Synthesis of pH-sensitive amphotericin B-poly(ethylene glycol) conjugates and study of their controlled release in vitro

New intravenous conjugates of amphotericin B (AMB) with poly(ethylene glycols) (PEG) (M=5000, 10,000, 20,000) have been synthesized and characterised. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AMB gave conjugates containing a pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. All types of the conjugates are relatively stable in phosphate buffer at physiological conditions of pH 7.4 (37 degrees C), less than 5 mol% AMB being split off from them within 24 h. For a model medium of afflicted tissue was used a phosphate buffer (pH 5.5, 37 degrees C), in which controlled release of AMB from the conjugates takes place. The imine linkage is split to give free AMB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights of the PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4 (37 degrees C) with half-lives of 2-5 h depending on molecular weights of the PEGs used (M = 5000, 10,000, 20,000). At first, the splitting of ester linkage produces the relatively stable pro-drug, that is, 4-carboxybenzylideniminoamphotericin B, which is decomposed to AMB and 4-formylbenzoic acid in a goal-directed manner only at pH 7 (t1/2 = 2 min, pH 5.5, 37 degrees C). A goal-directed release of AMB is only achieved by acid catalysed hydrolysis of imine linkage, either from the polymeric conjugate or from the pro-drug released thereof. The LD50 values determined in vivo (mouse) are 20.7 mg/kg and 40.5 mg/kg for the conjugates with ester linkage (M = 10,000 and 5000, respectively), which means that they are ca. 6-11 times less toxic than free AMB.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Alterations in the serum glycome due to metastatic prostate cancer

Glycomic profiles derived from human blood sera of 10 healthy males were compared to those from 24 prostate cancer patients. The profiles were acquired using MALDI-MS of permethylated N-glycans released from 10-microL sample aliquots. Quantitative permethylation was attained using solid-phase permethylation. Principal component analysis of the glycomic profiles revealed significant differences among the two sets, allowing their distinct clustering. The first principal component distinguished the 24 prostate cancer patients from the healthy individuals. It was determined that fucosylation of glycan structures is generally higher in cancer samples (ANOVA test p-value of 0.0006). Although more than 50 N-glycan structures were determined, 12 glycan structures, of which six were fucosylated, were significantly different between the two sample sets. Significant differences were confirmed through two independent statistical tests (ANOVA and ROC analyses). Ten of these structures had significantly higher relative intensities in the case of the cancer samples, while the other two were less abundant in the cancer samples. All 12 structures were statistically significant, as suggested by their very low ANOVA scores (<0.001) and ROC analysis, with area under the curve values close to 1 or 0. Accordingly, these structures can be considered as cancer-specific glycans and potential prostate cancer biomarkers. Therefore, serum glycomic profiling appears worthy of further investigation to define its role in cancer early detection and prognostication.     

Histone deacetylase inhibitors: overview and perspectives

Histone deacetylase inhibitors (HDACi) comprise structurally diverse compounds that are a group of targeted anticancer agents. The first of these new HDACi, vorinostat (suberoylanilide hydroxamic acid), has received Food and Drug Administration approval for treating patients with cutaneous T-cell lymphoma. This review focuses on the activities of the 11 zinc-containing HDACs, their histone and nonhistone protein substrates, and the different pathways by which HDACi induce transformed cell death. A hypothesis is presented to explain the relative resistance of normal cells to HDACi-induced cell death.     

Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.

The effects of aphidicolin, a specific inhibitor of DNA polymerase α, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 × 10⁻⁶ M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube-like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin-positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube-like elements may develop simply by cell fusion without cell division and DNA synthesis.     

Nano- and Micro-Scale Surface Modification of FCC Metal Using High Submerged Cavitating Water Jet

The aim of this paper is to establish a possible application of the cavitation phenomenon as an efficient method to modify surface properties at the nano and micro levels. Commercial-purity copper was subjected to high submerged cavitating jets under different initial conditions, for time periods between 15 and 1,800 s. The force generated by jet cavitation is employed to modify the surface roughness in the order of nano and micro scales. The target surface was analyzed with optical as well as scanning probe electron microscopy. The results showed the possibility to use cavitation bubbles to establish a nanofabrication method for the surface preparation, shoot-less surface peening (nano/micro level). Also, the cavitation is assumed to be one of the miniaturized testing methods that have to be developed to reliably measure mechanical properties in small dimensions and to identify the behavior caused by the size dependence. With optical, SEM, and atomic force microscopy observation techniques in this study, the deformation mechanism and the formation of planar or wavy slip were also studied. The results indicate that even at short exposure times, observed roughness having a characteristic “serpentine” configuration can be related to the start of the plastic deformation of the specimen surface. Longer exposure times inevitably result in a greater number of jet–specimen interactions leading to specimen fracture.     

Variability of the honey bee mite Varroa destructor in Serbia, based on mtDNA analysis

Only two mitochondrial haplotypes (Korea and Japan) of Varroa destructor, the ectoparasitic honey bee mite, are known to be capable of infesting and successfully reproducing in Apis mellifera colonies worldwide. Varroa destructor (then called Varroa jacobsoni) was observed in Serbia for the first time in 1976. In order to obtain insight into the genetic variability of the mites parasitizing A. mellifera we analyzed 45 adult female mites sampled from nine localities dispersed throughout Serbia. Four fragments within cox1, atp6, cox3 and cytb mtDNA genes were sequenced. The Korea haplotype of V. destructor was found to be present at all localities, but also two new haplotypes (Serbia 1 and Peshter 1) were revealed, based on cox1 and cytb sequence variability. The simultaneous occurrence of Korea and Serbia 1 haplotypes was observed at five localities, whereas Peshter 1 haplotype was identifed at only one place.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10^?5–10^?9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10^?4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect.Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells.While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls.These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.