G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton.

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein beta subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type beta subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

The NF-kappaB pathway blockade by the IKK inhibitor PS1145 can overcome imatinib resistance.

Imatinib represents at present the most attractive therapy for BCR-ABL positive leukemias, even though a percentage of CML patients develop resistance to this compound. For these resistant patients a therapeutic approach based on a combination of drugs is more likely to be effective. In the last years, constitutive NF-kappaB/Rel activity has been demonstrated in several hematological malignancies. As a result, NFkB/Rel-blocking approaches have been proposed as antineoplastic strategies. Furthermore, the identification of specific kinases within the NF-kappaB activation pathway offers a selective target to address tailored therapies. In the current study, we show that the IKK inhibitor PS1145 is able to inhibit the proliferation of CML cell lines and primary BM cells. Moreover, the addition of Imatinib increases the effects of PS1145 in resistant cell lines and BM cells from resistant patients, with a further increase of apoptosis and inhibition of proliferation and colony growth. Our data provide the rational for a new therapeutic approach, which combines Imatinib and the IKK inhibitor PS1145 in CML resistant patients.

RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes.

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64-69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP-RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP-RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Cell-cell signaling and adhesion in phagocytosis and early development of Dictyostelium.

Cell-cell signaling and adhesion regulate transition from the unicellular to the multicellular stage of development in the cellular slime mold Dictyostelium. Essential gene networks involved in these processes have been identified and their interplay dissected. Heterotrimeric G protein-linked signal transduction plays a key role in regulating expression of genes mediating chemotaxis or cell adhesion, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Two classes of cell adhesion molecules, one cadherin-like and the second belonging to the IgG superfamily, contribute to the strength of adhesion in Dictyostelium aggregates. The developmental role of genes involved in motility and adhesion, and their degree of redundancy, have been re-assessed by using novel developmental assay conditions which are closer to development in nature.

Cloning and transcriptional regulation of the gene encoding the vacuolar/H+ ATPase B subunit of Dictyostelium discoideum.

The main function of vacuolar H+ ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H+ ATPase (vatB) and analyzed its transcriptional regulation. The deduced protein comprises 493 amino acids with a calculated molecular mass of 54874 Da. The predicted protein sequence is highly homologous to previously determined V/H+ ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis, but not by fluid-phase endocytosis. Acidic and alkaline conditions affect vatB gene expression differently.

Polysiloxane: potential noncaloric fat substitute; effects on body composition of obese Zucker rats.

Phenylmethylpolysiloxane (PS), a noncaloric, nonabsorbable liquid oil, was studied for effects on body comparison as fat substitute in the diet. Two groups of female obese Zucker rats were fed either a control low-fat (LF) or an experimental diet containing PS (22% wt/wt) incorporated into LF. Two additional groups were fed either PS or cellulose (CE) in diet providing equivalent caloric dilution. Rats on PS lost weight whereas LF control rats gained. Dissectible fat and adipocyte size of PS were smaller than those of LF. Food intake, body water, and adipocyte number did not differ between PS and LF. Body protein on PS increased only in proportion to weight. When both diets were diluted, PS animals lost more weight than CE controls despite similar food intakes, suggesting absorption of calorigenic substances derived from partial digestion of CE but not PS by intestinal microflora. Obese rats did not compensate for caloric dilution with PS.

Enhanced thermogenesis and diminished deposition of fat in response to overfeeding with diet containing medium chain triglyceride.

The mechanism whereby overfeeding with diet containing medium chain triglyceride (MCT) results in diminished body weight and fat was studied. Fifteen male Sprague-Dawley rats were fitted under anesthesia with gastrostomy tubes and divided into two groups. One group was fed MCT diet, the other an isocaloric diet containing long chain triglyceride (LCT) in excess (150%) of spontaneous calorie intake. Both diets, fed for 6 wk, derived 50% of calories from fat. Basal and norepinephrine (25 micrograms/100 g) stimulated 02 consumption and CO2 production, as well as metabolic rate were measured. After the rats were killed, total dissectible fat and fat cell size and number were determined. MCT rats gained 15% less weight than LCT controls (p less than 0.001). Total dissectible fat was significantly lower (p less than 0.001) in MCT group, as was mean adipocyte size (p less than 0.001). Resting and maximal norepinephrine-stimulated 02 consumptions were 39.7 and 22.1% higher in MCT than in LCT group, respectively. Resting and norepinephrine-stimulated metabolic rates were 38.8 and 22.2% higher in MCT than LCT fed rats, respectively. Overfeeding MCT diet results in decreased body fat related to increased metabolic rate and thermogenesis.

