Exploring the Thermal-Oxidative Stability of Azithromycin Using a Thermoactivated Sensor Based on Cerium Molybdate and Multi-Walled Carbon Nanotubes.

The chemical stability of azithromycin (AZM) may be compromised depending on the imposed thermo-oxidative conditions. This report addresses evidence of this process under varying conditions of temperature (20-80 °C), exposure time to UV radiation (1-3 h irradiation at 257 nm), and air saturation (1-3 h saturation with atmospheric air at 1.2 L min-1 and 15 kPa) through electrochemical measurements performed with a thermoactivated cerium molybdate (Ce2(MoO4)3)/multi-walled carbon nanotubes (MWCNT)-based composite electrode. Thermal treatment at 120 °C led to coordinated water elimination in Ce2(MoO4)3, improving its electrocatalytic effect on antibiotic oxidation, while MWCNT were essential to reduce the charge-transfer resistance and promote signal amplification. Theoretical-experimental data revealed remarkable reactivity for the irreversible oxidation of AZM on the working sensor using phosphate buffer (pH = 8) prepared in CH3OH/H2O (10:90%, v/v). Highly sensitive (230 nM detection limit) and precise (RSD < 4.0%) measurements were recorded under these conditions. The results also showed that AZM reduces its half-life as the temperature, exposure time to UV radiation, and air saturation increase. This fact reinforces the need for continuous quality control of AZM-based pharmaceuticals, using conditions closer to those observed during their transport and storage, reducing impacts on consumers' health.

Multimode hybrid gold-silicon nanoantennas for tailored nanoscale optical confinement.

High-index dielectric nanoantennas, which provide an interplay between electric and magnetic modes, have been widely used as building blocks for a variety of devices and metasurfaces, both in linear and nonlinear regimes. Here, we investigate hybrid metal-semiconductor nanoantennas, consisting of a multimode silicon nanopillar core coated with a gold layer, that offer an enhanced degree of control over the mode selection and confinement, and emission of light on the nanoscale exploiting high-order electric and magnetic resonances. Cathodoluminescence spectra revealed a multitude of resonant modes supported by the nanoantennas due to hybridization of the Mie resonances of the core and the plasmonic resonances of the shell. Eigenmode analysis revealed the modes that exhibit enhanced field localization at the gold interface, together with high confinement within the nanopillar volume. Consequently, this architecture provides a flexible means of engineering nanoscale components with tailored optical modes and field confinement for a plethora of applications, including sensing, hot-electron photodetection and nanophotonics with cylindrical vector beams.

Growth and differentiation of mitochondria in the regenerating rat adrenal cortex.

Diameters of the circular profiles of spherical mitochondria in parenchymal cells of the zona fasciculata in rat adrenal cortex were measured for intact controls and for the regenerating adrenal cortex on electron micrographs recorded at random. The diameter data were then processed by Bach's method which deals with the sphere size distribution. The structural parameters of the mitochondria were computed with the aid of an electronic computer. The total number of mitochondria in all the parenchymal cells of the zona fasciculata were calculated. The surface area of the inner mitochondrial membrane was then determined stereologically. Biochemical parameters were obtained for the protein, the phospholipid, and the cytochrome P-450 content, per averaged mitochondrion. The number of cytochrome P-450 molecules contained in the inner membrane was determined in terms of the unit surface area and of the unit amount of phospholipid. These correlated biochemical and stereological parameters have led to the following conclusions. (a) The genesis of the mitochondria after the adrenal enucleation is almost completed within 10 days. (b) During the period of mitochondrial proliferation, the mitochondria are small in size and also immature both in the structure and in the function of their inner membrane, (c) These small and immature mitochondria grow through an increase of the phospholipid and protein, and this increase is accompanied by expansion of the area of the membrane surface, (d) An enrichment of the inner membrane with cytochrome P-450 molecules occurs, thus indicating the differentiation of adrenocortical mitochondria. The process of membrane differentiation is not tightly coupled with that of membrane growth.

Kinetics of suicide substrates. Steady-state treatments and computer-aided exact solutions.

