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.

Analysis of clinical AIDS-free interval after CD4+ cell counts fall below 200 x 10(6) L-1 in Japanese haemophiliacs infected with HIV-1.

We analysed the time from the date CD4+ cell counts fell below 200 x 10(6) L-1, defined as ti, to the onset of clinical AIDS, according to the 1987 Centers for Disease Control and Prevention case definition, in 129 Japanese haemophilia patients infected with HIV-1. The cumulative onset of clinical AIDS was analysed by the Kaplan-Meier method and proportional hazard model. Incorporated covariates were age of each patient at time ti, as well as CD4+ and CD8+ cell counts, serum levels of IgG, IgA, IgM, GOT and GPT at ti. The time of antiretroviral treatment initiation was also considered. The 50% AIDS-free interval after ti was 3.00 years (95% confidence interval (CI), range 0.49-5.51) and 1.71 years (95% CI, range 0.66-2.76) for the patients at CDC stage II and stage III, respectively (significantly different, P = 0.0013). Among the patients at CDC stage II at ti, higher levels of IgA were tightly associated with a shorter period from ti to onset of clinical AIDS (P < 0.0001), and relative hazard was 1.35 (95% CI, 1.11-1.64) with increase of IgA level by 1.0 g L-1. Thus there is a broad distribution in the time to onset of clinical AIDS in Japanese haemophiliacs even after CD4+ cell counts fall below 200 x 10(6) L-1. This should be taken into consideration in deciding upon the therapy and care of HIV-1 infected people.

SP-22 is a thioredoxin-dependent peroxide reductase in mitochondria.

SP-22 is a mitochondrial antioxidant protein in bovine adrenal cortex. The protein is homologous to thioredoxin peroxidase and other antioxidant proteins. It protects radical-sensitive enzymes from oxidative damage by a radical-generating system (Fe2+/dithiothreitol) in the presence of a small amount of serum. In this study we purified a second mitochondrial protein with Mr 11,777, which cooperates with SP-22 to protect glutamine synthetase and other proteins from Fe2+/dithiothreitol-mediated damage. Without SP-22, the protein had no protecting activity. We determined amino acid and nucleotide sequences of the protein and its cDNA, respectively, and found that it was a protein of the thioredoxin family. The protein, designated as mt-Trx (mitochondrial thioredoxin), had a presequence composed of 59 amino acids that seemed to be a mitochondrial targeting signal. Mitochondrial extract prepared from adrenal cortex contained NADPH-dependent 5,5'dithiobis(2-nitrobenzoic acid) (Nbs2) reductase activity. The enzyme was thought to have thioredoxin reductase activity, since the Nbs2-reducing activity was stimulated by mt-Trx. We partially purified the Nbs2 reductase from bovine adrenocortical mitochondria. In the presence of the partially purified reductase, mt-Trx, and NADPH, SP-22 showed the activity to protect oxyhemoglobin against ascorbate-induced damage. Furthermore, with the three protein components (Nbs2 reductase, mt-Trx, and SP-22) NADPH was oxidized in the presence of hydrogen peroxide or tert-butyl hydroperoxide. The oxidation of NADPH was concomitant with the disappearance of an equimolar amount of hydrogen peroxide. Without any one of the protein components no hemoglobin-protecting and peroxide-dependent NADPH-oxidizing activities were observed. From these results we concluded that SP-22 is thioredoxin-dependent peroxide reductase or so-called thioredoxin peroxidase in mitochondria from the adrenal cortex.

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.

Activation of cathepsin D by polyanionic compounds.

We examined the nature of the activation of cathepsin D by polyanionic compounds. Tripolyphosphate, a model compound for polyanions, decreased the Km value of porcine cathepsin D for bovine serum albumin without affecting VMAX. Half-maximal activation was achieved at 0.2 mM free tripolyphosphate. Electrophoretic mobility of cathepsin D decreased as the tripolyphosphate concentration was increased, and the enzyme had no net charge at 50 mM triP. The concentration for the half-maximal mobility change of cathepsin D (0.18 mM) was similar to that for half-maximal activation. These results suggest that tripolyphosphate increased affinity of the enzyme for its substrate by cancelling positive charges on cathepsin D and thus decreasing the electrostatic repulsion.

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.

Life time expectancy of hemophilia patients infected with HIV-1 with the risk of hepatocellular carcinoma after HCV infection.

