Nucleolar variation in response to nutritional condition in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

The variations of the nucleolar characteristics size and number were studied in the gill and liver of Odontesthes bonariensis under different feeding regimes in order to test the hypothesis that functional genomes at the nucleolus, which determine variation of size (heterogeneity) in juvenile fishes, are modulated by environmental factors such as food availability. The programme that controls genetic changes of the nucleolar characteristics of the diploid cells of O. bonariensis was different from that described in the diploid cells of cyprinid fishes. No variations of the mean nucleolar number per nucleus were observed in liver or gill cells in relation to the feeding regime. Conversely, significant reduction of the mean nucleolar volume per nucleus ( V _MN) and change in the shape of the frequency distribution of the total nucleolar volume per cell was evident in both cell types in response to starvation or food restriction. Tissue specificity was observed in relation to the absolute values of the nucleolar characteristics; however, the relative behaviour was comparable in digestive and non-digestive tissues. The time-course reduction of V _MN in gill and liver cells of restrictively fed fish followed a negative exponential. A good correlation between V _MN and the condition index of the fish ( K ) was observed after long-term food restriction; however, a faster response of V _MN was observed after short-term fasting events. Results supported the stated hypothesis and showed nucleolar activity as a direct, rapid and sensitive indicator of the nutritional status of the fish.     

In vivo monitoring of the therapeutic efficacy of a CXCR1/2 inhibitor with 18F-FDG PET/CT imaging in experimental head and neck carcinoma: A feasibility study

The chemokine receptors CXCR1/2 play a key role in the aggressiveness of several types of cancers including head and neck squamous cell carcinomas (HNSCCs). In HNSCCs, CXCR1/2 signaling promotes cell proliferation and angiogenesis leading to tumor growth and metastasis. The competitive inhibitor of CXCR1/2, C29, inhibits the growth of experimental HNSCCs in mice. However, a non-invasive tool to monitor treatment response is essential to implement the use of C29 in clinical practices. ¹⁸F-FDG PET/CT is a gold-standard tool for the staging and the post-therapy follow-up of HNSCCs patients. Our study aimed to perform the first in vivo monitoring of C29 efficacy by non-invasive ¹⁸F-FDG PET/CT imaging. Mice bearing experimental HNSCCs (CAL33) were injected with ¹⁸F-FDG (T0) and thereafter treated (n = 7 mice, 9 tumors, 50 mg/kg by gavage) or not (n = 7 mice, 10 tumors) with C29 for 4 consecutive days. Final ¹⁸F-FDG-tumor uptake was determined at day 4 (TF). The average relative change (TF-T0) in ¹⁸F-FDG tumor uptake was +25.85 ± 10.93 % in the control group vs ?5.72 ± 10.07 % in the C29-treated group (p < 0.01). These results were consistent with the decrease of the tumor burden and with the decrease of tumor proliferating Ki67+ cells. These results paved the way for the use of ¹⁸F-FDG to monitor tumor response following C29 treatment.     

Impact of Cadmium Exposure during Pregnancy on Hepatic Glucocorticoid Receptor Methylation and Expression in Rat Fetus

Adverse fetal environment due to maternal undernutrition or exposure to environmental chemicals alters glucocorticoid (GC) metabolism increasing the risk of metabolic disorders in adulthood. In this study, we investigated the effects of maternal exposure to cadmium (Cd, 50 ppm) during pregnancy in the methylation of fetal hepatic glucocorticoid receptor promoter (GR) and the correlation with its expression and that of the DNA methyltransferases (DNMT1a and 3a). We also studied the expression of liver phosphoenolpyruvate carboxykinase (PEPCK) and acyl-CoA oxidase (AOX), two enzymes involved in the metabolism of carbohydrates and lipids respectively. The methylation of the rat GR gene exon 1(10) (GR1(10)) in nucleotides -2536 to -2361 was analyzed by pyrosequencing. Quantitative real time PCR was used to assess hepatic GR, PEPCK and AOX mRNA, and their protein levels using Western blotting analysis. Differential methylation was noted across groups at all CpG sites in the GR exon 1(10) in a sex-dependent manner. In males, CpG were more methylated than the controls (185±21%, p<0.001) but only CpG sites 1,6,7 and 9 showed a significantly different extent of methylation. In addition, a lower expression of GR (mRNA and protein) was found. On the contrary, in females, CpG were less methylated than the controls (62±11%, p<0.05) and overexpressed, affecting PEPCK and AOX expression, which did not change in males. The GR methylation profile correlates with DNMT3a expression which may explain epigenetic sex-dependent changes on GR1(10) promoter induced by Cd treatment. In conclusion, Cd exposure during pregnancy affects fetal liver DNMT3a resulting in sex-dependent changes in methylation and expression of GR1(10). Although these effects do not seem to be directly involved in the low birth weight and height, they may have relevant implications for long-term health.     

Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure

Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 ?. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.     

Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb’hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb’wingless, Gb’dachshund, Gb’aristaless and Gb’Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb’hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb’hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb’hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.     

Hox gene sequences from the geophilomorph centipede Pachymerium ferrugineum (C. L. Koch, 1835) (Chilopoda: Geophilomorpha: Geophilidae): implications for the evolution of the Hox class genes of arthropods.

Here we report on a partial screen for Hox gene sequences from the geophilomorph centipede Pachymerium ferrugineum, resulting in 11 different sequences. All of these sequences could be homologized to specific Drosophila genes, yielding two representatives for the Dfd class and one each for the remaining classes. Phylogenetic analysis of these data with a broad sample of arthropod/onychophoran homologous sequences confirmed these results and provided further support for the monophyly of the Hox3/zen class. Conversely, the phylogenetic status of ftz-type genes remains uncertain. Our results complement the previous partial findings for two other centipedes (the scolopendromorph Ethmostigmus rubripes and the lithobiomorph Lithobius forficatus) and confirm the expectation that in myriapods, too, all Hox genes classes are present. This suggests that even the Chilopoda, with uniform trunk segments, have the same number of Hox genes as the more tagmatized Insecta.     

Specific strychnine binding sites on acrosome-associated membranes of golden hamster spermatozoa

This study demonstrates for the first time, that membrane vesicles originated from the hamster sperm head after the occurrence of the acrosome reaction, possess specific strychnine binding sites. [3H]Strychnine binding was saturable and reversible, being displaced by unlabeled strychnine (IC(50)=26.7+/-2.3 microM). Kinetic analysis revealed one binding site with K(d)=120nM and B(max)=142fmol/10(6) spermatozoa. Glycine receptor agonists beta-alanine and taurine inhibited strychnine binding by 20-30%. Surprisingly, glycine stimulated binding by about 40-50%. Results obtained in this study strongly suggest the presence of glycine receptors-with distinctive kinetic properties on the periacrosomal plasma membrane of hamster spermatozoa. Localization of this receptor fits well with its previously proposed role in acrosomal exocytosis during mammalian fertilization.