A steady-state differential equation that describes the kinetics of suicide substrate was derived for a scheme presented by Walsh et al. (Walsh, C., Cromartie, T., Marcotte, P. and Spencer, r. (1978) Methods Enzymol. 53, 437-488). Using its analytical solutions, the progress curves of substrate disappearance, product formation and enzyme inactivation were calculated for a hypothetical model system, and were compared with the exact solutions which were obtained by the numerical computation on a set of rate equations. The results obtained with the present analytical solutions were much more consistent with the exact solutions than those obtained using Waley's solution (Waley, S.G. (1980) Biochem. J. 185, 771-773). The most important factor for a system of suicide substrates was found to be the term (1 + r)mu as proposed by Waley, where r is the ratio of the rate constant of product formation to that of enzyme inactivation and mu is the ratio of initial concentration of enzyme to that of suicide substrate. In cases where this term has a value greater than unity, all the molecules of suicide substrate are used up leaving some enzyme molecule still active. To the contrary, in cases where the term has a value smaller than unity, all the enzyme molecules are inactivated with some molecules of suicide substrate being left unreacted. When the term is equal to unity, then all the enzyme molecules are inactivated and all the molecules of the suicide ar converted. Practical methods for estimating kinetic parameters are described.

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.

Suppressive effects of tranilast on the expression of inducible cyclooxygenase (COX2) in interleukin-1beta-stimulated fibroblasts.

We investigated the effects of tranilast on inducible cyclooxygenase (COX2)-mediated prostaglandin E2 (PGE2) production and enzyme induction in interleukin-lbeta (IL-1beta)-stimulated cultured dermal fibroblasts. IL-1beta enhanced PGE2 production in cultured fibroblasts. Tranilast did not affect constitutive cyclooxygenase (COX1) or COX2 activity in non-stimulated or IL-lbeta-stimulated fibroblasts. However, the COX2 expression induced by IL-1beta was inhibited by tranilast. This result, that IL-1beta-induced COX2 expression was suppressed by tranilast, was confirmed by immunohistochemical analysis. Thus, it is possible for tranilast to regulate PGE2 production by inhibiting COX2 induction.

Reversible aggregation and stability of lysosomal acid beta-galactosidase from porcine adrenal cortex.

1. Acid beta-galactosidase [EC 3.2.1.23] of porcine adrenocortical lysosomes, assayed for its activity towards p-nitrophenyl-beta-D-galactopyranoside, showed two activity peaks on gel filtration profile at pH 7.4, one corresponding to a molecular weight of approximately 270,000 (termed form A3) and the other about 65,000 (termed form A1). 2. Another form of acid beta-galactosidase with a molecular weight of about 130,000 (termed form A2) was found when the high speed extract or partially purified form A1 was chromatographed on Sephadex G-150 at pH 4.5. 3. In the presence of 0.1 M NaCl or saturating amounts of substrate at pH 4.5, the high speed extract showed the aggregation of form A2 yielding form A3. Dissociation of form A3 back to form A1 was observed on incubation at 37 degrees C in 0.02 M sodium phosphate buffer, pH 7.4, and that was followed by irreversible enzyme inactivation. 4. Dissociation of form A3 into form A1 and enzyme inactivation in phosphate buffer, pH 7.4, were prevented by addition of 0.1 M NaCl. 5. The interconvertible enzymic forms showed the same pH-activity profiles and Michaelis constants. 6. These results suggest that the lysosomal acid beta-galactosidase in the porcine adrenal cortex exists in vivo as the dimer, and that the dimer may further aggregate into the tetramer.

Characterization of cathepsin D in porcine adrenocortical lysosomes.

More than 95% of the apparent cathepsin D activity in the lysosomes of porcine adrenal cortex was due to a genuine cathepsin D that has a molecular weight of 42,800 +/- 800 (mean +/- standard deviation of the mean in 5 runs) as determined by Sephadex G-100 column chromatography. On CM-Sephadex C-50 column chromatography at pH 6.9, the enzyme was resolved into five peaks which were termed Fractions D1 through D5 in order of their elution from the column. Fraction D4 and Fraction D5 together constituted more than 70% of the total cathepsin D, and were purified through chromatographic procedures to constant specific activities. The content of lysosomal cathepsin D was estimated to be about 1% of the total cellular protein. On isoelectric focusing, Fraction D4 was resolved into one major band with pI of 7.34 (termed Form D4-1) and one minor band of 7.20 (Form D4-2), while Fraction D5 gave one major band with pI of 7.50 (Form D5-1) and two minor ones of 7.37 (Form D5-2) and of 7.20 (Form D5-3). All of these 5 bands were enzymatically active. On SDS-polyacrylamide gel electrophoresis, both Form D4-1 and Form D5-1 dissociated into three subunits with molecular weights of 27,400 +/- 500 (termed Subunit A), 24,600 +/- 400 (Subunit B) and 13,800 +/- 600 (Subunit C) (mean +/- standard deviation of the mean in 16 determinations). The three subunits all contained carbohydrates.

