Signaling and metabolic predispositions linked to the colorectal cancer

The setting up and the progression of the colorectal cancer (CCR) follow a sequence of events that are spatio-temporally rigorously orchestrated. The failures that specifically target the signaling pathways responsible for the cancerization of the colorectal mucosa have been well described and among these it seems that a dysregulation of the Wnt/β-catenin pathway is involved in the triggering of near 90 % of the cases. It has been also described that several risk factors linked to metabolic disorders (feeding, insulin resistance, metabolic syndrome, etc.) predispose individuals to CCR but no rational explanations were given. We propose that, since it is implicated in the control of the insulin pathway among other actions, the nutritional sensor O-GlcNAcylation may be the element linking these metabolic disorders to CCR.     

The P2Y12 Antagonists, 2-Methylthioadenosine 5��-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y₁ and P2Y₁₂. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y₁ receptors are involved in the initiation of platelet aggregation, P2Y₁₂ receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y₁₂ antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y₁₂ antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require G_i signaling or functional P2Y₁₂ receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving G_s. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP₂ receptors, which are known to couple to G_s. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y₁₂ receptors.Upon damage to the endothelial layer of the blood vessel wall, the underlying subendothelium is exposed to platelets in the blood, initiating a cascade of signaling events resulting in the transformation of “resting” platelets into “activated” platelets (1). One significant characteristic associated with these signaling events is the secretion of ADP from the platelet-dense granules (2). This released ADP acts to further amplify the platelet activation response by interacting with its G-protein-coupled receptors on the platelet surface, namely P2Y₁ (coupled to G_q) and P2Y₁₂ (coupled to G_i) (3–5). The consequence of platelet activation through ADP is a conformational change in the platelet membrane glycoprotein αIIbβ3 (6, 7), which then binds to fibrinogen present in the plasma. The binding of fibrinogen with αIIbβ3 on the surface of adjacent platelets results in fibrinogen-platelet cross-linking and the formation of a hemostatic plug at the site of vascular injury (8).Consequently, ADP is thought to play an integral role in the normal process of hemostasis. Of the two ADP-receptor signaling pathways in platelets, evidence has indicated that ADP-mediated P2Y₁₂ signaling appears to play a more prominent role in platelet activation than ADP-mediated P2Y₁ signaling (9, 10). For the most part, support for this notion derives from the use of the adenosine-based P2Y₁₂ antagonists (i.e. 2MeSAMP⁴ and ARC69931MX), which have a much broader inhibitory profile than P2Y₁ antagonists (e.g. A3P5P (adenosine-3′-phosphate-5′-phosphate) or MRS2179) (9). Thus, 2MeSAMP and ARC69931MX inhibit platelet aggregation in response to multiple agonists, such as thromboxane A₂, collagen, thrombin, etc. (11–13), whereas P2Y₁ antagonists do not. On the other hand, this general requirement for P2Y₁₂ signaling seems to be inconsistent with earlier reports indicating that activation of certain platelet receptors (e.g. thromboxane A₂ receptor) can cause aggregation through ADP-independent mechanisms (14, 15). Based on this apparent inconsistency in the contribution of P2Y₁₂ signaling to the overall platelet activation response, the present study investigated the possibility that the broad spectrum of inhibitory activity of this new generation of P2Y₁₂ antagonists (i.e. MeSAMP and ARC69931MX) may derive from an elevation in platelet cAMP levels.Our data demonstrated that both 2MeSAMP and ARC69931MX do in fact significantly raise human platelet cAMP. Furthermore, this pharmacological effect is independent of P2Y₁₂-G_i signaling and appears to proceed through activation of a separate G_s-coupled platelet receptor. Taken together, the results therefore indicate that these adenosine-based P2Y₁₂ antagonists can produce their inhibition of platelet function through a cAMP-mediated mechanism.     

Blood sampling by puncture in the cavernous sinus from hunted wild boar

A new method of sampling based on the extraction of blood from the cavernous sinus of the dura mater has been assessed in hunted wild boar. Blood from 139 animals was obtained by two different extraction methods: the harvesting from thoracic cavity (TC) and intracavernous venipuncture (IV). Sera obtained by the IV method had higher volume (mean 2.85 vs 1.85 ml), were less hemolytic (mean absorbance at 450 nm: 1.01 vs 2.41 nm). A higher number of samples and a higher proportion of sera collected by IV (90.6 %) compared to those obtained using the TC method (78.4 %), could be analyzed against Aujeszky’s disease using blocking ELISA. No statistically significant differences in seroprevalences between samples obtained using both extraction methods were observed. The results obtained indicate that the IV is an easy, fast, reliable, clean, and safe method to collect blood samples from hunted wild boar, proving a real alternative to the traditional collection method.     

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.     

Balanced translocation (10;13) in a father,ascertained through the study of meiosis in semen,and partial trisomy 10q in his son

Summary We describe a reciprocal translocation (10;13) in a man, ascertained through the study of meiosis in semen, and a partial trisomy 10q in his abnormal son. The phenotypic anomalies of the partial 10q trisomy syndrome are probably due to the presence in triplicate of the region q25qter of chromosome 10.     

Prevalence of ultrasonography proved polycystic ovaries in North Indian women with type 2 diabetes mellitus

Background  

Polycystic ovaries (PCO) and their clinical expression (the polycystic ovary syndrome [PCOS]) as well as type 2 diabetes mellitus (T2DM) are common medical conditions linked through insulin resistance. We studied the prevalence of PCO and PCOS in women with diet and/or oral hypoglycemic treated T2DM and non-diabetic control women.     

A Genetic and Mosaic Analysis of a Locus Involved in the Anesthesia Response of Drosophila Melanogaster

We describe a genetic and behavioral analysis of several alleles of har38, a mutant with altered sensitivity to the general anesthetic halothane. We obtained a P-element-induced allele of har38 and generated several excision alleles by remobilizing the P element. The mutants narrow abdomen (na) and har85 are confirmed to be allelic to har38. Besides a decreased sensitivity to halothane, all mutant alleles of this locus cause a characteristic walking behavior in the absence of anesthetics. We have quantified this behavior using a geotaxis apparatus. Responses of the mutant alleles to different inhalational anesthetics were tested. The results strongly favor a multipathway model for the onset of anesthesia. Mosaic flies were tested for their response to halothane and checked for their abnormal walking behavior. The analysis suggests that both the behaviors are exhibited only by such mosaics as have the entire head of mutant origin. It is likely that this focus represents an element of a common pathway in the anesthetic response to several inhalational anesthetics but not all. This result is the first demonstration of regional specificity in the CNS of any animal for general anesthetic action.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.