Role of signaling pathways in the regulation of folate transport in ethanol-fed rats

Folate is an essential cofactor for normal cellular proliferation and tissue regeneration. Alcohol-associated folate deficiency is common, primarily due to intestinal malabsorption, the mechanism of which needs attention. The aim of the present study was to evaluate the regulatory events of folate transport in experimental alcohol ingestion. For this, male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and folate transport was studied in isolated intestinal epithelial cells across the crypt-villus axis. The role of different signaling pathways in folate transport regulation was evaluated independently to that of reduced folate carrier (RFC) expression. The results showed that differentiated cells of villus possess high folate uptake activity as compared to mid villus and crypt base cells. During chronic ethanol ingestion, decrease in transport was observed all along the crypt-villus axis but was more pronounced at proliferating crypt base stem cells. Studying the effect of modulators of signaling pathways revealed the folate transport system to be under the regulation of cAMP-dependent protein kinase A (PKA), the activity of which was observed to decrease upon alcohol ingestion. In addition, protein kinase C might have a role in folate transport regulation during alcoholic conditions. The deregulation in the folate transport system was associated with a decrease in RFC expression, which may result in lower transport efficiency observed at absorptive surface in alcohol-fed rats. The study highlights the role that perturbed regulatory pathways and RFC expression play in the decreased folate transport at brush border surface during alcohol ingestion.     

Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure

Enzymatic and non-enzymatic antioxidants serve as an important biological defense against environmental oxidative stress. Information on antioxidant defense in fish is meager despite that fish are constantly exposed to a myriad of environmental stress including the oxidants. This study, therefore, assesses the activities of antioxidant enzymes viz., glutathione peroxidase, catalase and glutathione S-transferase and the non-enzymatic antioxidants viz., glutathione and metallothionein in various tissues of freshwater fish Channa punctatus (Bloch), in response to short-term and long-term exposures to paper mill effluent. The fish were exposed to the effluent at a concentration of 1.0% (v/v) for 15, 30, 60 and 90 days. The exposure caused a time-dependent increase in glutathione level (P < 0.001), activities of glutathione peroxidase (P < 0.05 to P < 0.001), glutathione S-transferase (P < 0.001) and a marginal initial decrease in catalase activity in the liver (P < 0.01 to P < 0.001). Metallothionein was induced in liver after 60 days of exposure. Two isoforms of metallothionein were detected. Catalase activity also increased 60 days afterwards. Antioxidant pattern was different in gill and kidney showing that liver was more resistant to oxidative damage as compared to gills and kidney. Our results demonstrate a pollutant-induced adaptive response in fish. In addition, levels of enzymatic and non-enzymatic tissue antioxidants may serve as surrogate markers of exposure to oxidant pollutants in fish.     

Synthesis and SAR comparative studies of 2-allyl-4-methoxy-1-alkoxybenzenes as 15-lipoxygenase inhibitors

A group of 2-alkoxy-5-methoxyallylbenzene were designed, synthesised and evaluated as potential inhibitors of the soybean 15-lipoxygenase (SLO) on the basis of the eugenol and esteragol structures. Compound 4d showed the best half maximal inhibitory concentration (IC??) for SLO inhibition (IC???=?5.9?±?0.6 μM). All the compounds were docked in the SLO active site retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB entry: 1IK3) and showed that the allyl group of the synthetic compounds similar to the linoleic acid double bond, were oriented toward the Fe3+-OH moiety in the active site of the enzyme and this conformation was especially fixed by the hydrophobic interaction of the 2-alkoxy group with Leu?1?, Trp?1?, Val??? and Ile??2. It was concluded that the molecular volume and shape of the alkoxy moiety was a major factor in the inhibitory potency variation of the synthetic compounds.     

Neurosecretion of the mediodorsal cells of the central nervous system of the snail Helisoma duryi

Summary The neurosecretory mediodorsal cells that produce a putative growth hormone of the snail Helisoma duryi were studied in fast-growing virgin snails and in slow-growing reproducing snails. There are about 60 mediodorsal cells in clusters on each side of the cerebral commissure of the central nervous system, and they contain dense-cored granules which are 100–200 nm in diameter. The cells of virgin snails have dense Golgi bodies, scattered ER cisternae, and few granules, while those of reproducing snails have pale Golgi bodies, stacked ER cisternae, and numerous granules. Thus the mediodorsal cells of the virgin snails appear to be more active synthetically than those of the reproducing snails. The cells near the endocrine dorsal bodies contain many dorsal body precesses in their membrane interdigitations. There are junction-like interactions between some of the interdigitations. Gap junction-like contacts are seen between mediodorsal cells and glial cells. The axon endings of the mediodorsal cells at the neurohemal area in the labial nerve show more release profiles in virgin snails than in reproducing snails. A daily pattern of release has been observed in reproducing snails, and rates of release are higher in the evening than in the morning.     

Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.     

Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry

The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus?×?Poncirus) nucleus and C. reticulata or C. aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.     

Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice

Background

Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).

Methods

Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.

Results

In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.

Conclusion

Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth

The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P₃) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P₃ levels in macrophages. The modulation of cellular PI(3,4,5)P₃/PIP₂ ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.     

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.