1. INTRODUCTION. The latest statistics how that Japanese hemophilia patients infected with HIV-through clotting factor concentrates may survive more than 10 years after HIV-infection without showing an onset of AIDS [1]. Unfortunately, however, results of the recent surveillance have revealed that about half of hemophilia patients had also been infected with the hepatitis C virus (HCV) [2]. Therefore, some hemophilia patients might suffer from hepatocellular carcinoma triggered by HCV after some latent periods. In the present study, we computed the life time expectancy of hemophilia patients with two risks of HIV-and HCV infections. 2. METHOD. We used the Weibull hazard function h(t) for the hazard rate from AIDS after infection with HIV-1. In order to describe the hazard rate arising from hepatocellular carcinoma after HCV infection, we utilized the theoretical function c(t) with two parameters that were obtained previously by ourselves from a case-controlled study on hepatocellular carcinoma patients infected with HCV through blood transfusion [3]. Substituting necessary parameters estimated for Japanese hemophilia patients into h(t), the life time expectancy t was computed by the following integration; tau=integral of exp(-integral of h(t')+c(t¿) dt') dt, where t' means the time after HIV-infection, and t¿ is a variable composed of tU and times of HIV-and HCV infections. 3. RESULTS AND DISCUSSION. Without hepatocellular carcinoma, the life time expectancy tau of the patients infected with HIV-1 at the age of 20 was computed as 15.0 years. On the other hand, for the same patients with the risk of hepatocellular carcinoma through HCV infection at the age of 10, 15, 20 and 25 years old, values of tau were computed at 12.2, 13.3, 14.1 and 14.4 years, respectively. Especially noticeable was the reduction of tau in cases of HCV infection was prior to HIV-infection. In the present computation, the two risks from HIV-1 and HCV infections were assumed to be additive because no explicit interaction between them has been reported yet. Various long term effects have been found with the development of HIV/HCV therapies; that should also take into account the survival estimation and therapy planning for HIV-1 infected patients in the coming years.

[The effects of GnRH agonist on steroidogenesis in the rat ovary].

The effects of GnRH agonist (buserelin) on in vitro ovarian steroidogenesis were studied using DES-treated immature rats and PMS-treated immature rats. The estradiol and progesterone secreted by the cultured ovarian cells and the activities of various enzymes of steroid-metabolism were examined with or without gonadotropins (FSH or hCG), and the effects of 10(-6)-10(-12) M of buserelin on those indices were observed for 3-72 hours. In addition, the kinetic study of ovarian GnRH receptor was performed using 125I-labelled buserelin and crude ovarian cell membrane fraction of PMS-treated rats. The Scatchard analysis revealed the specific high affinity and low capacity ovarian GnRH receptor (Kd = 0.92 nM and Bmax = 0.57 fmol/mg tissue). The FSH-stimulated cholesterol side chain cleavage enzyme (CSCC) activity of the DES-treated rats was suppressed in a dose-dependent manner by buserelin. Estradiol release and aromatase activity were increased by 10(-8) M buserelin within 48 hours from the beginning of the incubation of the PMS-treated rat ovarian cells, but were suppressed after 48 hours. Buserelin increased basal progesterone secretion and both activities of CSCC and of 3 beta-hydroxysteroid dehydrogenase of PMS-treated rat ovarian cells incubated without hCG, which were suppressed by buserelin co-incubated with 100 IU/ml of hCG. These results suggested that GnRH plays a physiological role in ovarian steroidogenesis binding the specific receptor and that GnRH promotes the development of the follicle through increased estrogen synthesis in the early stage of the folliculogenesis and the luteinization in the late stage of the follicular development through increased progesterone and decreased estradiol production and the luteolysis in the luteinized cells by hCG through decreased progesterone secretion.

[Dopamine increases the ovarian progesterone synthesis of PMSG-treated rats by regulating 3 beta-HSD activity].

Little is known about the dopamine system in the ovary. The present study has been undertaken to investigate the effect of dopamine (DA) on the ovarian steroidogenic enzymes of pregnant mare serum gonadotropin (PMSG)-treated immature rats. Ovarian cells from PMSG-treated rats were cultured for 1-5 hours with or without DA, D1 agonists or bulbocapnine (Bul)(D1 antagonist). Progesterone (P) and estradiol (E2) in the media were assayed by specific RIAs. The enzyme activities were assayed by adding radioactive substrates in the media before incubation. DA and D1 agonists increased P in the media which was caused by the increment of 3 beta -hydroxysteroid dehydrogenase (3 beta -HSD) activity because cholesterol side chain cleavage enzyme (CSCC) activity showed no significant change. The stimulating effects of DA and DA agonists on P and 3 beta -HSD activity were inhibited by Bul. DA showed no effect on 17 alpha-hydroxylase activity. DA decreased 17.20 lyase activity, but this decrement was probably a non specific effect. DA alone did not affect the E2 level in the media and aromatase activity. The present results suggest that DA mainly stimulated 3 beta -HSD activity of the PMSG-treated rat ovary which regulated P synthesis.

