Food source availability and interspecific dominance as structural mechanisms of ant-plant-hemipteran multitrophic networks

Extrafloral nectar of plants and honeydew of hemipterans is a food source extensively explored by ants. Although basically a sugary liquid food, nectar and honeydew are composed of different nutrients and offered in distinct ways; thus, ants must interact differently with plants and hemipterans. In this study we assessed the availability and dominance of nectar of extrafloral nectaries and honeydew of sap-sucking hemipterans (i.e., sugar-based resources) as mechanisms regulating interaction frequency and structuring ant-plant-hemipteran networks. We studied 12 plant species (240 shrubs, 20 per species) and 12 hemipteran species (240 aggregations, 20 per species) that interacted with 26 ant species in an area of Rupestrian Fields (Rocky Montane Savannah), Brazil. We observed that the 7 ant species that collected honeydew were a subset of the 25 ant species feeding on nectar, but the highly interacted species Camponotus crassus was the same for both subnetworks. The ant-plant subnetwork exhibited a nested pattern of interaction with a low degree of specialization, while the ant-hemipteran subnetwork exhibited lower nestedness but higher specialization. We found a positive relationship between the offer of EFNs and the number of interactions with ants, probably resulting from reduced competition in plants with high availability of EFNs. However, hemipteran species that were most abundant did not interact with more species of ants, probably because of the numerical dominance of the species tending all hemipteran aggregations, regardless of size. However, segregation between ant species was higher than expected by chance for both plants and hemipterans, confirming a deterministic factor (i.e., competition between ant species) regulating the frequency of interactions. In summary, the availability of ENFs seems to be an important mechanism regulating ant-plant interactions, while numerical dominance seems to be an important mechanism structuring ant-hemipteran interactions.     

A preliminary study of beta endorphin during chronic naltrexone maintenance treatment in ex-opiate addicts

Because opioid antagonists acutely produce rises in serum beta endorphin, we studied beta endorphin levels in 21 former opiate addicts chronically taking naltrexone. The mean AM (19.5 pg/ml) beta endorphin level was higher than the AM mean for 39 normals under 40 years old (12.1 pg/ml) (t = 3.2, p less than 0.001); the mean PM level for the naltrexone treated patients was 13.6 pg/ml. Four patients had beta endorphin levels more than 2 S.D. above the mean for the normals (greater than 26.4 pg/ml), and six others had relatively elevated PM levels. Thus, 47% (10/21) had abnormal patterns of beta endorphin levels. We had previously reported abnormally high cortisol levels in these patients, and AM cortisol correlated with AM beta endorphin levels (r = 0.7, p less than 0.001). We concluded that sustained beta endorphin elevations may occur during chronic naltrexone treatment.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

Naltrexone precipitated opiate withdrawal in methadone addicted human subjects: Evidence for noradrenergic hyperactivity

The noradrenergic alpha-2 receptor agonist, clonidine, suppresses many of the effects of opiate withdrawal in both humans and other animals and this is consistent with the data indicating important interactions of opiate and noradrenergic systems in brain. This evidence supports the hypothesis that central noradrenergic hyperactivity is involved in the expression of major signs and symptoms of the opiate withdrawal syndrome, but to date clinical studies have not provided biochemical data consistent with this idea. In order to assess whether naltrexone precipitated opiate withdrawal in methadone addicted human subjects is associated with changes in noradrenergic function, a double-blind study was completed in which 15 methadone-dependent subjects received naltrexone and 8 subjects received placebo. Signs and symptoms of the opiate abstinence syndrome increased significantly in the 15 subjects who received naltrexone compared with the 8 who received placebo. Plasma concentrations of free MHPG also increased significantly in those subjects who took naltrexone compared to the placebo treated subjects. In addition, withdrawal signs and symptoms were significantly correlated with plasma MHPG concentration. Since several compounds which suppress central noradrenergic activity also reduce the severity of the withdrawal syndrome, the noradrenergic hyperactivity suggested by the present clinical study may be a functional mechanism for at least part of the opiate withdrawal syndrome.     

