Association between cervical lesion grade and micronucleus frequency in the Papanicolaou test

The aim of this study was to evaluate the association between the frequency of micronuclei (MN) and the cellular changes detected in the conventional Papanicolaou test. One hundred and seventy-four Papanicolaou test smears with cellular changes were examined. MN screening was done in cytopathological smears by counting 1,000 cervical cells in a light microscope. MN frequencies were significantly higher in the group with cellular changes compared to the control group (p < 0.001). The mean MN frequencies were 0.95 ± 1.12 (mean ± SD) in the control group (n = 223), 2.98 ± 1.20 in individuals with atypical squamous cells of undetermined significance (ASC-US) (n = 50), 4.04 ± 1.45 in cervical intraepithelial neoplasia (CIN) I (n = 52), 5.97 ± 1.83 in CIN II (n = 30), 7.29 ± 1.55 in CIN III (n = 17) and 8.64 ± 1.55 in invasive cancer (n = 25). These findings suggest that MN monitoring should be included as an additional criterion for the early detection of cytogenetic damage in routine examinations. This monitoring should be done in the same smear as used for cytopathological examination. More specific and systematic studies are necessary to confirm this proposal.     

Ontogenetic changes in the carapace shape of the non-marine ostracod Eucypris virens (Jurine)

Developmental changes in carapace form (size+shape) during ontogeny have been explored in Eucypris virens (Crustacea, Ostracoda) using elliptic Fourier analysis. Clones from different geographic localities raised under controlled constant conditions (temperature and photoperiod) were used to characterize developmental pathways in the species. A larger data set including field populations and laboratory populations cultured under a range of environmental conditions were used to infer influence of environmental factors on carapace shape changes during ontogeny. Size changes between consecutive juvenile stages support empirical laws describing the doubling of ostracod volume at each moult. Ontogenetic changes point out the remarkable influence of environmental conditions on carapace shape.     

Evidence of an insulin generated pyruvate dehydrogenase stimulating factor in rat brain plasma membranes

1. The results of this study indicates that the binding of insulin to brain plasma membranes activates a membrane protease which, by a trypsin like mechanism, produces a soluble factor that modulates the PDH behaviour when added to brain mitochondria. 2. The supernatant from brain plasma membranes incubated with 0.5 mg/ml trypsin added to mitochondria increases PDH activity levels and cancels PDH inhibition by NaF, as has already been seen when the plasma membranes are incubated with 25 microU/ml insulin. No such effects are obtained when the incubation is run out with 0.5 mg/ml chymotrypsin. 3. The supernatants from insulin or trypsin treated plasma membranes retain their activating properties on mitochondrial PDH also after dansylation; from these preparations a dansylated active on PDH material was separated by monodimensional chromatography on HPTLC silica Gel plates, using chloroform/1-butanol (93:7 v/v) as a solvent. 4. Insulin incubation of plasma membranes pretreated with protease inhibitors (leupeptin, phenylmethylsulfonylfluoride) or with exogenous trypsin, but not chymotrypsin substrates (esters of arginine and tyrosine) yields an inactive supernatant on PDH. 5. Insulin treated plasma membrane supernatants lose all stimulating properties on PDH after incubation for 1 hr with 2 mg/ml trypsin or chymotrypsin.     

Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial–mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.     

The effect of leptin on Lep expression is tissue-specific and nutritionally regulated.

Leptin, the product of the Obese (Lep) gene, orchestrates behavioral and metabolic responses to nutrient intake. Here, we demonstrate tissue-specific autoregulation of Lep. Moderate increases in circulating leptin considerably decreased Lep expression in adipose tissue and induced lep expression in skeletal muscle, a tissue that normally does not express this gene. Changes in nutrient availability resulted in rapid alterations in Lep autoregulation. These findings demonstrate negative feedback regulation of Lep in fat, and indicate that leptin secretion can function as a vehicle of 'cross-talk' between adipose tissue and skeletal muscle, leading to tissue-specific modulation of the 'leptin signal'.     

