In vivo isomerization of all-trans- to 11-cis-retinoids in the eye occurs at the alcohol oxidation state

The vertebrate biochemical pathway for regeneration of visual pigments in the living eye after bleaching is largely uncharacterized. Since isomerization of an all-trans-retinoid to an 11-cis-retinoid could conceivably occur via the aldehyde, alcohol, or ester forms of vitamin A, it is important to determine the oxidation state of the retinoid that is isomerized in vivo. To address this problem, light-adapted rats and frogs were injected intraperitoneally with a mixture of [15-3H]-all-trans-retinol and [15-14C]-all-trans-retinol. After 4 or 24 h of dark adaptation, labeled retinoids in the animal's eyes were analyzed. All rats had the expected 50% loss of 3H label (relative to 14C) in 11-cis-retinal, a loss of 3H that must occur when [15-3H]retinol is oxidized to retinal. 11-cis-Retinyl esters in the rats' eyes at 4 h retained 67% of the 3H label, and this could be increased to 81% when the rats were pretreated with 4-methylpyrazole, an alcohol dehydrogenase inhibitor known to inhibit dark adaptation. This result demonstrates that retinoid isomerization occurs at the alcohol oxidation state in the rat eye. Had it occurred at the aldehyde oxidation state, at least 50% of the 3H in the 11-cis-retinyl esters would have been lost. The importance of this isomerization pathway is emphasized by the observation that dark-adapting rats whose alcohol dehydrogenase(s) had been inhibited by 4-methylpyrazole had increased amounts of 11-cis-retinyl ester in their eyes relative to control rat eyes, a result that is understandable only if retinoids are isomerized in vivo at the alcohol oxidation state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific inactivation of isomerohydrolase activity by 11-cis-retinoids

The endergonic trans-->cis isomerization of retinoids is an essential element in rhodopsin regeneration in vertebrates. All-trans-retinyl esters, which are generated by lecithin retinol acyltransferase (LRAT), are on the isomerization pathway. The critical isomerohydrolase activity, which catalyzes the trans-->cis isomerization/hydrolysis reaction of all-trans-retinyl esters, remains to be identified. It is demonstrated here that 11-cis-retinyl bromoacetate (cRBA) is a potent and specific inactivator of the bovine retinyl pigment epithelial (RPE) isomerohydrolase activity, with a measured K(I)=0.19 microM and a pseudo-first-order rate of inactivation k(inh)=1.83 x 10(-3) s(-1). This demonstrates that the isomerization is indeed enzyme-mediated. This inactivator should facilitate the identification and study of isomerohydrolase, or at least an essential component of it. Labeling of crude RPE membranes with 3H-cRBA reveals the presence of several labeled bands that may be isomerohydrolase candidates.     

Biosynthesis of 11-cis-retinoids and retinyl esters by bovine pigment epithelium membranes

Previously, we have shown that retina/pigment epithelium membranes from the amphibian can synthesize 11-cis-retinoids from added all-trans-retinol [Bernstein, P.S., Law, W.C., & Rando, R.R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853]. The activity was largely localized to the pigment epithelium. Here it is shown that, in the bovine system, the activity resides exclusively in the membranes of the pigment epithelium. Subcellular fractionation does not reveal a particular organelle where the activity resides. Washed bovine pigment epithelium membranes, which are devoid of retinoid redox activity, convert added all-trans-retinol to a mixture of 11-cis-retinol and its palmitate ester. all-trans-Retinal and all-trans-retinyl palmitate are not converted into 11-cis-retinoids by the membranes. The membranes show substantial ester synthetase activity, producing large amounts of all-trans-retinyl palmitate. Diverse chemical reagents, such as ethanol, hydroxylamine, and p-(hydroxymercuri)benzoate, inhibit both ester synthetase and isomerase activities in a roughly parallel fashion, suggesting a possible functional linkage between the two activities.     

Stereochemical inversion at C-15 accompanies the enzymatic isomerization of all-trans- to 11-cis-retinoids

