Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death

The altered metabolism of cancer can render cells dependent on the availability of metabolic substrates for viability. Investigating the signaling mechanisms underlying cell death in cells dependent upon glucose for survival, we demonstrate that glucose withdrawal rapidly induces supra‐physiological levels of phospho‐tyrosine signaling, even in cells expressing constitutively active tyrosine kinases. Using unbiased mass spectrometry‐based phospho‐proteomics, we show that glucose withdrawal initiates a unique signature of phospho‐tyrosine activation that is associated with focal adhesions. Building upon this observation, we demonstrate that glucose withdrawal activates a positive feedback loop involving generation of reactive oxygen species (ROS) by NADPH oxidase and mitochondria, inhibition of protein tyrosine phosphatases by oxidation, and increased tyrosine kinase signaling. In cells dependent on glucose for survival, glucose withdrawal‐induced ROS generation and tyrosine kinase signaling synergize to amplify ROS levels, ultimately resulting in ROS‐mediated cell death. Taken together, these findings illustrate the systems‐level cross‐talk between metabolism and signaling in the maintenance of cancer cell homeostasis.     

A concise synthesis of 3-fluoro-5-thio-xylo- and glucopyranoses, useful precursors towards their corresponding pyranonucleoside derivatives

The chemical synthesis of 1,2,4-tri-O-acetyl-3-deoxy-3-fluoro-5-thio-D-xylopyranose, 1,2,4,6-tetra-O-acetyl-3-deoxy-3-fluoro-5-thio-alpha-D-glucopyranose and their corresponding nucleosides of thymine is described. Treatment of 3-fluoro-5-S-acetyl-5-thio-D-xylofuranose, obtained by hydrolysis of the isopropylidene group of 3-fluoro-1,2-O-isopropylidene-5-S-acetyl-5-thio-D-xylofuranose, with methanolic ammonia and direct acetylation, led to triacetylated 3-deoxy-3-fluoro-5-thio-D-xylopyranose. Condensation of acetylated 3-fluoro-5-thio-D-xylopyranose with silylated thymine afforded the corresponding nucleoside. Selective benzoylation and direct methanesulfonylation of 3-fluoro-1,2-O-isopropylidene-alpha-D-glucofuranose gave the 6-O-benzoyl-5-O-methylsulfonyl derivative, which on treatment with sodium methoxide afforded the 5,6-anhydro derivative. Treatment of the latter with thiourea, followed by acetolysis, gave the 3-fluoro-5-S-acetyl-6-O-acetyl-1,2-O-isopropylidene-5-thio-alpha-D-glucofuranose. 3-fluoro-5-S-acetyl-6-O-acetyl-5-thio-D-glucofuranose, obtained after hydrolysis of 5-thiofuranose isopropylidene, was treated with ammonia in methanol and directly acetylated, giving tetraacetylated 3-deoxy-3-fluoro-5-thio-alpha-D-glucopyranose. Condensation of the latter with silylated thymine afforded the desired 3-deoxy-3-fluoro-5-thio-beta-D-glucopyranonucleoside analogue.     

Breast metastasis from uterine leiomyosarcoma diagnosed by fine needle aspiration: a case report

BACKGROUND: Leiomyosarcomas of the breast are a rare subgroup of primary breast sarcomas. Even more rare is breast metastasis of an extramammary leiomyosarcoma. To date, only 4 cases have been reported in the literature. CASE: We report a case of breast metastatic leiomyosarcoma in a 58-year-old woman with a prior history of uterine leiomyosarcoma, resected 18 months earlier. The breast mass was palpable and a fine needle aspiration (FNA) was performed. The microscopic examination showed cellular smears composed of loosely structured clusters and tissue fragments of spindle-shaped and polygonal or rounded malignant cells in disorderly arrangement. The tumor cells were medium- or large-sized, with basophilic cytoplasm and enlarged, irregular, hyperchromatic nuclei with nucleoli. Tumor giant cells and multinucleation were also present. The morphologic features along with immunocytochemical positivity for desmin, muscle-specific actin and vimentin indicated the diagnosis of a metastatic leiomyosarcoma. CONCLUSION: FNA cytology can be a reliable method for the diagnosis of leiomyosarcoma. The morphologic criteria in combination Swith the clinical history and the immunocytochemical findings can indicate a definitive diagnosis and avoid additional painful and time-consuming diagnostic procedures for the appropriate patient's further clinical management.     

Liquid-based cytology in breast fine needle aspiration. Comparison with the conventional smear

OBJECTIVE: To compare the various cytologic features on AutoCyte Prep (ACP) (AutoCyte, Inc., Burlington, North Carolina, U.S.A.) and conventional preparation (CP) specimens from breast fine needle aspiration cytology material with a semi-quantitative scoring system. STUDY DESIGN: A total of 100 randomized cases were studied. In each case, 2 passes were performed. One pass was used for CPs (Giemsa and Papanicolaou stain). The other pass produced material for the ACP technique and Papanicolaou stain. Both the conventional and liquid-based preparations were studied independently by two observers and compared for cellularity, obscuring and/or informing background, representative diagnostic material, preservation of cytomorphologic features, presence of monolayer cells and architectural arrangement. RESULTS: Comparing the two preparations, the results were as follows: (1) ACP was superior to CP in 2 features, lack of obscuring background and presence of monolayer arrangement with preservation of cell architecture; (2) ACP was inferior to CP in 1 feature, lack of informing background; and (3) ACP was equal, with small deviations, to CP in the rest of the features evaluated: cellularity, representative diagnostic material, preservation of cell morphology and architectural arrangement. CONCLUSION: The new technology of liquid-based cytology in breast FNA showed a good correlation with CP plus the advantages of: (1) easier and less time consuming evaluation of cell morphology (clear background, no overlapping, smaller area to screen); (2) reproducibility, a factor of great importance to quality control; and (3) possibility of adjunctive investigations (immunocytology, flow cytometry) on the same material.     