Overfeeding with medium-chain triglyceride diet results in diminished deposition of fat.

The study was designed to determine whether overfeeding rats with a diet containing medium-chain triglyceride (MCT) as the major fat source (45% of calories) would impede the expected gain in weight and body fat as compared to rats overfed with isocaloric amounts of diet containing long-chain triglyceride (LCT). For 6 wk rats were fed either MCT diet or LCT diet twice daily via a gastrostomy tube. MCT-fed rats gained 20% less weight (P less than 0.001) and possessed fat depots weighing 23% less (p less than 0.001) than LCT)-fed rats. Mean adipocyte size was smaller (p less than 0.005) in MCT- than in LCT-fed rats. Weights of carcass protein and water were similar for both groups as were concentrations of serum insulin and levels of physical activity. The decreased deposition of fat in the MCT-fed rats may have resulted from obligatory oxidation of MCT-derived fatty acids in the liver after being transported there via the portal vein, leaving almost no MCT derivatives for incorporation into body fat. MCT may have potential for dietary prevention of human obesity.

TOBEC methodology for body composition assessment: a cross-validation study.

The accuracy of prediction equations for estimating lean body mass (LBM) from total-body electrical conductivity (TOBEC) was examined by cross-validation. Two samples of adults, aged 18-35 yr, were drawn from separate geographic locations. LBM was determined by densitometry and TOBEC was measured with TOBEC II instrument. LBM and TOBEC were highly correlated in both samples (r = 0.96 and 0.97). Cross-validation of LBM prediction equations was accomplished by exchanging equations and comparing predicted LBM values. There was a mean difference of 0.974 kg LBM between the two equations (p less than 0.0001). Thus, data from 157 subjects were pooled and one equation was developed that incorporated height (cm), sex (males = 0, females = 1), and the zero-, first-, and second-order Fourier coefficients (FC0, FC1, and FC2) of the TOBEC phase value: LBM, kg = -36.410 + (-1.324 X sex) + (0.01185 X (FC1(0.5)xht)) + (12.347 X FC2(0.5)) + (0.0627 X FC0)-(0.9232 X FC2) This equation, developed from 157 subjects, accounted for 96% of the variability in LBM and had a standard error of estimate of 2.17 kg LBM.

Naltrexone and human eating behavior: a dose-ranging inpatient trial in moderately obese men.

To investigate the effects of the long-acting opiate antagonist naltrexone on spontaneous human eating behavior, eight moderately obese male paid volunteers were housed in a hospital metabolic unit for 28 days and offered palatable foods ad lib by a platter service method. Under double-blind conditions, equally divided doses of 100, 200 and 300 mg naltrexone, or an acetaminophen placebo, were administered twice daily in tablet form for 3-day periods each, according to a Latin Square design. The doses of naltrexone resulted in decreases of daily caloric intake from placebo level, but these reductions were neither statistically significant nor dose-related. When the averaged effects of the doses were compared to placebo, five subjects showed intake reductions but the overall intake reduction of 301.5 +/- 198.1 kcal/day (mean +/- SEM) was not statistically significant. Naltrexone administration failed to selectively alter intakes of individual meals and snacks or macronutrient consumption patterns. During active drug periods, subjects lost 0.62 +/- 0.22 lb over 3 days, while during the placebo period, subjects gained 0.46 +/- 0.68 lb. However, there was no reliable change of basal metabolic rate as a function of naltrexone administration. The present results, which indicate that naltrexone administration is relatively ineffective in reducing food intake and inducing body weight loss in obese humans, are thus in contrast with reports that administration of opiate antagonist agents promote significant reductions of food intake and attenuations of body weight gain in experimental animals.

Role of brown adipose tissue in thermogenesis induced by overfeeding a diet containing medium chain triglyceride.

