Mammalian cell defence mechanisms against the cytotoxicity of NaYF4:(Er,Yb,Gd) nanoparticles.

Water-soluble upconversion nanoparticles (UCNPs), based on polyvinylpyrrolidone (PVP)-coated NaYF4:Er3+,Yb3+,Gd3+, with various concentrations of Gd3+ ions and relatively high upconversion efficiencies, were synthesized. The internalization and cytotoxicity of the thus obtained UCNPs were evaluated in three cell lines (HeLa, HEK293 and astrocytes). No cytotoxicity was observed even at concentrations of UCNPs up to 50 µg ml-1. The fate of the UCNPs within the cells was studied by examining their upconversion emission spectra with confocal microscopy and confirming these observations with transmission electron microscopy. It was found that the cellular uptake of the UCNPs occurred primarily by clathrin-mediated endocytosis, whereas they were secreted from the cells via lysosomal exocytosis. The results of this study, focused on the mechanisms of the cellular uptake, localization and secretion of UCNPs, demonstrate, for the first time, the co-localization of UCNPs within discrete cell organelles.

Novel ZnO/MgO/Fe2O3 composite optomagnetic nanoparticles.

A facile sol-gel synthesis of novel ZnO/MgO/Fe2O3 nanoparticles (NPs) is reported and their performance is compared to that of ZnO/MgO. Powder x-ray diffraction (XRD) patterns reveal the crystal structure of the prepared samples. The average particle size of the sample was found to be 4.8 nm. The optical properties were determined by UV-vis absorption and fluorescence measurements. The NPs are stable in biologically relevant solutions (phosphate buffered saline (PBS), 20 mM, pH = 7.0) contrary to ZnO/MgO NPs which degrade in the presence of inorganic phosphate. Superparamagnetic properties were determined with a superconducting quantum interference device (SQUID). Biocompatible and stable in PBS ZnO/MgO/Fe2O3 core/shell composite nanocrystals show luminescent and magnetic properties confined to a single NP at room temperature (19-24 ° C), which may render the material to be potentially useful for biomedical applications.

Oxidative and hydrolytic properties of beta-amyloid.

beta-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733-738]. In the present work we compare the ability of beta-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of beta-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human beta-amyloid (1-40), containing three amino-acid substitutions (Arg5-->Gly, Tyr10-->Phe and His13-->Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that beta-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of beta-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to beta-amyloid toxicity.

Implication of novel biochemical property of beta-amyloid.

Alzheimer disease (AD) is a heterogeneous disorder with a variety of molecular pathologies converging predominantly on abnormal amyloid deposition particularly in the brain. beta-Amyloid aggregation into senile plaques is one of the pathological hallmarks of AD. beta-Amyloid is generated by a proteolytic cleavage of a large membrane protein, amyloid precursor protein (APP). We have observed a new property of beta-amyloid. The amyloid 1-42 beta fragment, when aggregated, possesses proteolytic and esterase-like activity, in vitro. Three independent methods were used to test the new property of beta-amyloid. While esterase activity involves imidazole catalysis, proteolytic activity is consistent with participation of a serine peptidase triad: catalytic Ser, His and Glu (or Asp). Although the amino acid triad is a necessary requirement for the protease reactivity, it is not sufficient since the secondary structure of the protein significantly contributes to the proteolytic activity. The ability of beta-amyloid to cleave peptide or ester bonds could be thus responsible for either inactivation of other proteins and/or APP proteolysis itself. This property may be responsible for early pathogenesis of AD since there is emerging evidence that non-plaque amyloid is elevated in Alzheimer patients.

The structure-based design of ATP-site directed protein kinase inhibitors.

The protein kinase family represents both a huge opportunity and a challenge for drug development. The conservation of structural features within the ATP binding cleft initially led to the belief that specificity would be difficult to achieve. This dogma has now been clearly dispelled with the discovery and clinical testing of a group of first generation compounds, which are characterized by a high degree of selectivity towards a variety of oncology targets. The structural basis for selectivity and potency has now been clarified with the crystallization of a number of such targets in complex with inhibitors. The protein kinase inhibitor field is now ripe for the structure based exploitation of additional highly validated targets from a variety of therapeutic areas.

Thermal imaging of receptor-activated heat production in single cells.

Changes in enthalpy (i.e., heat content) occur during the diverse intracellular chemical and biophysical interactions that take place in the life cycle of biological cells. Such changes have previously been measured for cell suspensions or cell-free biochemical extracts by using microcalorimetry, thermocouples, or pyroelectric films, all of which afford minimal spatial or temporal resolution. Here we present a novel thermal imaging method that combines both diffraction-limited spatial (approximately 300 nm) and sampling-rate-limited time resolution, using the temperature-dependent phosphorescence intensity of the rare earth chelate Eu-TTA (europium (III) thenoyltrifluoro-acetonate). With this thermosensitive dye, we imaged intracellular heat waves evoked in Chinese hamster ovary cells after activation of the metabotropic m1-muscarinic receptor. Fast application of acetylcholine onto the cells evoked a biphasic heat wave that was blocked by atropine, and after a brief delay was followed by a calcium wave. Atropine applied by itself produced a monophasic heat wave in the cells, suggesting that its interactions with the receptor activate some intracellular metabolic pathways. The thermal imaging technique introduced here should provide new insights into cellular functions by resolving the location, kinetics, and quantity of intracellular heat production.

Simultaneous detection of hepatitis C virus and human immunodeficiency virus RNA in serum using amplicor PCR tests.

