FITC-labeled enzyme with a K0.5 = 0.85 +/- 0.3 mM and a saturable binding capacity of 2121 pmol of 86Rb+/mg of protein and quenched the FITC fluorescence with a K0.5(Rb+) of 3.6 +/- 0.3 mM. The K(+)-competitive inhibitor, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ), also quenched FITC fluorescence with a K0.5(MDPQ) of 24.5 +/- 0.6 microM and competitively inhibited 86Rb+ binding with a K*0.5 = 35.8 microM (MDPQ). The MDPQ-induced quench of FITC fluorescence at Lys517 within the cytoplasmic M4/M5 nucleotide domain and displacement of 86Rb+ from a functionally defined extracytoplasmic binding domain indicate that structural determinants of the E2 conformational state exist within both cytoplasmic and extracytoplasmic domains of the H,K-ATPase and thus provide evidence of concerted conformational changes between the nucleotide and cation binding domains within the FITC-labeled H,K-ATPase. Membrane-bound fragments of the H,K-ATPase were prepared by tryptic hydrolysis in KCl medium. Tryptic digestion rapidly inactivated the phosphoenzyme site with a time course where k = 0.25 +/- 0.04 min-1 but both 86Rb+ binding and MDPQ fluorescence responses were retained. The concentration dependence of 86Rb+ binding and Rb(+)-dependent MDPQ fluorescence responses in the trypsin-digested membranes were described by a single class of binding sites where K0.5 = 1.2 +/- 0.3 and 0.73 +/- 0.09 mM, respectively. The stability of the Rb+ and MDPQ sites suggest their locations are near or within the membrane and are inaccessible to trypsin attack.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Fragmentos de Péptidos/metabolismo , Rubidio/metabolismo , Secuencia de Aminoácidos , Aminoquinolinas/farmacología , Animales , Unión Competitiva , Fluoresceína-5-Isotiocianato , Cinética , Ligandos , Microsomas/enzimología , Peso Molecular , Inhibidores de la Bomba de Protones , Análisis de Regresión , Radioisótopos de Rubidio , Espectrometría de Fluorescencia , TripsinaRESUMEN
To enhance the intrinsic potency of dihydropyrimidine calcium channel blockers, we have modified the structure of previously described 2-heteroalkyl-1,4-dihydropyrimidines 2 to 3-substituted 1,4-dihydropyrimidines 3. Structure-activity studies using potassium-depolarized rabbit aorta show that ortho, meta-disubstituted aryl derivatives are more potent than either ortho- or meta-monosubstituted compounds. While vasorelaxant activity was critically dependent on the size of the C5 ester group, isopropyl ester being the best, a variety of substituents (carbamate, acyl, sulfonyl, alkyl) were tolerated at N3. Our results show dihydropyrimidines 3 are significantly more potent than corresponding 2-heteroalkyl-1,4-dihydropyrimidines 2 and only slightly less potent than similarly substituted 2-heteroalkyl-1,4-dihydropyridines 4 and 5. Whereas dihydropyridine enantiomers usually show 10-15-fold difference in activity, the enantiomers of dihydropyrimidine 3j show more than a 1000-fold difference in activity. These results strengthen the requirement of an enamino ester for binding to the dihydropyridine receptor and indicate a nonspecific role for the N3-substituent.