The role of brown adipose tissue in the mechanism of medium chain triglyceride (MCT)-induced thermogenesis was investigated. Under anesthesia, the interscapular brown adipose tissue (IBAT) was excised in male Sprague-Dawley rats, and the animals were fitted with gastrostomy tubes. After a 10-day recovery period, the animals were divided into two groups: one group received a diet containing MCT as 50% of calories, and the other group received an isocaloric diet containing long chain triglyceride (LCT). The diets were fed for 6 wk at a level of calorie intake that was 150% of the ad libitum intake of a parallel control group. During the last week of the study, resting and norepinephrine (NE)-stimulated O2 consumption and CO2 production were measured in a Noyons diaferometer. At the end of 6 wk, the animals were weighed and killed. The individual fat pads were dissected and weighed, and an aliquot of the right retroperitoneal fat pad was used to measure adipocyte size and number. The results showed that body weight and adipocyte size (but not adipocyte number) were significantly smaller in the MCT-fed compared to the LCT-fed animals. Resting as well as maximal NE-stimulated oxygen consumption values were significantly higher in the MCT-fed than the LCT-fed rats. It is concluded that the enhanced thermogenesis induced by MCT persists despite the absence of IBAT and that the phenomenon is likely related to more extensive oxidation of MCT- in contrast to LCT-derived fatty acids, thus leading to increased oxygen consumption, enhanced dissipation of energy as heat and diminished efficiency of weight gain and deposition of body fat.

[Vascular access in hemodialysis. Surveillance and role of percutaneous transluminal angioplasty]. / Accessi vascolari in emodialisi. Sorveglianza e ruolo dell'angioplastica percutanea transluminale.

The function of vascular shunts in hemodialysis plays a vital role for the efficiency and effectiveness of replacement therapy. A study was performed in 147 patients undergoing periodical hemodialysis with distal FAV (no = 86), proximal FAV (no = 33), PTFE grafts (no = 23), Canaud-Tesio catheters (no = 7). A protocol for function evaluation was developed which also included the calculation of overall recirculation (R), that was found to be 10.8 + 7% (using the three blood sample method). In 28/143 patients the monitoring protocol recommended the use of angiography which identified abnormalities in 78% of cases, before the onset of thrombotic phenomena. In particular, surgical radiology was able to resolve 94% of cases in which angiography revealed a stenosis using percutaneous transluminal angioplasty and/or the insertion of one or more stents.

Low cost reclamation using the Advanced Integrated Wastewater Pond Systems Technology and reverse osmosis.

The sustainability of wastewater reclamation and reuse schemes is often limited by the increase in salt concentration that occurs with each water use. In this pilot study, we show that the cost of reclaiming wastewater and removing salt can be dramatically decreased by integrating recent advances in wastewater pond design, solids separation equipment, and membrane technology. Effluent from an AIWPS Facility was clarified in a Krofta Supracell Dissolved Air Flotation (DAF) unit and a Slow Sand Filter (SSF) prior to final treatment in an Expertise S.r.l. reverse osmosis (RO) unit. The ponds of the AIWPS Facility removed an average of 82% of soluble BOD and 80% of soluble nitrogen. Following clarification, filtration, and RO treatment, the pollutant removals were > 99% for soluble BOD, > 99% for soluble nitrogen, and 98% for TDS. Based on membrane fouling rate data, the cleaning interval for the RO membranes in a full-scale AIWPS-RO Facility would be over 100 days. This interval is on par with that typically seen in full-scale reclamation facilities treating secondary activated sludge effluent with microfiltration prior to reverse osmosis. A 4-MLD AIWPS-RO Facility is expected to produce permeate water at substantially lower cost and lower energy consumption (US $698 and 443 kWh per million liters treated) than a system of equal capacity using conventional activated sludge secondary treatment followed by microfiltration and reverse osmosis (US $1274 and 911 kWh per million litres treated). This cost and energy differential is attributable to the lower capital and operating expenses of the AIWPS Technology in comparison with activated sludge.

WITHDRAWN: Proteinase 3 (PR3) gene is highly expressed in CBF leukemias and codes for a protein with abnormal nuclear localization that confers drug sensitivity.

Core-binding factor (CBF) leukemias are characterized by a high degree of sensitivity to high-dose cytarabine (ARA-C) treatment and by a relatively favorable prognosis compared with most other forms of adult acute myeloid leukemia (AML). The molecular basis of the response to chemotherapy is still being analyzed. The proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells, and when it is abnormally expressed, it confers factor-independent growth to hematopoietic cells. In this study, we analyzed the expression levels of PR3 in 113 AML patients. PR3 is highly expressed in AML, mainly in CBF leukemias in which PR3 is not only expressed, but also abnormally localized within the nuclear compartment. Nuclear PR3 results in cleavage of nuclear factor (NF)-kappaB p65 into an inactive p56 subunit lacking any transcriptional activity. The nuclear localization of PR3 is responsible for increased proliferation, apoptosis arrest and increased sensitivity to high-dose ARA-C. This study provides a new molecular mechanism that is responsible for NF-kappaB inactivation and increased sensitivity to chemotherapy in CBF leukemias.Leukemia advance online publication, 14 January 2010; doi:10.1038/leu.2009.207.