Several tests are currently available to assist in the diagnosis of the hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Tests that actually detect or quantify these viruses are based on the polymerase chain reaction (PCR) technique. However, the application of PCR is limited by the cost, labor, time-consumption, and potential for contamination. In this article we describe some procedures developed to reduce these limitations. We have developed and validated simultaneous detection methods for HIV RNA and HCV RNA in single serum samples using Amplicor PCR tests. The sensitivity and specificity of this method are comparable with the results obtained with commercial reverse transcription polymerase chain reaction (RT-PCR) techniques for HIV and HCV RNA detection. In addition we have modified the HIV Amplicor test for the RT-PCR procedure and the Chomczynski's method of RNA isolation. We hope that our method can find same applications in HIV and HCV coinfection research, blood screening, and medical diagnosis.

Unexpected binding mode of the sulfonamide fluorophore 5-dimethylamino-1-naphthalene sulfonamide to human carbonic anhydrase II. Implications for the development of a zinc biosensor.

The three-dimensional structure of human carbonic anhydrase II (CAII) complexed with the sulfonamide fluorophore 5-dimethylamino-1-naphthalene sulfonamide (dansylamide) has been determined to 2.1-A resolution by x-ray crystallographic methods. Unlike other arylsulfonamide inhibitors of CAII, the naphthyl ring of dansylamide binds in a hydrophobic pocket in the active site, making van der Waals contacts with Val-121, Phe-131, Val-143, Leu-198, and Trp-209. Interestingly, a conformational change of Leu-198 is required to accommodate dansylamide binding, which rationalizes the enhanced dansylamide affinity measured for certain Leu-198 variants (Nair, S. K., Krebs, J.F., Christianson, D. W., and Fierke, C. A. (1995) Biochemistry 34, 3981-3989). Modeling studies indicate that a second binding mode, in which the fused aromatic ring is rotated out of the hydrophobic pocket, is sterically feasible. Both experimentally observed and modeled binding modes have implications for new leads in the design of avid CAII inhibitors. Finally, the structure of the CAII-dansylamide complex has implications for its exploitation in zinc biosensor applications, and possible routes toward the optimization of fluorophore design are considered on the basis on this structure.

Increased incidence of cutaneous carcinomas in patients with congenital ichthyosis.

It is well known that patients with heritable cutaneous diseases may have excess tumors during their lifetime. This relationship has been shown for such diverse conditions as xeroderma pigmentosum, Bloom syndrome, basal cell nevus syndrome, and psoriasis. However, multiple cutaneous tumors have not been previously described in people with lamellar ichthyosis. This article describes the cases of two such patients, possible treatment, and a previously undescribed ichthyosis syndrome.

Comparison of the stability of topical isotretinoin and topical tretinoin and their efficacy in acne.

After 4 hours of exposure to incandescent light, 80% of 0.05% topical isotretinoin and 60% of 0.05% topical tretinoin preparations remained in their original form. In contrast, after 2 hours of exposure to fluorescent light only 25% of topical tretinoin and possibly 60% of topical isotretinoin remained in their original forms. Longer exposure to fluorescent light did not result in further breakdown, and the final breakdown of both preparations was similar. A 12-week, double-blind clinical trial comparing isotretinoin (0.05%) with tretinoin (0.05%) applied topically to patients with moderate acne was carried out. Both preparations caused significant reductions in papules and pustules. However, neither treatment was significantly superior to the other in the reduction of acne lesions. This may be because both preparations are rapidly broken down to similar products when exposed to fluorescent light.

Interactions among red cell membrane proteins.

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.

Properties of red cell membrane proteins: mechanism of spectrin and band 4.1 interaction.

Interactions between human red cell's band 4.1 and spectrin were studied by fluorescence resonance energy transfer and batch microcalorimetry techniques. The association constant (Ka = 8.6 X 10(7) M-1), the stoichiometry (one molecule of band 4.1 to one molecule of spectrin), the reversibility, and the enthalpy (delta H = -6 kcal/mol) were determined. A proton uptake was observed to take place as a result of the spectrin-band 4.1 complex formation. In addition to the protonation of the reaction products, the entropic contribution (-T delta S) has been observed to be responsible for approximately 50% of the binding free energy. We concluded that the environment plays a significant role in the stabilization of the complex. Since band 4.1 has been required for the maintenance of the cytoskeletal stability, small alterations of the binding energies or the degree of interaction could have a pronounced effect on the structure of the erythrocyte membrane.

Modulation of actin polymerization by the spectrin-band 4.1 complex.

The effect of human erythrocyte spectrin dimer and band 4.1 on the polymerization of actin was studied by two independent methods: by following the increase in fluorescence of actin covalently conjugated to N-pyrenyl-iodoacetamide (pyrenylactin) and by following the increase in light scattered by actin polymers. Both techniques indicated that the complex of spectrin dimer and band 4.1, but neither spectrin nor band 4.1 alone, stimulates the rate of nucleation (decreases the lag phase of polymerization) and stabilizes oligomers of F-actin. While the band 4.1-spectrin complex, but not spectrin alone, had no immediate effect on the rate of polymerization after the lag phase, it eventually decreases the rate of actin filament growth when the molecular ratio of actin-spectrin-band 4.1 approaches the physiological range. The complex has no detectable effect on the critical actin concentration and does not significantly alter the apparent order of the nucleation reaction.

Bullae in a patient with toxic shock syndrome.

A case of toxic shock syndrome presenting with macular erythema and subepidermal bullae is described. The patient meets the Centers for Disease Control criteria for toxic shock syndrome. A biopsy of the bullae and macular erythema revealed, respectively, subepidermal bullae and superficial perivascular infiltration. This case demonstrates the potential difficulty in differentiating between toxic shock syndrome and toxic epidermal necrolysis.

Effect of hemoglobin A and S on human erythrocyte ghosts.

The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.