Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, function, and trafficking of the G protein-coupled cholecystokinin receptor

The lipid microenvironment of receptors can influence their conformation, function, and regulation. Cholecystokinin (CCK)-stimulated signaling is abnormal in some forms of hyperlipidemia, suggesting the possibility of unique sensitivity to its lipid environment. Here we examined the influence of cholesterol and sphingolipids on CCK receptors in model Chinese hamster ovary cell systems having lipid levels modified. Cholesterol was modulated chemically or metabolically, and sphingolipids were modulated using a temperature-sensitive cell line (SPB-1). Receptor conformation was probed with a fluorescent full agonist ligand, Alexa 488-conjugated Gly-[Nle(28,31)]CCK-(26-33), shown previously to decrease in anisotropy and lifetime when occupying a receptor in the active conformation (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Anisotropy and lifetime of this probe were increased and prolonged with cholesterol enrichment, and decreased and shortened with depletion of cholesterol or sphingolipids. The increase in these parameters with cholesterol enrichment may reflect change in CCK receptor conformation toward its inactive, uncoupled state. Indeed, cholesterol enrichment resulted in nonproductive agonist ligand binding, with affinity of binding higher than normal and calcium signaling in response to this reduced. In cholesterol- and sphingolipid-depleted states, the receptor moved into conformations that were less than optimal. With cholesterol depletion, both ligand binding and signaling were decreased, yet internalization and trafficking were unperturbed. With sphingolipid depletion, ligand binding and signaling were normal, but internalization and trafficking were markedly inhibited. Of note, normal transferrin receptor trafficking through the same clathrin-dependent pathway was maintained under these conditions. Thus, lipid microenvironment of the CCK receptor is particularly important, with different lipids having distinct effects.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

Enzyme Promiscuity: Engine of Evolutionary Innovation

Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions. Action on multiple substrates (substrate ambiguity) can be harnessed for performance of functions in the cell that supersede catalysis of a single metabolite. These functions include proofreading, scavenging of nutrients, removal of antimetabolites, balancing of metabolite pools, and establishing system redundancy. In this review, we present examples of enzymes that perform these cellular roles by leveraging substrate ambiguity and then present the structural features that support both specificity and ambiguity. We focus on the phosphatases of the haloalkanoate dehalogenase superfamily and the thioesterases of the hotdog fold superfamily.     

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH^−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH^−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH^−/− iPS cell lines, we aggregated FAH^−/−-iPS cells with tetraploid embryos and obtained entirely FAH^−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH^−/− mice. Then, we transduced FAH cDNA into the FAH^−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.     

Maternal hyperinsulinemia predisposes rat fetuses for hyperinsulinemia, and adult-onset obesity and maternal mild food restriction reverses this phenotype

We have previously shown that artificial rearing of newborn female rat pups on a high-carbohydrate (HC) milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype) and that the maternal HC phenotype was transmitted to their progeny (2-HC rats) because of fetal development in the HC female rat. The aims of this study were to investigate 1) the fetal adaptations that predisposed the progeny for the expression of the HC phenotype in adulthood and 2) whether the transfer of the HC phenotype to the progeny could be reversed by maternal food restriction. Fetal parameters such as plasma insulin and glucose levels, mRNA level of preproinsulin gene, pancreatic insulin content, and islet insulin secretory response in vitro were determined. On gestational day 21, 2-HC fetuses were hyperinsulinemic, had increased insulin content and mRNA level of the preproinsulin gene in their pancreata and demonstrated an altered glucose-stimulated insulin secretory response by isolated islets. Modification of the intrauterine environment in HC female rats was achieved by pair feeding them to the amount of diet consumed by age-matched control rats from the time of their weaning. This mild dietary restriction reversed their HC phenotype and also prevented the development of the HC phenotype in their progeny. These findings show that mal-programming of the progeny of the hyperinsulinemic-obese HC female for the expression of the HC phenotype is initiated in utero and that normalization of the maternal environment in HC female rats by mild food restriction resulted in the normal phenotype in their progeny.     

Decolorisation, biodegradation and detoxification of benzidine based azo dye

The present study deals with the decolorisation, biodegradation and detoxification of Direct Black-38, a benzidine based azo dye, by a mixed microbial culture isolated from an aerobic bioreactor treating textile wastewater. The studies revealed a biotransformation of Direct Black-38 into benzidine and 4-aminobiphenyl followed by complete decolorisation and biodegradation of these toxic intermediates. From cytotoxicity studies, it was concluded that detoxification of the dye took place after degradation of the toxic intermediates by the culture.     

Three New Species of Brevitobrilus (Nematoda) with a Discussion on Relationships within the Genus

Three new species of the genus BrevitobriusTsalolikhin, 1981 are described. Brevitobrilus glandulatus n. sp. is characterized by conspicuous sphincter between pars dilatata and uterus; two pairs of vaginal glands; spicules having elliptical capitula with small proximal stiffening piece; proximally-arcuate gubernaculum; S3 and S4 smaller than other supplements; S6 out of spicular range and 57-60 micropapillae. Brevitobrilus dimorphicus n. sp. is diagnosed by sexual dimorphism in labial sensilla and amphids; thick-walled rectum with a diverticulum protruding into intestinal lumen and males with boat-shaped spicules and S6 occasionally slightly smaller than other supplements. Brevitobrilus allahabadensis n. sp. possesses large amphids of 28-33% of corresponding labial diameter in both sexes; vagina and uterus with muscular, plicate walls; well developed sphincter between vas deferens and ejaculatory duct; capitulate spicules with sloping ventral and angular dorsal walls; S3, S4 and S6 smaller than other supplements, S6 close to cloaca and 28-37 micropapillae. The relationships of the species of genus Brevitobrilus have been assessed using morphological characters subjected to parsimony and a non cladistic key to identification of species is given.     

Sex‐specific effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic/steroid‐metabolizing enzymes and antioxidant status

Liver has evolved complex enzymatic mechanisms to detoxify a wide array of xenobiotic substances, ranging from dietary components to environmental toxins to pharmaceuticals. Activities of many steroid‐metabolizing enzymes in adult rat liver microsomes are sexually differentiated. Toxic effects of lead and cadmium on hepatic tissue have been well established in our earlier studies. We thus monitored the effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic‐ and steroid‐metabolizing enzyme activities in both male and female F1 generation postnatal day (PND) 56 rats. Adult pregnant female rats were treated subcutaneously [0.05 mg/(kg body wt. day)] with sodium acetate (control group), lead acetate, and cadmium acetate separately and in combination throughout the gestational and lactational period. Hepatic phase I xenobiotic‐metabolizing enzymes (NADPH‐ and NADH‐cytochrome c reductase) activities significantly decreased significantly in all the metal‐treated groups in both PND 56 male and female rats as compared with the control group. Hepatic phase II enzymes (uridine diphosphate‐glucuronosyl transferase, γ‐glutamyl transpeptidase, glutathione‐S‐transferase, 17‐β‐hydroxysteroid oxidoreductase) were also highly susceptible to all the metal‐treated groups. The observed alterations in the oxidative stress and biochemical parameters in the liver of F1 generation male and female rats resulted from an independent effect of lead and/or cadmium and also from their interaction. Results suggest that early developmental exposure to lead and cadmium both alone and in combination can suppress the hepatic xenobiotic‐metabolizing enzyme activities in the liver of F1 generation male and female rats in a sex‐dependent manner. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:419–431, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20305