Characterization of the alimentary canal of the aphidophagous ladybird,Adalia bipunctata (Coleoptera: Coccinellidae): anatomical and histological approaches

The alimentary canal of the two‐spot ladybird Adalia bipunctata (Linnaeus) (Coleoptera: Coccinellidae) presents the foregut (stomodeum), the midgut (mesenteron) and the hindgut (proctodeum). The shortest region is the foregut and the longest is the midgut. The relative proportions of the main regions were found to be similar for males and females. In the foregut it was possible to distinguish the pharynx, the esophagus and the proventriculus but no crop. The hindgut is composed of the ileum, rectum and rectal canal. Generally the organ width is similar for males and females, but females presented a wider proventriculus. The epithelium of the foregut varied from squamous to simple cuboidal and columnar. In the midgut the epithelium is simple columnar with goblet and regenerative cells. The epithelium of the hindgut varied from simple cuboidal to squamous. Females presented thicker midgut epithelium whereas males presented thicker epithelium in the esophagus. The anatomy of the alimentary canal of A. bipunctata seems to conform to its carnivorous and recent phylogenetic status within the family Coccinellidae.     

Gamma-Carboxylation and Fragmentation of Osteocalcin in Human Serum Defined by Mass Spectrometry

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0–3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.Osteocalcin (Oc)¹ is a member of the family of vitamin K-dependent gamma (γ)-carboxylated proteins. The formation of γ-carboxyglutamic acid (Gla) occurs via the carboxylation of three specific glutamic acid residues in the mid-molecular region of Oc (E17, E21, and E24) and results in the binding of Oc to hydroxyapatite in bone (1). In circulation Oc is a highly specific bone marker that has been used for assessing relative degrees of bone turnover in clinical studies (2). Based on immunological methods, a general notion has been that the major forms of circulating Oc are the intact molecule and a large N-terminal-mid molecule fragment encompassing residues 1–43, thought to be the result of trypsin-like activity in serum or poor sample handling (3). However, the precise sequence has never been clearly defined and considerable inconsistency is evident when comparing values from different laboratories or commercial kits because of differences in antibody specificity.Carboxylated variants of Oc are also found both in human bone and in circulation. Initial observational studies that reported an association between poor vitamin K status and bone loss attributed the finding to an increased proportion of Oc in circulation that was not carboxylated, reflecting a nonfunctional protein in bone (4–6). However, assays for total Oc are indifferent to carboxylation status, and methods used to measure the carboxylation state of circulating Oc do not distinguish among the fully, partially, or uncarboxylated forms (7, 8). Therefore, even though the amount of undercarboxylated Oc relative to the total in circulation (%ucOC) is a biomarker of vitamin K status in bone, there is no consensus on the precise amount in circulation or how many of the three potential Gla residues are carboxylated.Recently, mouse models have indicated that circulating Oc also serves as an endocrine hormone with a positive role in glucose metabolism (9). Paradoxically, the active form is un- or undercarboxylated, whereas the carboxylated form is inactive (10). A growing number of human studies have examined associations between total Oc and baseline or changing levels of fasting glucose, insulin, or HOMA-IR (11). However, few have directly measured the putative active form of the protein or taken into account that the carboxylation of Oc is very sensitive to daily fluctuations in intakes of vitamin K provided in such food sources as plant oils such as olive, canola and soybean, and green vegetables, such as broccoli, spinach, and kale (12, 13).Based on results provided by mass spectrometric immunoassay (MSIA), we herein report new qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual blood plasma and serum samples. We further present molecular details on the responses of specific carboxylated forms and fragments of Oc in plasma of free-living older adults who received different amounts of vitamin K under controlled conditions. Such determinations of Oc γ-carboxylation in individual serum samples will ultimately be necessary to translate the functional significance of fluctuating levels of Oc in human health and disease.     

Expression and variability of molecular chaperones in the sugarcane expressome

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study.     

Coupling of M3 acetylcholine receptor to Gq16 activates a natriuretic peptide receptor guanylyl cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M(3)AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Galpha(i/o) subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Galpha(q16), whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Galpha(q15/16) stimulated the NPR-GC. Coupling of alpha(q16) to M(3)AChR is supported by MAS decreased [(3)H]QNB binding, being abolished after M(3)AChR-4-DAMP-alkylation. Anti-i(3)M(3)AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i(3)M(3)AChR (M(3)P) was more potent than MAS increasing GTPgamma [(35)S] and decreasing the [(3)H]QNB activities. Coupling between NPR-GC and Galpha(q16) was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Galpha(q16), also showing an immunoreactive heterotrimeric-G-beta-subunit. These data support the existence of a novel transducing cascade, involving Galpha(q16)beta gamma coupling M(3)AChR to NPR-GC.     

Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring

Background  

The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals.     

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii

Mycopathologia - Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix...     

Early Interaction of Alternaria infectoria Conidia with Macrophages

Mycopathologia - Fungi of the genus Alternaria are ubiquitous indoor and outdoor airborne agents, and individuals are daily exposed to their spores. Although its importance in human infections and,...     

Exposure to Leishmania braziliensis Triggers Neutrophil Activation and Apoptosis

BackgroundNeutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate.ConclusionsWe show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.