Anabolic signaling and protein deposition are enhanced by intermittent compared with continuous feeding in skeletal muscle of neonates

Orogastric tube feeding is indicated for neonates with impaired ability to ingest and can be administered by intermittent bolus or continuous schedule. Our aim was to determine whether feeding modalities affect muscle protein deposition and to identify mechanisms involved. Neonatal pigs were overnight fasted (FAS) or fed the same amount of food continuously (CON) or intermittently (INT; 7 × 4 h meals) for 29 h. For 8 h, between hours 20 and 28, pigs were infused with [(2)H(5)]phenylalanine and [(2)H(2)]tyrosine, and amino acid (AA) net balances were measured across the hindquarters. Insulin, branched-chain AA, phenylalanine, and tyrosine arterial concentrations and whole body phenylalanine and tyrosine fluxes were greater for INT after the meal than for CON or FAS. The activation of signaling proteins leading to initiation of mRNA translation, including eukaryotic initiation factor (eIF)4E·eIF4G complex formation in muscle, was enhanced by INT compared with CON feeding or FAS. Signaling proteins of protein degradation were not affected by feeding modalities except for microtubule-associated protein light chain 3-II, which was highest in the FAS. Across the hindquarters, AA net removal increased for INT but not for CON or FAS, with protein deposition greater for INT. This was because protein synthesis increased following feeding for INT but remained unchanged for CON and FAS, whereas there was no change in protein degradation across any dietary treatment. These results suggest that muscle protein accretion in neonates is enhanced with intermittent bolus to a greater extent than continuous feeding, mainly by increased protein synthesis.     

Pharmacological characterization of the ghrelin receptor mediating its inhibitory action on inflammatory pain in rats

Recent research suggests a role for ghrelin in the modulation of inflammatory disorders. However, the type of ghrelin receptor (GHS-R) involved in both the anti-inflammatory and anti-hyperalgesic actions of ghrelin remains to be characterized. In this study, we examined whether the inhibitory effect of ghrelin in the development of hyperalgesia and edema induced by intraplantar carrageenan administration depends on an interaction with GHS-R1a. Both central (1 nmol/rat, i.c.v.) and peripheral (40 nmol/kg, i.p.) administration of the selective GHS-R1a agonist EP1572 had no effect on carrageenan-induced hyperalgesia measured by Randall–Selitto test and paw edema. Furthermore, pre-treatment with the selective GHS-R1a antagonist, d-lys³-GHRP-6 (3 nmol/rat, i.c.v.) failed to prevent the anti-hyperalgesic and anti-inflammatory effects exerted by central ghrelin administration (1 nmol/rat), thus indicating that the type 1a GHS-R is not involved in these peptide activities. Accordingly, both central (1 and 2 nmol/rat, i.c.v.) and peripheral (40 and 80 nmol/kg, i.p.) administration of desacyl-ghrelin (DAG), which did not bind GHS-R1a, induced a significant reduction of the hyperalgesic and edematous activities of carrageenan. In conclusion, we have shown for the first time that DAG shares with ghrelin an inhibitory role in the development of hyperalgesia, as well as the paw edema induced by carrageenan and that a ghrelin receptor different from type 1a is involved in the anti-inflammatory activities of the peptide.     

Vimentin regulates peripheral nerve myelination

Myelination is a complex process that requires coordinated Schwann cell-axon interactions during development and regeneration. Positive and negative regulators of myelination have been recently described, and can belong either to Schwann cells or neurons. Vimentin is a fibrous component present in both Schwann cell and neuron cytoskeleton, the expression of which is timely and spatially regulated during development and regeneration. We now report that vimentin negatively regulates myelination, as loss of vimentin results in peripheral nerve hypermyelination, owing to increased myelin thickness in vivo, in transgenic mice and in vitro in a myelinating co-culture system. We also show that this is due to a neuron-autonomous increase in the levels of axonal neuregulin 1 (NRG1) type III. Accordingly, genetic reduction of NRG1 type III in vimentin-null mice rescues hypermyelination. Finally, we demonstrate that vimentin acts synergistically with TACE, a negative regulator of NRG1 type III activity, as shown by hypermyelination of double Vim/Tace heterozygous mice. Our results reveal a novel role for the intermediate filament vimentin in myelination, and indicate vimentin as a regulator of NRG1 type III function.     

Diversity of locally useful tropical forest wild-plants as a function of species richness and informant culture

Different conservation values and perspectives can lead to divergent conservation objectives. Understanding such differences is crucial to developing more comprehensive and inclusive conservation approaches. Using plots, we assessed how numbers of useful species as reported by indigenous forest-dwelling people relate to plant species richness. We used 173 plots recording both trees and herbaceous vegetation and the knowledge of both Merap- and Punan-dominated communities in Malinau, Kalimantan (Indonesian Borneo). We used general linear models (GLMs) to characterise the relationships. Useful species increase with species richness in all cases. The relationship varied across culture and community and was not always linear. The proportion of tree species reported as useful by Merap (primarily agriculturalist) informants was not constant but declined significantly as plot diversity increased; this was not the case for Punan (primarily hunter-gatherer) informants. There was no decline for the reported proportion of useful herbs as richness increases, as assessed by either ethnic group. Communities with less wealth and less schooling generally reported a higher proportion of the useful species. We interpret these results in terms of how landscape patterns of plant diversity are experienced. Understanding of these relationships can help us develop a more explicit approach to weighing and reconciling different conservation values and management objectives in changing forest landscapes.     