Conversion of D-carnitine into L-carnitine with stereospecific carnitine dehydrogenases

D-Carnitine was converted to L-carnitine by resting and permeabilized cells as well as with purified stereospecific carnitine dehydrogenases from Agrobacterium sp. With permeabilized cells only 11% of D-carnitine was converted into L-carnitine. Using highly stereospecific D- and L-carnitine dehydrogenases from Agrobacterium sp. (pH 8.5, 50 mM D-carnitine, 1 mM NAD + , 0.1 mM NADH, 25-fold excess of L-carnitine dehydrogenase) almost 50% of the D-carnitine could be converted into L-carnitine.     

Role of opioid antagonists in treating intravenous cocaine abuse

Intravenous cocaine abuse is a major probel in opioid abusers including those treated in methadone maintenance. Studying 138 opioid addicts, we found that speedballing by combining opioid agonists with cocaine may be blocked by opioid antagonists such as naltrexone and by partial antagonists such as buprenorphine. With both these treatments cocaine abuse was five to eight times less than with methadone treatment.     

CD95 promotes metastatic spread via Sck in pancreatic ductal adenocarcinoma

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial–mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95''s induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.Recent analysis of the cellular heterogeneity within the tumour mass revealed the existence of cells that share characteristics with stem cells of the tissue of origin.¹ These cells are responsible for the tumour''s resistance to current therapies and therefore provide new perspectives in cancer treatment. Cancer stem cells (CSCs) or tumour-initiating cells (TICs) are characterized by their self-renewal and differentiation capacity, which are assessed by their ability to generate a heterogeneous tumour in immunocompromised mice in serial transplantations.² In pancreatic cancer, those properties were initially shown by cells expressing CD24, CD44 and ESA (epithelial surface antigen).³Pancreatic cancer is the fourth leading cause of cancer-related death in the United States of America.⁴ The highly malignant phenotype of pancreatic ductal adenocarcinoma (PDAC) results from aggressive invasion and early metastatic potential. Epithelial–mesenchymal transition (EMT) is considered to be the first step of metastatic spread. During this process, the tumour cells master the ability to detach from their neighbours and gain motile and invasive properties enabling them to spread via blood or lymph vessels.⁵ As cells undergo EMT, they lose their epithelial features including sheet-like architecture, polarity and E-cadherin expression and gradually gain motility and expression of mesenchymal markers such as N-cadherin, fibronectin and vimentin. Recent studies have uncovered a link between the EMT and the acquisition of stem cell characteristics.^{6, 7} Most growth factors such as TGF-β, HGF, EGF, IGF and FGF are known to trigger EMT.⁸ Interestingly, there is growing evidence that the so-called ‘death receptor'' CD95 (Fas/Apo-1) behaves like a growth factor receptor in cancer cells.^{9, 10, 11}CD95 was first discovered as the initiator of programmed cell death by forming death-inducing signalling complex (DISC, including Fas-associated death domain, FADD and caspase-8/10) upon stimulation with CD95 ligand (CD95L).¹² However, mitogen-activated protein kinases (MAPKs), leading to p38, JNK or extracellular signal-regulated kinase (ERK) 1/2 activation, were also reported to be driven by CD95.^{13, 14} In glioblastoma multiforme (GBM), CD95-induced migration depends on the formation of the so-called phosphatidyl-inositol 3-kinase (PI3K) activation complex (PAC),^{11, 12} consisting of the Src family kinase (SFK), Yes and p85, the regulatory subunit of PI3K. PAC components, however, differ between cell types, encompassing also other SFKs or the Syk tyrosine kinase.^{15, 16}Here, we show that the expression of CD95 increases in primary PDACs as compared with non-tumour-bearing pancreas and is higher in metastatic pancreatic cells than in primary PDAC. In CSCs isolated from primary PDAC surgical specimens, the expression of CD95 positively correlates with EMT markers. We also identified Sck as the molecular link between CD95 and activation of the PI3K and MAPK pathways. Neutralization of the CD95L reduces PDAC growth and metastasis. The present study defines CD95 and its downstream signalling pathway components as new targets for PDAC therapy.     

Structure-activity profiles of Ab-derived TNF fusion proteins

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD(50) of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.