Quercetin is a substrate for the transmembrane oxidoreductase Dcytb

Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O₂. We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin–Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb⁺) or Dcytb-null MDCK cells. In Dcytb⁺ cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (K_m = 6.53 ± 1.57 μM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential.     

Inflammation,Endothelial Dysfunction and Increased Left Ventricular Mass in Chronic Kidney Disease (CKD) Patients: A Longitudinal Study

Introduction

Within this longitudinal study we investigated the association of inflammation markers C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) and endothelial dysfunction markers intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) with left ventricular mass indexed for height²·⁷¹ (LVMI) in hypertensive predialysis CKD patients.

Material and Methods

From 2004 to 2005, 182 incident consecutive adult patients from the outpatient CKD clinics of two hospitals in Greece with CKD and hypertension or using antihypertensive medication, were included. Of these, 107 patients underwent CRP (mg/l) and LVMI (g/height²·⁷¹) measurements annually for three years.

Results

In the longitudinal analyses, using linear mixed modeling, a higher IL-6 (ß = 1.9 (95%ci:0.38;3.5), inflammation score based on CRP, IL-6 and TNF-α (ß = 5.0 (95%ci:0.72; 9.4) and VCAM-1 (ß = 0.01 (95%ci:0.005;0.02) were associated with higher LVMI. These models were adjusted for age, gender and primary renal disease, and for confounders that on top changed the beta with ≥10%, i.e. diuretic use (for IL-6 and inflammation score).

Conclusion

The results suggest that in predialysis CKD patients, inflammation as well as endothelial dysfunction may play an important role towards the increase in LVMI.     

Decreased levels of alpha‐synuclein in cerebrospinal fluid of patients with clinically isolated syndrome and multiple sclerosis

Cerebrospinal fluid (CSF) α‐synuclein (ASYN) levels are emerging as a possible biomarker in a number of neurodegenerative conditions; however, there has been little study of such levels in demyelinating conditions with neurodegeneration such as multiple sclerosis (MS). In this study, we aimed to assess CSF ASYN levels in MS spectrum [clinically isolated syndrome (CIS) and MS] patients and compare them to those obtained in control subjects with benign neurological conditions (BNC). We used a recently developed, ultra‐sensitive sandwich enzyme‐linked immunosorbent assay to measure and compare CSF ASYN levels in three categories of subjects: BNC (n = 38), CIS (n = 36) and MS [Relapsing Remitting (RRMS, n = 22) and Primary Progressive (PPMS, n = 15)]. We also performed secondary analyses, including relationship of CSF ASYN levels to aging, gender, presence of CSF oligoclonal bands (OB) and gadolinium (Gd)‐enhancing demyelinating lesions on T1‐weighted MRIs. CSF ASYN levels were found to be significantly lower in the CIS (78.2 ± 7.5 pg/mL), RRMS (76.8 ± 5.1 pg/mL), and PPMS (76.3 ± 6.7 pg/mL) groups compared to the BNC (125.7 ± 13.6 pg/mL) group. Secondary analyses did not reveal additional correlations. Our results suggest that in a cohort of CIS and MS patients, CSF ASYN levels are decreased, thus providing another possible link between MS and neurodegeneration. Future studies will need to be performed to confirm and extend these findings, to lead to a fuller understanding of the possible biological link between ASYN and MS.

     

Distinct signaling pathways regulate differential opioid effects on actin cytoskeleton in malignant MCF7 and nonmalignant MCF12A human breast epithelial cells

The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and nonmalignant (MCF12A) breast epithelial cells expressing functional opioid receptors. Exposure of MCF7 cells to the opioid agonist alpha(s1) casomorphin induced important actin assembly and reorganization, including the formation of filopodia and lamellipodia. In contrast, incubation of MCF12A cells with alpha(s1) casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analyses showed rapid phosphorylation of focal adhesion kinase (FAK) and vinculin in opioid-treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pretreatment of MCF7 cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of Rac1 and actin reorganization, while the opioid-induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid-treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of actin regulating the protein villin. Finally, opioids differentially inhibited cell motility in each cell line. Our data suggest a distinct, opioid-induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.     

Spatial averaging and disturbance lead to high productivity in aquatic metacommunities

Dispersal in heterogeneous ecosystems, such as coastal metacommunities, is a major driver of diversity and productivity. According to theory, both species richness and spatial averaging shape a unimodal relationship of productivity with dispersal. We experimentally tested the hypothesis that disturbances acting on local patches would buffer the loss of productivity at high dispersal by preventing synchronized species oscillations. To simulate these disturbances, our experimental assemblages involved species that self‐organized in isolation under three inflow pulsing frequencies, where hydraulic displacement and nutrient loading affected assemblage diversity and composition. At steady‐state, the emerging isolated assemblages were connected at three levels of dispersal creating three metacommunities of different connectivity. Consistent with theory, as dispersal increased, species richness in the metacommunity declined; productivity however remained high. This occurred because the most productive species in our study (which dominated the isolated patch of intermediate inflow pulsing frequency) dominated all three patches (low, intermediate and high inflow pulsing frequencies) after dispersal commenced in our metacommunities. This experimental result provides empirical support for the mechanism of spatial averaging. Furthermore, disturbances, in the form of localized pulsed inflows, prevented population oscillation synchrony caused by homogenization. Overall, our observations suggest that localized environmental fluctuations and the identity of species seem to be more influential than dispersal in shaping the diversity and composition of phytoplankton assemblages and stabilizing productivity.