Arginine-aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro.
(33/15061)
HIV gene expression is crucially dependent on binding of the viral Tat protein to the transactivation RNA response element. A number of synthetic Tat-transactivation responsive element interaction inhibitors of peptide/peptoid nature were described as potential antiviral drug prototypes. We present a new class of peptidomimetic inhibitors, conjugates of L-arginine with aminoglycosides. Using a gel-shift assay and affinity chromatography on an L-arginine column we found that these compounds bind specifically to the transactivation responsive element RNA in vitro with Kd values in the range of 20-400 nM, which is comparable to the Kd of native Tat bound to the transactivation responsive element (10-12 nM). Confocal microscopy studies demonstrated that fluorescein-labelled conjugate penetrates into live cells. High affinity to the transactivation responsive element, low toxicity, and relative simplicity of synthesis make these compounds attractive candidates for antiviral drug design. (+info)
The effect of mannitol versus dimethyl thiourea at attenuating ischemia/reperfusion-induced injury to skeletal muscle.
(34/15061)
OBJECTIVE: Mannitol is used as a treatment for skeletal muscle ischemia/reperfusion (I/R) injury in humans, despite the fact that its effectiveness in vivo is still disputed. The purpose of this study was to determine the efficacy of mannitol in attenuating I/R injury at the microcirculatory level. METHODS: The study was designed as an experimental study with male Wistar rats. The main outcome measures were intravital microscopy, which was used to measure capillary perfusion, capillary and venular red blood cell velocity (VRBC), and leukocyte-endothelial interactions in the extensor digitorum longus muscle of the rat hind limb before and after ischemia. In addition, tissue injury was assessed during reperfusion with the fluorescent vital dyes bisbenzimide and ethidium bromide. Dimethyl thiourea (DMTU), a highly effective therapeutic agent of experimental I/R injury, was used as a positive control. RESULTS: No-flow ischemia (2 hour) resulted in a 40% drop in capillary perfusion, a decline in capillary and venular VRBC, and increased leukocyte venular adherence and tissue infiltration. Tissue injury increased to a constant level during reperfusion. Mannitol attenuated capillary malperfusion during the first 60 minutes of reperfusion and prevented a decline in capillary VRBC. However, mannitol did not reduce tissue injury or leukocyte adherence and infiltration during reperfusion. By comparison, DMTU not only prevented the perfusion deficits and the increases in leukocyte venular adherence and tissue infiltration but significantly reduced the magnitude of tissue injury. CONCLUSION: Our findings suggest that mannitol may be of limited value for the prevention of early reperfusion-induced injury after no-flow ischemia in skeletal muscle. By comparison, DMTU was highly efficacious by not only reducing microvascular perfusion deficits but by also reducing leukocyte-endothelial cell interactions and the incidence of cellular injury. (+info)
Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.
(35/15061)
Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units. (+info)
Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging.
(36/15061)
The fluorescence of quinolinium-based Cl- indicators such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl- by a collisional mechanism without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl--sensitive moiety [excitation wavelength (lambdaex) 350 nm, emission wavelength (lambdaem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl--insensitive moiety because of its different spectral characteristics (lambdaex 380 nm, lambdaem 546 nm), insensitivity to Cl-, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl--sensitive indicators were alpha-MQ-alpha'-dimethyl-AQ-xylene dichloride and trans-1, 2-bis(4-[1-alpha'-MQ-1'-alpha'-dimethyl-AQ-xylyl]-pyridinium)ethyl ene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl- sensitive; Stern-Volmer constants 82 and 98 M-1) and 565 nm (Cl- insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 microM, 10 min). Changes in Cl- concentration in response to Cl-/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl- indicators for ratiometric measurement of Cl- concentration. (+info)
Distribution of 5-chloromethylfluorescein diacetate staining during meiotic maturation and fertilization in vitro of mouse oocytes.
(37/15061)
The aim of this confocal microscopy study was to determine whether the pattern of CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) staining changes during meiotic maturation and fertilization in vitro of mouse oocytes. At different times during meiotic maturation and fertilization, oocytes, zygotes and two-cell embryos were stained with CMFDA to demonstrate intracellular glutathione S-transferase activity. After washing in CMFDA-free medium, most oocytes, zygotes and embryos were stained with dihydroethidium (HE) to visualize DNA structures. Meiotic maturation and fertilization in vitro of mouse oocytes were associated with changes in the pattern of intracellular CMFDA staining. In particular, accumulations of CMFDA-positive membranes were observed around the nucleus of germinal vesicle (GV) oocytes, overlaying the sperm nucleus as well as overlaying the first mitotic spindle if this approached the plasma membrane. Staining of oocytes and zygotes with the probes 3,3'-dihexyloxacarbocyanine iodine [DiOC6(3)], which stains all the intracellular membranes, and rhodamine 123, which stains active mitochondria, demonstrated that the intracellular structures evidenced by CMFDA staining did not correspond to accumulations of mitochondria. Exposure of oocytes and zygotes to the microtubule-disrupting agent nocodazole or the actin-depolymerizing drug cytochalasin D revealed an autonomous microfilament-dependent transport and relocation of CMFDA-positive membranes during meiotic maturation and fertilization. Such a transport of CMFDA-positive membranes may be envisaged as a protective shield built to prevent damage to DNA from endogenous and exogenous mutagen metabolites. (+info)
Characterization of nodular neuronal heterotopia in children.