In situ analysis of native microbial communities in complex samples with high particulate loads

In the present study a procedure combining a cell extraction method and Fluorescence In Situ Hybridization (FISH) for molecular monitoring and quantification of bacteria in soil and aquifer samples is presented. FISH was applied to bacterial cells extracted from the matrix by density gradient centrifugation. This separation method was applied to soil and aquifer samples and produced high cell recovery of 76.5%+/-4.4 and 78.0%+/-3.2, respectively. FISH, performed on the harvested cells, permitted a perfect visualization and quantification of bacteria. This approach is therefore promising for in situ detection of indigenous bacterial communities in complex samples.     

Phosphorylated p40PHOX as a negative regulator of NADPH oxidase

The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.     

Synergistic anti-proliferative and pro-apoptotic activity of combined therapy with bortezomib, a proteasome inhibitor, with anti-epidermal growth factor receptor (EGFR) drugs in human cancer cells

The proteasome plays a pivotal role in the turnover of regulatory transduction proteins induced by activated cell membrane growth factor receptors. The epidermal growth factor receptor (EGFR) pathway is crucial in the development and progression of human epithelial cancers. Proteasome inhibition may sensitize human cancer cell lines to EGFR inhibitors. We investigated the growth inhibitory and pro-apoptotic effects of the proteasome inhibitor bortezomib in combination with anti-EGFR drugs, such as gefitinib, vandetanib, and cetuximab in EGFR-expressing human cancer cell lines. Bortezomib determined dose-dependent growth inhibition in a nine cancer cell line panel (IC(50) values, range 6-42 nM). A significant synergistic growth inhibitory effect was observed with the combination of bortezomib and each EGFR inhibitor in all cell lines (combination index, CI, range 0.10-0.55), which was accompanied by a significant induction in apoptosis by the combined treatment with bortezomib, cetuximab and vandetanib. In HCT-116 colon cancer and A549 lung adenocarcinoma cells, bortezomib plus EGFR inhibitor treatment induced a more effective inhibition of EGFR-activated down-stream signals, including a marked suppression in activated, phosphorylated Akt (P-Akt). In contrast, overexpression of a constitutively active P-Akt protected A549 cells by cell growth inhibition and apoptosis following treatment with bortezomib and EGFR inhibitors. The combined treatment with bortezomib and EGFR inhibitors has a synergistic growth inhibitory and pro-apoptotic activity in different human cancer cells which possess a functional EGFR-dependent autocrine growth pathway through to a more efficient and sustained inhibition of Akt.     

Hyperglycemia modulates angiotensinogen gene expression

Elevated plasma angiotensinogen (AGT) levels have been demonstrated in insulin-resistant states such as obesity and type 2 diabetes mellitus (DM2), conditions that are directly correlated to hypertension. We examined whether hyperinsulinemia or hyperglycemia may modulate fat and liver AGT gene expression and whether obesity and insulin resistance are associated with abnormal AGT regulation. In addition, because the hexosamine biosynthetic pathway is considered to function as a biochemical sensor of intracellular nutrient availability, we hypothesized that activation of this pathway would acutely mediate in vivo the induction of AGT gene expression in fat and liver. We studied chronically catheterized lean (approximately 300 g) and obese (approximately 450 g) Sprague-Dawley rats in four clamp studies (n = 3/group), creating physiological hyperinsulinemia (approximately 60 microU/ml, by an insulin clamp), hyperglycemia (approximately 18 mM, by a pancreatic clamp using somatostatin to prevent endogenous insulin secretion), or euglycemia with glucosamine infusion (GlcN; 30 micromol. kg(-1). min(-1)) and equivalent saline infusions (as a control). Although insulin infusion suppressed AGT gene expression in fat and liver of lean rats, the obese rats demonstrated resistance to this effect of insulin. In contrast, hyperglycemia at basal insulin levels activated AGT gene expression in fat and liver by approximately threefold in both lean and obese rats (P < 0.001). Finally, GlcN infusion simulated the effects of hyperglycemia on fat and liver AGT gene expression (2-fold increase, P < 0.001). Our results support the hypothesis that physiological nutrient "pulses" may acutely induce AGT gene expression in both adipose tissue and liver through the activation of the hexosamine biosynthetic pathway. Resistance to the suppressive effect of insulin on AGT expression in obese rats may potentiate the effect of nutrients on AGT gene expression. We propose that increased AGT gene expression and possibly its production may provide another link between obesity/insulin resistance and hypertension.