Carbonic anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity for sulfonamide inhibitors

Human carbonic anhydrase (CA, EC 4.2.1.1) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Here we report an unexpected S-glutathionylation of hCA VII which has also been observed earlier in vivo for hCA III, another cytosolic isoform. Cys183 and Cys217 were found to be the residues involved in reaction with glutathione for hCA VII. The two reactive cysteines were then mutated and the corresponding variant (C183S/C217S) expressed. The native enzyme, the variant and the S-glutathionylated adduct (sgCA VII) as well as hCA III were fully characterized for their CO(2) hydration, esterase/phosphatase activities, and inhibition with sulfonamides. Our findings suggest that hCA VII could use the in vivo S-glutathionylation to function as an oxygen radical scavenger for protecting cells from oxidative damage, as the activity and affinity for inhibitors of the modified enzyme are similar to those of the wild type.     

Cell-to-cell signaling influences the fate of prostate cancer stem cells and their potential to generate more aggressive tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.     

Mechanism-based screen establishes signalling framework for DNA damage-associated G1 checkpoint response

DNA damage activates checkpoint controls which block progression of cells through the division cycle. Several different checkpoints exist that control transit at different positions in the cell cycle. A role for checkpoint activation in providing resistance of cells to genotoxic anticancer therapy, including chemotherapy and ionizing radiation, is widely recognized. Although the core molecular functions that execute different damage activated checkpoints are known, the signals that control checkpoint activation are far from understood. We used a kinome-spanning RNA interference screen to delineate signalling required for radiation-mediated retinoblastoma protein activation, the recognized executor of G(1) checkpoint control. Our results corroborate the involvement of the p53 tumour suppressor (TP53) and its downstream targets p21(CIP1/WAF1) but infer lack of involvement of canonical double strand break (DSB) recognition known for its role in activating TP53 in damaged cells. Instead our results predict signalling involving the known TP53 phosphorylating kinase PRPK/TP53RK and the JNK/p38MAPK activating kinase STK4/MST1, both hitherto unrecognised for their contribution to DNA damage G1 checkpoint signalling. Our results further predict a network topology whereby induction of p21(CIP1/WAF1) is required but not sufficient to elicit checkpoint activation. Our experiments document a role of the kinases identified in radiation protection proposing their pharmacological inhibition as a potential strategy to increase radiation sensitivity in proliferating cancer cells.     

Differential Effects of Lovastatin on Cisplatin Responses in Normal Human Mesothelial Cells versus Cancer Cells: Implication for Therapy

The cancer killing efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is limited by their side effects to normal tissues. Therefore, research efforts optimizing the safety and efficacy of those agents are clinically relevant. We did screen for agents that specifically protect normal human mesothelial cells against CDDP without reducing the cancer cell killing efficacy. Lovastatin was identified from the screen. Lovastatin at a pharmacologically relevant concentration strongly arrested the proliferation of normal cells, whereas cancer cells were less affected. CDDP-induced DNA damage response was not activated and normal cells showed enhanced tolerance to CDDP when normal cells were treated with the combination of CDDP and lovastatin. We demonstrate that interfering with protein geranylgeranylation is involved in the lovastatin-mediated CDDP protective effect in normal cells. In contrast to normal cells, in cancer cells lovastatin did not change the CDDP-induced response, and cancer cells were not protected by lovastatin. Furthermore, lovastatin at the pharmacological relevant concentration per se induced DNA damage, oxidative stress and autophagy in cancer cells but not in normal mesothelial cells. Therefore, our data suggest that lovastatin has a potential to improve the therapeutic index of cisplatin-based therapy.     

Arsenic trioxide and ascorbic acid interfere with the BCL2 family genes in patients with myelodysplastic syndromes: an ex-vivo study

ABSTRACT: BACKGROUND: Arsenic Trioxide (ATO) is effective in about 20% of patients with myelodysplasia (MDS); its mechanisms of action have already been evaluated in vitro, but the in vivo activity is still not fully understood. Since ATO induces apoptosis in in vitro models, we compared the expression of 93 apoptotic genes in patients' bone marrow before and after ATO treatment. For this analysis, we selected 12 patients affected by MDS who received ATO in combination with Ascorbic Acid in the context of the Italian clinical trial NCT00803530, EudracT Number 2005-001321-28. METHODS: Real-time PCR quantitative assays for genes involved in apoptosis were performed using TaqMan(R) Assays in 384-Well Microfluidic Cards "TaqMan(R) Human Apoptosis Array". Quantitative RT-PCR for expression of EVI1 and WT1 genes was also performed. Gene expression values (Ct) were normalized to the median expression of 3 housekeeping genes present in the card (18S, ACTB and GAPDH). RESULTS: ATO treatment induced up-regulation of some pro-apoptotic genes, such as HRK, BAK1, CASPASE-5, BAD, TNFRSF1A, and BCL2L14 and down-regulation of ICEBERG. In the majority of cases with stable disease, apoptotic gene expression profile did not change, whereas in cases with advanced MDS more frequently pro-apoptotic genes were upregulated. Two patients achieved a major response: in the patient with refractory anemia the treatment down-regulated 69% of the pro-apoptotic genes, whereas 91% of the pro-apoptotic genes were up-regulated in the patient affected by refractory anemia with excess of blasts-1. Responsive patients showed a higher induction of BAD than those with stable disease. Finally, WT1 gene expression was down-regulated by the treatment in responsive cases. CONCLUSIONS: These results represent the basis for a possible association of ATO with other biological compounds able to modify the apoptotic pathways, such as inhibitors of the BCL2 family.