Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.     

Immunohistochemical Localization of Mitochondrial Fatty Acid ��-Oxidation Enzymes in Rat Testis

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid β-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid β-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid β-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis. (J Histochem Cytochem 58:195–206, 2010)     

TGFβ and Retinoic Acid Intersect in Immune-Regulation

Transforming growth factor (TGFβ) prevents TH1 and TH2 differentiation and converts naïve CD4 cells into Foxp3-expressing T regulatory (Treg) cell1, 2. In sharp contrast, in the presence of pro-inflammatory cytokines, including IL-6, TGFβ not only inhibits Foxp3 expression but also promotes the differentiation of pro-inflammatory IL17-producing CD4 effector T (TH17) cells3-5. This reciprocal TGFβ-dependent differentiation imposes a critical dilemma between pro- and anti-inflammatory immunity and suggests that a sensitive regulatory mechanism must exist to control TGFβ-driven TH17 effector and Treg differentiation. A vitamin A metabolite, retinoic acid (RA), was recently identified as a key modulator of TGFβ-driven immune deviation capable of suppressing TH17 differentiation while promoting Foxp3+Treg generation 6-10.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of beta-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of beta-carotene (mg beta-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

Cytochemical Localization of Pectinase Activity in Pollen Mother Cells of Tobacco During Meiotic ProphaseⅠand Its Relation to the Formation of Secondary Plasmodesmata and Cytoplasmic Channels

Pectinase activity was localized at the ultrastructural level in pollen mother cells of tobacco (Nicotiana tabacum L.) during meiotic prophaseⅠto elucidate its role in the biogenesis of secondary plasmodesma (sPD) and cytoplasmic channel (CC). At the leptotene stage the enzyme was mainly present in the cisternae of smooth endoplasmic reticulum (SER) and their derived vesicles, but absent in the Golgi body and Golgi vesicles. Later at the zygotene stage, when sPDs and CCs were actively formed, strong pectinase activity was observed not only in the SER cisternae and their derived vesicles but also in the cell wall, especially in the vicinity of or within both simple and branched plasmodesmata, notably along the middle lamellae, which also characterized the sites of CCs being formed. The presence of exocytotic vesicles containing reaction products suggests that pectinase shares the same excretive pathway as that used by cellulase for its delivery into the wall, i.e. in active form via smooth endoplasmic reticulum (ER) and its derived vesicles by exocytosis. In combination with cellulase, pectinase also promotes the secondary formation of plasmodesmata and CCs by specifically digesting the pectin in middle lamella.     

Localization of Cholesterol and Fatty Acid in a Model Lipid Membrane: A?Neutron Diffraction Approach

The intercellular lipid matrix of the skin’s stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.     

Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

Microarray analyses of gene expression are widely used, but reports of the same analyses by different groups give widely divergent results, and raise questions regarding reproducibility and reliability. We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes. These reports show substantial differences in their results. In this article, we review the methodology, results, and potential causes of differences in these applications of microarrays. Finally, we suggest practices to improve the reliability and reproducibility of microarray experiments.