Increasing the expression of calcium-permeable TRPC3 and TRPC7 channels enhances constitutive secretion

The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane

Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans

The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing

Metabolic Profiling of Hypoxic Cells Revealed a Catabolic Signature Required for Cell Survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography–mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network

Functional validation of the truncated UNC-63 acetylcholine receptor subunit in levamisole resistance

International audienceLevamisole is a broad-spectrum anthelmintic which permanently activates cholinergic receptors from nematodes, inducing a spastic paralysis of the worms. Whereas this molecule is widely used to control parasitic nematodes impacting livestock, its efficacy is compromised by the emergence of drug-resistant parasites. In that respect, there is an urgent need to identify and validate molecular markers associated with resistance. Previous transcriptomic analyses revealed truncated cholinergic receptor subunits as potential levamisole resistance markers in the trichostrongylid nematodes Haemonchus contortus, Telodorsagia circumcincta and Trichostrongylus colubriformis. In the present study we used the Xenopus oocyte, as well as the free-living model nematode Caenorhabditis elegans, as heterologous expression systems to functionally investigate truncated isoforms of the levamisole-sensitive acetylcholine receptor (L-AChR) UNC-63 subunit. In the Xenopus oocyte, we report that truncated UNC-63 from C. elegans has a strong dominant negative effect on the expression of the recombinant C. elegans L-AChRs. In addition, we show that when expressed in C. elegans muscle cells, truncated UNC-63 induces a drastic reduction in levamisole susceptibility in transgenic worms, thus providing the first known functional validation for this molecular marker in vivo

Database marketing management for agricultural lenders

Many ag lenders are using databases to improve marketing techniques and customer segmentation in the competitive agricultural lending environment. Continual awareness of needs and perceptions of the customers is a critical element of the success of a progressive lending institution. Lenders are examining product and service use by agricultural customers to determine relationship between business and personal characteristics. The latter method is an attempt to gain a competitive edge by courting a customer at point of anticipation rather than point of sale

Database marketing management for agricultural lenders

Many ag lenders are using databases to improve marketing techniques and customer segmentation in the competitive agricultural lending environment. Continual awareness of needs and perceptions of the customers is a critical element of the success of a progressive lending institution. Lenders are examining product and service use by agricultural customers to determine relationship between business and personal characteristics. The latter method is an attempt to gain a competitive edge by courting a customer at point of anticipation rather than point of sale

Studies on glc-3, a potential target of Ivermectin in parasitic nematodes

International audienceThe free-living nematode Caenorhabditis elegans has been used for many years as an expression system for genes from parasitic species. We wished to further develop and improve this system by using CRISPR/Cas9 to delete specific genes from C. elegans and replace them with single copies of orthologous genes from the parasite, Haemonchus contortus. Initial experiments focussed on glc-3 which encodes a subunit of the glutamate-gated chloride channels, the target of the avermectin/milbemycin family of anthelmintics. We cloned the promoters from the glc-3 genes of both species and compared the expression patterns of mCherry under the control of both promoters. The C. elegans glc-3 promoter drove expression in a subset of head interneurons, as previously reported whereas the H. contortus promoter drove expression in a pharyngeal motoneuron, M4. We were able to generate heterozygous worms in which one copy of glc-3 was deleted, but we could never obtain homozygous knock-outs. Further investigation of the mRNAs encoded by glc-3 revealed a novel transcript, glc-3T, which encodes a severely truncated form of GLC-3. The presence of such truncated transcripts may explain the unexpected difficulties encountered in attempting to knock out ion channel genes in C. elegans

Overexpression of TGN38/41 leads to mislocalisation of γ-adaptin

AbstractTGN38 and TGN41 are isoforms of a monotopic integral membrane protein which recycles between the trans Golgi network (TGN) and the cell surface, but which, at steady state, is predominantly located in the TGN. Full-length and truncated versions of rat TGN38/41 have been expressed in monkey (COS) and human (Heb7a) cells under the control of the heavy metal inducible Metallothionein IIA promoter. This has allowed the regulated expression of TGN38/41 protein constructs to different levels in the transfected cells. These studies show that (i) controlled overexpression of TGN38/41 results in mislocalisation to parts of the endocytic pathway, (ii) a truncated version of TGN38/41, lacking the cytoplasmic domain, remains in the TGN, and (iii) there is a direct or indirect interaction between the cytoplasmic domain of TGN38/41 and γ-adaptin