Characterization of porcine adrenocortical lysosomes.

Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase were found exclusively in the lysosomes, while alpha-mannosidase and beta-glucuronidase were in both the lysosomal and microsomal fractions. The activity of cathepsin D was remarkably high, amounting to more than 6 times that in porcine liver and to more than 10 times that in liver of Sprague-Dawley rats in terms of units per g wet tissue. Porcine adrenocortical lysosomes showed a modal isopycnic density value of 1.155, but mitochondria a value of 1.145. The validity of these values was studied by investigating the possibilities of agglutination of organelles, damage to lysosomal membranes, disruption of mitochondria due to the hydrostatic pressure and by applying the same procedures of isopycnic centrifugation to hog and rat livers. After these validity tests, porcine adrenocortical lysosomes were concluded to be unique in their strikingly high content of cathepsin D as well as in their low modal isopycnic density which is very close to that of porcine adrenocortical mitochondria.

Purification and characterization of a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex.

We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.

In vitro degradation of mitochondrial proteins by ATP-dependent protease in bovine adrenal cortex.

We have examined in vitro degradation of mitochondrial proteins by ATP-dependent protease in bovine adrenal cortex. Purified ATP-dependent protease degraded at least five proteins in vitro in the presence of ATP and Triton X-100 using mitochondrial fraction as a substrate. Two of the degraded proteins were identified as P-450scc and adrenodoxin reductase by immunoblotting. No degradation of P-45011 beta or adrenodoxin was detected. Purified P-450scc and adrenodoxin reductase were also degraded. By analyzing degradation products of P-450scc we identified 49 peptides and 59 cleavage sites. The size of the products was between 3 and 18 amino acid residues. The protease preferentially cleaved the carboxy-side of Leu, Phe, Ala, Val, Met, and some other amino acids. Since the protease (a matrix enzyme) is accessible to P-450scc and adrenodoxin reductase localized on the matrix side of inner membrane, the present results suggest that the protease might participate in the turnover of these two proteins and some other mitochondrial proteins.

Unique biochemical and biological features of cathepsin D in rodent lymphoid tissues.

Cathepsin D, an enzyme consistently found to be lysosomal in many cells, has an unusual localization in rat thoracic duct lymphocytes (TDL). After fractionation of homogenates of rat TDL, most of the enzyme activity, as measured at pH 3.6 on denatured bovine hemoglobin, is distributed differently from the other lysosomal enzymes. The enzyme also has some unique properties: it is not inhibited by an antiserum inhibitory for rat liver cathepsin D; it exists in two molecular weight forms (approximately 45,000 and approximately 95,000) both of which have a higher specific activity than rat liver cathepsin D, as determined by studies using the irreversible inhibitor, sodium pepstatin; the high molecular weight form converts to the low molecular weight form after treatment with beta-mercaptoethanol without any loss in activity. These enzymes appear to be restricted to rodent lymphoid tissues. Reasons for considering them to be a type of cathepsin D are given in the text.

The cDNA sequence encoding bovine SP-22, a new defence system against reactive oxygen species in mitochondria.

We have isolated cDNA clones coding SP-22, an antioxidant protein in mitochondria, from a bovine adrenal medulla cDNA library constructed with (lambda)gt11. The largest clone contained the entire coding sequence for mature SP-22. Since the isolated cDNA clones lacked 5'- and 3'-ends, we determined the sequences of both ends by the "Rapid Amplification of cDNA Ends (RACE)" tecnique. The deduced amino acid sequence of the mature protein region was the same as that determined by protein sequencing. Since SP-22 had a mitochondrial targetting signal, its mitochondrial localization was confirmed.