Reproductive system morphology of Lightiella magdalenina (Crustacea,Cephalocarida): functional and adaptive implications

The reproductive systems of adults and larvae of Lightiella magdalenina were examined. Lightiella magdalenina, similar to the best-known cephalocarida species Hutchinsoniella macracantha, is a simultaneous hermaphrodite. Although the morphology of their reproductive system is similar, L. magdalenina differs from H. macracantha in exhibiting reduced fecundity: it lays one egg, not two, per reproductive event. This is due to asynchronous development of the oocytes inside the paired female reproductive structures, which determines the maturation of a single egg at a time. The reduced fecundity of L. magdalenina could be offset by the precocious release of oocytes from the germarium, which begins the vitellogenetic process during the last larval stages. Due to this process, after their last moult, reproductive adults can have a large number of advanced vitellogenic oocytes, reducing the time required for their maturation. A possible adaptive relationship between the halved fecundity with pre- and post-hatching parental care is discussed.     

Inorganic phosphate enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin via a p53‐dependent pathway

Osteosarcoma is the most common malignant primary bone tumor in children and adolescents. The clinical outcome for osteosarcoma remains discouraging despite aggressive surgery and intensive radiotherapy and chemotherapy regimens. Thus, novel therapeutic approaches are needed. Previously, we have shown that inorganic phosphate (Pi) inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. In this study, we investigated whether Pi could affect chemosensitivity of osteosarcoma cells and the underlying molecular mechanisms. Here, we report that Pi inhibits proliferation of p53‐wild type U2OS cells (and not of p53‐null Saos and p53‐mutant MG63 cells) by slowing‐down cell cycle progression, without apoptosis occurrence. Interestingly, we found that Pi strongly enhances doxorubicin‐induced cytotoxicity in U2OS, and not in Saos and MG63 cells, by apoptosis induction, as revealed by a marked increase of sub‐G1 population, Bcl‐2 downregulation, caspase‐3 activation, and PARP cleavage. Remarkably, Pi/doxorubicin combination‐induced cytotoxicity was accompanied by an increase of p53 protein levels and of p53 target genes mdm2, p21 and Bax, and was significantly reduced by the p53 inhibitor pifithrine‐alpha. Moreover, the doxorubicin‐induced cytotoxicity was associated with ERK1/2 pathway inhibition in response to Pi. Altogether, our data enforce the evidence of Pi as a novel signaling molecule capable of inhibiting ERK pathway and inducing sensitization to doxorubicin of osteosarcoma cells by p53‐dependent apoptosis, implying that targeting Pi levels might represent a rational strategy for improving osteosarcoma therapy. J. Cell. Physiol. 228: 198–206, 2013. © 2012 Wiley Periodicals, Inc.     

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.     

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD⁺-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.     

The kinetic deuterium isotope effect as applied to metabolic deactivation of imatinib to the des-methyl metabolite,CGP74588

There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation to the significantly less active CGP74588. Since deuterium–carbon bonds are stronger than hydrogen–carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties, and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl analogue would maintain target inhibition and show a reduced propensity for N-demethylation in in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased exposure of the N-trideuteromethyl analogue following intravenous administration of the compound to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from either parent drug.     

The synthesis and biological testing of bacilysin analogues

A series of compounds based on the structure of bacilysin were synthesised and tested for antibacterial activity. The key steps in the syntheses are the coupling of an iodide to a diketopiperazine (DKP) and mono-lactim ether scaffold, respectively. The diastereoselectivity of the coupling reactions was dependant on the scaffold, with selectivity for DKP of about 4:1 and mono-lactim ether exceeding 98:2. Subsequent elaboration of the compounds to give open chain dipeptides and DKPs that mimic the structure of bacilysin but substitute the epoxy ketone for a saturated or unsaturated ketone is described. Overall yield from coupling to final product was between 5 and 21 %, with the yield of the saturated products notably higher. The open chain dipeptides demonstrated moderate antibacterial and antifungal activity.     

Melatonin reduces migratory restlessness in <Emphasis Type="Italic">Sylvia</Emphasis> warblers during autumnal migration

Introduction

A remarkable aspect of bird migration is its nocturnality, particularly common in Passeriformes. The switch in activity from purely diurnal to also nocturnal is evident even in caged birds that during migratory periods develop an intense nocturnal restlessness, termed Zugunruhe. The mechanisms that control this major change in activity are mostly unknown. Previous work with Sylvia warblers suggested an involvement of melatonin, a hormone associated with day-night cycles in most vertebrates. In a recent study we found no effects of melatonin administration on Zugunruhe during spring migration. However, previous studies indicated that the response to melatonin manipulation could differ between spring and autumn migration, which are in fact separate life history stages. Here we tested whether a non-invasive treatment with melatonin can alter Zugunruhe in wild garden warblers S. borin and blackcaps S. atricapilla subject to temporary captivity at an autumnal stopover site. Food availability in the cage (yes/no) was added as a second factor because previous work showed that it enhanced Zugunruhe.

Results

The melatonin treatment significantly decreased the amount of Zugunruhe, while the availability of food only tended to increase the amount of Zugunruhe. Fuel deposits also had a strong effect on the amount of nocturnal activity: lean birds with a fat score of 1 showed significantly less Zugunruhe than fatter birds. The change in body mass during the time spent in the recording cage depended on food availability, but not on any of the other factors.

Conclusions

This study shows that the migratory programme of two Sylvia warblers can be manipulated by administration of exogenous melatonin and confirms that this hormone is involved in the control of migratory behaviour. To our knowledge, this is one of the first demonstrations that the autumn migratory programme can be altered by hormonal manipulation in migrating birds. The comparison with a similar study carried out with the same modalities during spring migration suggests that there are seasonal differences in the sensitivity of the migratory programme to hormonal factors. In birds breeding in the northern hemisphere, the importance of a timely arrival to the breeding sites could explain why the control of the migratory programme is more rigid in spring.