(38/15061)
Neuronal heterotopia are seen in various pathologies and are associated with intractable epilepsy. We examined brain tissue from four children with subcortical or periventricular nodular heterotopia of different aetiologies: one with severe epilepsy following focal brain trauma at 17 weeks gestation, one with hemimegalencephaly and intractable epilepsy, one with focal cortical dysplasia and intractable epilepsy, and one dysmorphic term infant with associated hydrocephalus and polymicrogyria. The connectivity of nodules was investigated using histological and carbocyanine dye (DiI) tracing techniques. DiI crystal placement adjacent to heterotopic nodules revealed numerous DiI-labelled fibres within a 2-3 mm radius of the crystals. Although we observed labelled fibres closely surrounding nodules, the majority did not penetrate them. Placement of DiI crystals within nodules also identified a limited number of projections out of the nodules and in one case there was evidence for connectivity between adjacent nodules. The cellular and neurochemical composition of nodules was also examined using immunohistochemistry for calretinin and neuropeptide Y (NPY), which are normally expressed in GABAergic cortical interneurons. Within heterotopic nodules from all cases, numerous calretinin-positive neurons were identified, along with a few cell bodies and many processes positive for NPY. Calretinin-positive neurons within nodules were less morphologically complex than those in the cortex, which may reflect incomplete differentiation into an inhibitory neuronal phenotype. There were also abnormal clusters of calretinin-positive cells in the overlying cortical plate, indicating that the migratory defect which produces heterotopic nodules also affects development of the cortex itself. Thus, heterotopic nodules consisting of multiple neuronal cell types are associated with malformation in the overlying cortical plate, and have limited connectivity with other brain regions. This abnormal development of connectivity may affect neuronal maturation and consequently the balance of excitation and inhibition in neuronal circuits, leading to their epileptogenic potential. (+info)
Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis.
(39/15061)
Porcine alveolar macrophages (AMphi) are the dominant cell type that supports the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro. In order to determine the characteristics of the virus-receptor interaction, the attachment of PRRSV to cells was examined by using biotinylated virus in a series of flow cytometric assays. PRRSV bound specifically to AMphi in a dose-dependent manner. Binding of PRRSV to AMphi increased gradually and reached a maximum within 60 min at 4 degrees C. By confocal microscopy, it was shown that different degrees of PRRSV binding exist and that entry is by endocytosis. Virus uptake in vesicles is a clathrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH4Cl and chloroquine, it was demonstrated that PRRSV uses a low pH-dependent entry pathway. In the presence of these reagents, input virions accumulated in large vacuoles, indicating that uncoating was prevented. These results indicate that PRRSV entry into AMphi involves attachment to a specific virus receptor(s) followed by a process of endocytosis, by which virions are taken into the cell within vesicles by a clathrin-dependent pathway. A subsequent drop in pH is required for proper virus replication. (+info)
Identification of megalin/gp330 as a receptor for lipoprotein(a) in vitro.
(40/15061)
Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein of unknown physiological function. The mechanism of Lp(a) atherogenicity as well as its catabolic pathways are only incompletely understood at present. In this report, we show that the low density lipoprotein receptor (LDLR) gene family member megalin/glycoprotein (gp) 330 is capable of binding and mediating the cellular uptake and degradation of Lp(a) in vitro. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient in LDLR-related protein (LRP) and a control cell line carrying a double knockout for both LRP and megalin/gp330 were compared with regard to their ability to bind, internalize, and degrade dioctadecyltetramethylindocarbocyanine perchlorate (DiI)-fluorescence-labeled Lp(a) as well as equimolar amounts of 125I-labeled Lp(a) and LDL. Uptake and degradation of radiolabeled Lp(a) by the megalin/gp330-expressing cells were, on average, 2-fold higher than that of control cells. This difference could be completely abolished by addition of the receptor-associated protein, an inhibitor of ligand binding to megalin/gp330. Mutual suppression of the uptake of 125I-Lp(a) and of 125I-LDL by both unlabeled Lp(a) and LDL suggested that Lp(a) uptake is mediated at least partially by apolipoprotein B100. Binding and uptake of DiI-Lp(a) resulted in strong signals on megalin/gp330-expressing cells versus background only on control cells. In addition, we show that purified megalin/gp330, immobilized on a sensor chip, directly binds Lp(a) in a Ca2+-dependent manner with an affinity similar to that for LDL. We conclude that megalin/gp330 binds Lp(a) in vitro and is capable of mediating its cellular uptake and degradation. (+info)