Increase sensitivity to chemotherapeutical agents and cytoplasmatic interaction between NPM leukemic mutant and NF-kappaB in AML carrying NPM1 mutations.

Mutations in nucleophosmin (NPM) exon 12 and the resulting delocalization of NPM into the cytoplasm are the most specific and frequent cellular events in acute myeloid leukemia patients (AML) with normal karyotype. Cytoplasmatic NPM (NPMc+) is associated with responsiveness to chemotherapy and better prognosis. The activation of nuclear factor-kappaB (NF-kappaB) has been demonstrated to occur in a subset of AML patients and is thought to induce resistance to many chemotherapeutical agents. In this study, we demonstrate the increased in vitro sensitivity of NPMc+ cells to chemotherapeutical agents and their reduced NF-kappaB activity. Furthermore, we provide evidence of the interaction between NPMc+ and NF-kappaB in the cytoplasm, resulting in the sequestration and inactivation of NF-kappaB. The cytosolic localization and consequent inactivation of NF-kappaB justifies the reduced NF-kappaB DNA-binding activity observed in NPMc+ patients. These data, taken together, may provide a possible explanation for the increased rate of chemosensitivity observed among the NPMc+ patients.

NF-kB inhibition as a strategy to enhance etoposide-induced apoptosis in K562 cell line.

NF-kB is a transcription factor that mediates antiapoptotic signals in several cancer cell lines. Here we have demonstrated that the cytotoxic drug, Etoposide, activates NF-kB in K562, a chronic myeloid leukemia blast crisis cell line. Treatment with the NF-kB inhibitors MG-132, Bay11-7082, and Resveratrol impedes Etoposide-induced NF-kB activation, rendering K562 sensitive to Etoposide-induced apoptosis. Stable expression of mutant form of IkB-alpha, which retains NF-kB inactive in the cytoplasm of cells, confirmed the data obtained with molecular inhibitors. Both inhibitors and stable expression of SR-IkB are associated with down-modulation of the antiapoptotic protein Bcl-xL, suggesting that the survival pathway activated by Etoposide involves NF-kB-mediated Bcl-xL expression.

Detection of subtle phenotypes: the case of the cell adhesion molecule csA in Dictyostelium.

Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell-cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell-cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.

Insulin increases body fat despite control of food intake and physical activity.

The effect of experimentally induced hyperinsulinemia on body composition was studied in rats with food intakes precisely controlled by intragastric feeding and physical activity manipulated by sedation with chlordiazepoxide (CDP). Male Sprague-Dawley rats (n = 38) were fitted surgically with gastrostomy tubes. After 8 days the animals were divided into four groups. Group 1 received daily injections of protamine-zinc insulin; group 2 received daily injections of saline; group 3 received the same insulin doses as group 1 plus daily administration of CDP mixed with the diet; group 4 received daily injections of saline plus CDP in the diet. All groups were tubefed identical amounts of semiliquid diet via gastrostomy. Physical activity was measured by electronic monitor. After 4 wk the rats were killed. The insulin-treated groups (1 and 3) had significantly larger fat depots and larger mean fat cell size than the noninsulin-treated groups (2 and 4). This increase in fat occurred concurrently with a decrease in carcass protein and water. Physical activity, as measured, was unaltered by insulin but was significantly reduced by CDP. Treatment with CDP only increased the dorsal fat depot and liver weight but had no significant effect on total dissected fat depots and had a reductive effect on carcass protein. In conclusion insulin treatment enhanced the efficiency of conversion of energy intake into fat energy stores.

A new method for estimation of body composition in the live rat.

Measurement of total body electrical conductivity (TOBEC) has been used to estimate lean and fat content of meat based on the principle that electrical conductivity of lean tissue is far greater than that of fat. This approach was used to estimate body composition of live rats. An instrument designed for commercial analysis of ground meat (DjMe 100) was used to measure TOBEC in 30 male Sprague-Dawley rats (197-433 g). Individual TOBEC values were obtained in 20 seconds and repeated twice for each rat. The animals were then killed with ether, hair was shaved, lungs collapsed and body density measured hydrostatically. Carcasses were homogenized and analyzed for fat, nitrogen, and water. A high correlation was found between TOBEC and lean body mass by densitometry (r = .97) and between TOBEC and fat-free mass derived from direct carcass analysis (r = .97). Rats weighing up to 450 g could be accommodated in this particular instrument. Measurement of TOBEC should prove useful in estimating body composition and monitoring its changes in live rats and other small laboratory animals.