all-trans-Retinol (vitamin A) is processed by membranes from the pigment epithelium of the amphibian or bovine eye to form 11-cis-retinoids. When the isomerization reaction is performed with either [15(S)-3H,14C]-all-trans-retinol or [15(R)-3H,14C]-all-trans-retinol as substrate, the resultant 11-cis-retinals, formed by the in vitro enzymatic oxidation of the retinols, retain their 3H in the former case and lose it in the latter. The ocular all-trans- (pro-R specific) and 11-cis-retinol (pro-S specific) dehydrogenases operate with different stereochemistries with respect to the prochiral methylene hydroxyl centers of their substrates. Inversion of stereochemistry at the prochiral retinol centers was shown to accompany the isomerization process in both the amphibian and bovine systems. The 11-cis-retinol formed from [15(S)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(R)-3H,14C]-all-trans-retinol. The 11-cis-retinol formed from [15(R)-3H,14C]-all-trans-retinol was chemically isomerized with I2 to produce [15(S)-3H,14C]-all-trans-retinol. The stereochemistry at the prochiral center of retinol is not affected by the I2-catalyzed double-bond isomerization process and, hence, inversion of stereochemistry at C-15 must accompany isomerization. The same inverted stereochemistry was found with the associated retinyl palmitates. Possible mechanistic reasons for the observed inversion of stereochemistry during isomerization are discussed.     

Inhibitory effect of combined administration with CPT-11 and 5-fluorouracil in vitro and in vivo

Combined antitumor activity of CPT-11 and 5-fluorouracil (5-FU) was evaluated in a human cultured cell line derived from lung cancer. After 24 h culture with SN-38 followed by 5-FU 24 h, synergistic effect was observed in the cell line. In addition, the antitumor effect of this combination was studied in in vivo experiments using Donryu rat with Yoshida sarcoma cells. CPT-11 and 5-FU synergistically inhibited tumor growth. There was no significant increase of toxicity as assessed by the body weights. These results might support for the combination with 5-FU and CPT-11 in a chemotherapy for cancer.     

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.     

Microbial conversion of bilirubin to urobilins in vitro and in vivo

No urobilins are formed from bilirubin in germ-free rats. To isolate and investigate the strains of intestinal microorganisms responsible for this transformation, a suitable test medium was adopted. The strength of the medium and a rather high initial pH were found to be of importance. In this medium, suspensions of rat faeces and a single strain, Cl. ramosum (G62), converted bilirubin to urobilins. Cultivations of Cl. ramosum (G62) together with E. coli significantly enhanced the conversion, whereas addition of 4 other bacterial strains was without the influence. The highest in vitro formation of the urobilins was about 10% of the bilirubin present. When the 6 strains investigated in vitro were established in EXG rats, the in vivo conversion of bilirubin to urobilins was found to be about 15%, compared to 70% in CONV rats.     

Adhesion of lactobacilli to polymer surfaces in vivo and in vitro

The ability of bacteria to attach to surfaces has been recognized as an important phenomenon, particularly for pathogenic organisms that utilize this capacity to initiate disease. The present investigation was undertaken to determine whether indigenous urogenital bacteria, lactobacilli, colonized prosthetic devices in vivo and in vitro and attached to specific polymer surfaces in vitro. Polyethylene intrauterine devices (IUDs) in place for 2 years were removed from six women who were asymptomatic and free of signs of cervical or uterine infection. Lactobacilli were found attached to the IUDs, as determined by culture, and fluorescent antibody and acridine orange staining techniques. This demonstrated that bacterial biofilms consisting of indigenous bacteria can occur on prosthetic devices without inducing a symptomatic infection. In vitro studies were then undertaken with well-documented lactobacilli strainsL. acidophilus T-13,L. casei GR-1, GR-2, and RC-17, andL. fermentum A-60. These organisms were found to adhere to IUDs and urinary catheters within 24 hours. A quantitative assay was designed to examine the mechanisms of adhesion ofL. acidophilus T-13 to specific polymer surfaces that are commonly used as prosthetic devices. The lactobacilli adhered optimally to fluorinated ethylene propylene when 10⁸ bacteria were incubated for 9 hours at 37°C in phosphate buffered saline, pH 7.1. Additional experiments verified that the lactobacilli adhered to polyethyleneterephthalate, polystyrene, and sulfonated polystyrene and to silkolatex catheter material. There was a linear relationship found between polymer hydrophobicity and bacterial adherence. These results demonstrate that lactobacilli bind to various surfaces in vivo and in vitro, and that the nature of the substratum can affect the colonization.     

Binding of actinomycin D to mRNA in vivo and in vitro

1. When rats received an intraperitoneal injection of [3H]actinomycin D, it bound to the RNA moiety of free and bound polysomes of rat liver. The labeling increased gradually up to 6 h or 6 + 6 h. The poly(A)-containing mRNA showed definite radioactivity and its specific activity was higher than that of rRNA, although the total radioactivity of rRNA was markedly higher than that of poly(A)-containing mRNAs. 2. An equilibrium dialysis method using rabbit globin mRNA showed that the binding constant of actinomycin D to globin mRNA was 0.056 x 10(6) M-1, and globin mRNA had 2 binding sites per mol for actinomycin D. 3. From the results of the present experiments and those described in the preceding paper, it is suggested that one of the mechanisms by which actinomycin D treatment in vivo and in vitro stimulates the template activity of mRNA may be binding to mRNA, which alters the conformation of mRNA.     

