Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Egg-cases and size at hatching of Sympterygia acuta in the south-western Atlantic

Ten egg-cases of Sympterygia acuta were collected from the surf line at Cassino Beach, Rio Grande do Sul, Brazil, and incubated in the laboratory. The egg-cases and neonates (77–84 mm total length) are described.     

Arundifungin, a novel antifungal compound produced by fungi: biological activity and taxonomy of the producing organisms

Echinocandins, the lipopeptide class of glucan synthase inhibitors, are an alternative to ergosterol-synthesis inhibitors to treat candidiasis and aspergillosis. Their oral absorption, however, is low and they can only be used parenterally. During a natural product screening program for novel types of glucan synthesis inhibitors with improved bioavailability, a fungal extract was found that inhibited the growth of both a wild-type Saccharomyces cerevisiae strain and the null mutant of the FKS1 gene (fks1::HIS). The mutant strain was more sensitive to growth inhibition, suggesting that the fungal extract could contain an inhibitor of glucan synthesis. A novel acidic steroid, named arundifungin, was purified from a fungal extract obtained from a liquid culture of Arthrinium arundinis collected in Costa Rica. Arundifungin caused the same pattern of hallmark morphological alterations in Aspergillus fumigatus hyphae as echinocandins, further supporting the idea that arundifungin belongs to a new class of glucan synthesis inhibitors. Moreover, its antifungal spectrum was comparable to those of echinocandins and papulacandins, preferentially inhibiting the growth of Candida and Aspergillus strains, with very poor activity against Cryptococcus. Arundifungin was also detected in nine other fungal isolates which were ecologically and taxonomically unrelated, as assessed by sequencing of the ITS1 region. Further, it was also found in two more Arthrinium spp from tropical and temperate regions, in five psychrotolerant conspecific isolates collected on Macquarie Island (South Pacific) and belonging to the Leotiales, and in two endophytes collected in central Spain (a sterile fungus belonging to the Leotiales and an undetermined coelomycete).     

Improvement in the titer of echinocandin-type antibiotics: A magnesium-limited medium supporting the biphasic production of pneumocandins A0 and B0

Summary We have developed a liquid fermentation medium for the submerged culture of the fungus,Zalerion arboricola, which supports the rapid production of an echinocandin-type antibiotic, pneumocandin A₀ (formerly L-671, 329), in yields increased at least 4-fold over those reported previously. The improvements were achieved through medium simplification, substitution of high levels of mannitol for glycerol as the major source of carbon, and restriction of available magnesium. Antibiotic formation in batch cultures with this mannitol-based medium is not confined to the idiophase; rather production appears to be biphasic, with synthesis beginning during growth (i.e., at day 3) and increasing in rate at day 11, well after rapid growth has ended. Accumulation of antibiotic continues beyond 14 days, and by 21 days titers greater than 500 g/ml are attained. For the synthesis of a related compound, pneumocandin B₀, by a mutant strain ofZ. arboricola, the medium gives similar production kinetics and a titer of 800 g/ml. Although supplementation of the medium with magnesium ions stimulates growth, it decreases titer by preferentially affecting the second phase of antibiotic synthesis. This decline in synthesis in the magnesium-supplemented medium is explained by the depletion of mannitol before the second phase of synthesis can begin. In contrast, mannitol in the magnesium-limited medium is used more slowly with approximately half still available at day 11 to support continued antibiotic formation.     

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.     

Monitoring of HAB species in the Mediterranean Sea through molecular methods

A qualitative and semi-quantitative polymerase chain reaction(PCR)-based assay was developed for the detection of severalpotentially Harmful Algal Bloom (HAB) species and genera belongingto Dinophyceae, Bacillariophyceae and Raphydophyceae. Oligonucleotideprimers were designed based on Internal Transcribed Spacer (ITS)–5.8Sribosomal DNA (rDNA) sequences available in public databaseor identified in this study. The specificity and sensitivityof the PCR assay were validated using clonal cultures and thennatural seawater samples, as well as the known copy number ofplasmids containing the target ITS–5.8S rDNA regions.A filter system for collecting mixed phytoplankton cells coupledto a target species-specific PCR assay was performed on spatialand temporal series of net and surface seawater samples duringcoastal water monitoring carried out in several localities ofthe Mediterranean Sea. The application of PCR allowed a rapiddetection of various genera and species-specific potential HABtaxa in all examined natural samples. Qualitative and semi-quantitativePCR results obtained from field samples were compared with microscopic[light microscope (LM)] examinations. The two methods gave comparableresults, and the molecular assay was able to detect HAB targetcells at concentrations not detectable by microscopy or thoseof uncertain identity. The highest values of positive detectionof potential HAB taxon presence obtained by PCR assay comparedwith the microscopic examination ranged from 67 to 8.0%.     

VEGETATIVE AND SEXUAL ASPECTS OF DINOPHYSIS PAVILLARDI (DINOPHYCEAE)

Phases in the life history of the dinoflagellate Dinophysis pavillardi Schroeder from cultured phytoplankton assemblages are described. Under stressful conditions, induced in the laboratory through substantial thermic and nutritive changes, vegetative cells divided repeatedly. Scanning electron and light microscopy of dividing specimens showed that thecal fission began with the separation of the sulcal and ventral epithecal plates and the simultaneous dislocation of the pore plates from the right cell half. The posterior progression of the division led to pairs of cells attached antapically, which produced a new wall of reduced size. This phase of the life cycle coincided with the appearance and development of small forms of D. pavillardi, which displayed cytological features and behavior typical of male gametes, suggesting a process of gametogenesis through depauperating mitotic divisions. Anisogamy occurred at the time of the maximum production of small cells and involved the shedding of thecal components by the smaller gamete and subsequent cytoplasmic fusion and formation of planozygotes. Although the dormancy aspects of this species remain unknown, these observations provide the first evidence of sexuality .