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.

Effects of gonadotropin-releasing hormone agonist on steroidogenesis in the rat ovary.

To assess the regulatory roles of gonadotropin-releasing hormone (GnRH) in ovarian function, the kinetics of the ovarian GnRH receptor and the effects of the GnRH superagonist buserelin on steroidogenesis in ovarian cell culture were examined. Scatchard analysis of buserelin-binding to crude ovarian cell membrane revealed a specific high-affinity GnRH receptor. Buserelin together with follicle-stimulating hormone stimulated estradiol (E2) production in immature follicles in hypophysectomized and DES-treated rats. On the other hand, applied to developing follicles of rats treated with pregnant-mare-serum gonadotropin buserelin suppressed E2 production to terminate follicle maturation and simultaneously stimulated progesterone (P4) production to induce luteinization. With ovarian cells luteinized by human chorionic gonadotropin in vitro, buserelin suppressed production of both P4 and E2, leading to luteolysis. Buserelin affected steroid production by modulating activities of key enzymes in steroid synthesis. These findings indicate that buserelin action depended on the gonadotropin priming of ovarian cells, and suggest the possible involvement of GnRH in the regulation of steroidogenesis throughout the ovulatory cycle.

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.

[Activity of ATP-dependent protease and steroid metabolism in the human placenta]. / "Aktivität der ATP-abhängigen Protease und Steroidmetabolismus in der menschlichen Plazenta".

Placenta play the important role of nourishment of fetus and for the regulation of fetal and maternal steroid metabolism. Steroid metabolism is synthesized in mitochondria and levels of enzymes in steroid metabolism are regulated by the rate of synthesis and degradation of these enzymes. Therefore, we studied protease in human placental mitochondria to clarify regulation mechanism of steroid-synthesizing enzymes. 50 micrograms of human early and term placenta homogenate, proteins were analyzed by immunoblotting technique after SDS-polyacrylamid gel electrophoresis. ATP-dependent protease was detected using antiserum raised against purified bovine adrenocortical ATP-dependent protease and avidin-biotin complex method. ATP-dependent protease activity was assayed using 14C-Caseins a substrate. Five cell fractions of homogenate placenta were electrophorated and antibody-stained using ATP-dependent protease, cytochrome P-450 scc and adrenodoxin antibody after the blotting, and the quantity was analyzed by the densitometry and the method of De Douve. We had the following results: 1) ATP dependent protease is present in human placenta and localized in mitochondria. 2) ATP dependent protease decomposes adrenodoxin reductase in human placenta. 3) ATP dependent protease present in almost same consistent in early and term placenta and there is no significant difference. It is suggested as follows that the ATP dependent protease in human placenta participates in the regulation of steroid hormone through the metabolism of steroid synthetic enzyme.

Growth dynamics of the heart from perinatal period to childhood.

In order to obtain information useful for the diagnosis of fetus and newborn heart disease, we established a theoretical model of perinatal cardiac growth. We measured with the use of ultrasonic cross-section imaging system the mitral valve ring dimension, tricuspid valve ring dimension, and total cardiac dimension as morphological parameters of the heart in 45 cases composed of fetuses and children. The obtained data were entered into a computer. With the use of these data, simulation was made on the basis of the general theory of biological development. The present simulation showed that from the fetal stage to childhood the growth rates of the foregoing three morphological parameters mutually differ, but during the period of growth to the age of 12 years of the present study, they all demonstrated continuous growth up to 3 1/2 years after birth when they reached a growth saturation level.

Comparative biochemistry of acid proteinase from animal origins.

Acid proteinases from 17 tissues of 12 animal species were compared with respect to molecular weight, inhibition by pepstatin and activation by tripolyphosphate. Gel filtration of acid proteinases from protochordates and vertebrates showed a common elution profile and three peaks with mol. wts of -20,000, -45,000 and above 150,000 were detected with acid-denatured hemoglobin as substrate at pH 3.6. The main component of vertebrate acid proteinases was identified as cathepsin D. In the invertebrate acid proteinases, the elution profiles through gel filtration were characteristic to the tissues examined, and were not so distinct as those of vertebrates. Through a biochemical survey, the animal acid proteinase was discussed from a comparative point of view.