Isomerization of trans-diethylstilbestrol to pseudo-diethylstilbestrol

This study reports the formation and isolation of a diethyl-stilbestrol-dimethylsulfoxide (DES-DMSO) adjunct and Z-3, 4-di(p-hydroxyphenyl)-2-hexene (ψ-DES) from $\text{trans}$-DES. The presence of ψ-DES was indicated by NMR and mass spectrometry and confirmed by direct comparison to a reference sample. High resolution NMR(360 MHz) along with the comparison of the chemical shift values of methine and methyl protons attached to carbon-carbon double bonds in Z and E isomers of 3-substituted-2-pentenes and dienestrol derivatives were used in postulating the Z-stereochemistry for ψ-DES. A NMR additive increment method was useful for the comparison of the chemical shift values of methine protons in ψ-DES and other literature compounds. Nuclear Overhauser Enhancement (NOE) confirmed the Z-stereochemistry of ψ-DES.     

all-trans-retinoids and dihydroretinoids as probes of the role of chromophore structure in rhodopsin activation

The absorption of a photon of light by rhodopsin results in the cis to trans isomerization of the 11-cis-retinal Schiff base chromophore. In the studies reported here, an attempt is made to determine the mechanism of the energization of rhodopsin as it relates to the chemistry of the isomerization process and the geometrical state of the chromophore. Studies were performed with vitamin A analogues to probe this mechanism. Both 11-cis-7,8-dihydroretinal and 9-cis-7,8-dihydroretinal form bleachable pigments when combined with opsin. Photolysis of these pigments in the presence of G-protein results in the activation of the latter as revealed by its GTPase activity. Phosphodiesterase is also activated when it is included in the incubation. Therefore, the possibility that rhodopsin is energized by mechanisms involving photochemically induced charge transfer from the protonated Schiff base to the beta-ionone ring can be discarded. Further studies were conducted with all-trans-vitamin A derivatives to determine if these compounds can form the GTPase-activating state R*, a situation that is possible, in principle, by microscopic reversibility. Neither all-trans-retinal nor its oxime, when incubated with bovine opsin in the dark, caused activation of the GTPase, requiring at least a 5 kcal/mol energy gap between them. Furthermore, stoichiometric adducts of all-trans-retinoids and opsin were also unable to mediate activation of the GTPase. Since both all-trans-15,16-dihydroretinylopsin and all-trans-retinoylopsin possess an all-trans-retinoid permanently adducted to opsin, it can be concluded that the all-trans-retinoid chromophore-opsin linkage may be necessary but not sufficient to achieve activation of the visual pigment.     

Synthesis and evaluation in vitro and in vivo of a 11C-labeled cyclooxygenase-2 (COX-2) inhibitor

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.     

Gene delivery to pancreatic exocrine cells in vivo and in vitro

Background

Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein).

Results

The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1?C2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17?-oestradiol (E₂), the synthetic oestrogen 17??- ethinyloestradiol (EE₂), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE₂; 10?ng/L, E₂; 100?ng/L, after 72?h exposures). For the NP exposures, GFP expression was observed at 10???g NP/L after 72?h (100???g NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally.

Conclusion

Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.     

Sperm factors related to in vitro and in vivo porcine fertility

The prediction of sperm fertilizing ability has great economic importance for breeding herds when artificial insemination is used. Classical methods of semen evaluation generally measure the sperm concentration, progressive motility, percentage of viable cells, and acrosome morphology. These assays are poor in predicting sperm fertility, because only the samples with markedly poor quality can be detected. The development of new sperm tests that measure certain sperm functions is an attempt to solve this problem. On the other hand, the binding and penetration of the zona pellucida is one of the most important barriers the spermatozoa must overcome in the fertilization process. Also, the interaction with the oocyte plasma membrane appears to explain much of the variability in sperm fertilizing potential among fertile boars. Thus, the study of the relationship between sperm factors and in vitro fertility may be a good strategy and assays that include a study of gamete interaction may lead to a better way to predict male fertility than the routine laboratory evaluation of semen. This review will discuss the relationships between sperm factors and fertility in vitro and in vivo (AI trial) with both diluted and frozen-thawed semen. We will also try to analyze the problems and limitations related to the interpretation of boar sperm tests.