A chitin-like component in Aedes aegypti eggshells, eggs and ovaries

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.     

Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.     

Transmembrane proteins are not required for early stages of nuclear envelope assembly

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.     

Osteogenesis promoted by calcium phosphate N,N-dicarboxymethyl chitosan

The effects of N,N-dicarboxymethyl chitosan (DCMC) on the precipitation of insoluble calcium salts, namely phosphate, sulfate, oxalate, carbonate, bicarbonate and fluoride, and magnesium salts, namely phosphate and carbonate, were studied. Results indicated that the chelating ability of DCMC interfered effectively with the well-known physico-chemical behaviour of magnesium and calcium salts. Dicarboxymethyl chitosan formed self-sustaining gels upon mixing with calcium acetate, as a consequence of calcium chelation. DCMC mixed with calcium acetate and with disodium hydrogen phosphate in appropriate ratios (molar ratio Ca/DCMC close to 2.4) yielded a clear solution, from which, after dialysis and freeze-drying, an amorphous material was isolated containing an inorganic component about one half its weight. This compound was used for the treatment of bone lesions in experimental surgery and in dentistry. Bone tissue regeneration was promoted in sheep, leading to complete healing of otherwise non-healing surgical defects. Radiographic evidence of bone regeneration was observed in human patients undergoing apicectomies and avulsions. The DCMC–CaP chelate favoured osteogenesis while promoting bone mineralization.     

Functional and molecular insights into KSRP function in mRNA decay

KSRP is a single strand nucleic acid binding protein that controls gene expression at multiple levels. In this review we focus on the recent molecular, cellular, and structural insights into the mRNA decay promoting function of KSRP. We discuss also some aspects of KSRP-dependent microRNA maturation from precursors that are related to its mRNA destabilizing function. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Reproductive measures and chick provisioning of Cory's Shearwater Calonectris diomedea borealis in the Azores

Capsule Timing of breeding influenced wing-length at fledging, and egg size may be an indicator of fledging weight and the amount of food received by chicks.

Aims To investigate chick growth, temporal patterns of chick food provisioning and the importance of indices of parental condition or quality, egg size and hatching date, to predict nestling body mass and wing-length at fledging, and compare breeding and chick feeding characteristics between colonies in the northeast Atlantic.

Methods A survey of Cory's Shearwater nests was carried out at Vila islet. A sample of 52 chicks, ringed and weighed at hatching, was selected to study chick growth and food provisioning.

Results Hatching success (51%) was much lower than fledging success (87%). Both hatching date and egg size contributed to explain wing-length at fledging, but hatching date, which was negatively correlated with wing-length at fledging, had the most important contribution (22%). There was some indication that egg size may explain variation in fledging weight and the amount of food received by chicks. Food delivery and feeding frequency of chicks varied throughout the chick development stage and three phases were distinguished: (1) 0–29 days, the highest feeding frequency values and a linear increase in food delivery; (2) 30–69 days, an oscillation in food delivery and medium feeding frequencies; (3) 70–90+ days, a sharp decrease in both food delivery and feeding frequency.

Conclusion Variation in food availability did not seem sufficient to override the overall importance of indices of parental quality in determining reproductive measures and chick provisioning. Breeding and feeding characteristics were similar between colonies in the northeast Atlantic, with variability in chick provisioning higher further south.     

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.     

Expression of DNA methyltransferases is involved in Quercus suber cork quality

Cork oak (Quercus suber) is an important Portuguese species, mainly due to the economic value of the cork it produces. Cork results from phellogen, a meristematic tissue, which can locally produce lenticels or have discontinuities, originating “defects”: pores and nail inclusions that are detrimental to cork industrial use. Epigenetic processes control plant development and its deregulation can lead to altered phenotypes; therefore, the study of epigenetic players in the phellogen is important to understand the emergence of cork's defects. DNA methyltransferases (DNMTs) and one protein associated to MET1 (DMAP1) were characterized in Q. suber, and their gene expression was analyzed in phellogen and contiguous differentiating cell layers of trees producing high and low quality cork, after the evaluation of their defects by physical and image analysis methods. All classes of DNMTs (MET, DRM, and CMT) with the respective canonical motifs were identified in Q. suber. The expression analyses of these genes showed that QsDRM2 was the most active methyltransferases in the cells analyzed, and that all the genes were differentially expressed in trees with distinct cork quality, with a tendency for higher expression levels in low quality producers. Interestingly, the global methylation level was higher in cells with low expression of DNA methyltransferases. A positive and significant correlation was obtained between QsDMAP1 gene expression and the percentage of cork defects. This work provides the first evidence that cork quality in Q. suber is likely influenced by epigenetic mechanisms.