Access to Enantiomerically Pure cis‐ and trans‐β‐Phenylproline by High‐Performance Liquid Chromatography Resolution

The preparation of all four stereoisomers of the proline analog that bears a phenyl group attached to the β carbon either cis or trans to the carboxylic acid (cis‐ and trans‐β‐phenylproline, respectively) has been addressed. The methodology developed allows access to multigram quantities of the target amino acids in enantiomerically pure form and suitably protected for use in peptide synthesis. Racemic precursors of cis‐β‐phenylproline and trans‐β‐phenylproline were prepared from easily available starting materials and subjected to high‐performance liquid chromatography enantioseparation. Semipreparative columns (250 × 20 mm) containing chiral stationary phases based on amylose (Chiralpak IA) (Daicel‐Chiral Technologies Europe, Illkirch, France) or cellulose (Chiralpak IC) were used respectively for the resolution of the cis‐ and trans‐β‐phenylproline precursors. Chirality, 24:1082‐1091, 2012. © 2012 Wiley Periodicals, Inc.     

Effects of 13-cis-retinoic acid on hindbrain and craniofacial morphogenesis in long-tailed macaques (Macaca fascicularis)

Hindbrain and craniofacial development during early organogenesis was studied in normal and retinoic acid-exposed Macaca fascicularis embryos. 13-cis-retinoic acid impaired hindbrain segmentation as evidenced by compression of rhombomeres 1 to 5. Immunolocalization with the Hoxb-1 gene product along with quantitative measurements demonstrated that rhombomere 4 was particularly vulnerable to size reduction. Accompanying malformations of cranial neural crest cell migration patterns involved reduction and/or delay in pre- and post-otic placode crest cell populations that contribute to the pharyngeal arches and provide the developmental framework for the craniofacial region. The first and second pharyngeal arches were partially fused and the second arch was markedly reduced in size. The otocyst was delayed in development and shifted rostrolaterally relative to the hindbrain. These combined changes in the hindbrain, neural crest, and pharyngeal arches contribute to the craniofacial malformations observed in the retinoic acid malformation syndrome manifested in the macaque fetus.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Simultaneous determination of all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites and all-trans-retinol in human plasma by high-performance liquid chromatography

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.     

Vibrational circular dichroism as a probe of solid‐state organisation of derivatives of cyclic β‐amino acids: Cis‐ and trans‐2‐aminocyclobutane‐1‐carboxylic acid

Peptide models built from cis‐ and trans‐2‐aminocyclobutane‐1‐carboxylic acids (ACBCs) are studied in the solid phase by combining Fourier‐transform infrared spectroscopy (FTIR) absorption spectroscopy, vibrational circular dichroism (VCD), and quantum chemical calculations using density functional theory (DFT). The studied systems are N‐tert‐butyloxycarbonyl (Boc) derivatives of 2‐aminocyclobutanecarboxylic acid (ACBC) benzylamides, namely Boc?(cis‐ACBC)?NH?Bn and Boc?(trans‐ACBC)?NH?Bn. These two diastereomers show very different VCD signatures and intensities, which of the trans‐ACBC derivative being one order of magnitude larger in the region of the ν (CO) stretch. The spectral signature of the cis‐ACBC derivative is satisfactorily reproduced by that of the monomer extracted from the solid‐state geometry of related ACBC derivatives, which shows that no long‐range effects are implicated for this system. In terms of hydrogen bonds, the geometry of this monomer is intermediate between the C6 and C8 structures (exhibiting a 6‐ or 8‐membered cyclic NH?O hydrogen bond) previously evidenced in the gas phase. The benzyl group must be in an extended geometry to reproduce satisfactorily the shape of the VCD spectrum in the ν (CO) range, which qualifies VCD as a potential probe of dispersion interaction. In contrast, reproducing the IR and VCD spectrum of the trans‐ACBC derivative requires clusters larger than four units, exhibiting strong intermolecular H‐bonding patterns. A qualitative agreement is obtained for a tetramer, although the intensity enhancement is not reproduced. These results underline the sensitivity of VCD to the long‐range organisation in the crystal.     

cis‐Regulatory analysis for later phase of anterior neuroectoderm‐specific foxQ2 expression in sea urchin embryos

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis‐regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis‐regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half‐life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4–9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension‐class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.     

Cell‐autonomous signal transduction in the Xenopus egg Wnt/β‐catenin pathway

Wnt proteins are thought to bind to their receptors on the cell surfaces of neighboring cells. Wnt8 likely substitutes for the dorsal determinants in Xenopus embryos to dorsalize early embryos via the Wnt/β‐catenin pathway. Here, we show that Wnt8 can dorsalize Xenopus embryos working cell autonomously. Wnt8 mRNA was injected into a cleavage‐stage blastomere, and the subcellular distribution of Wnt8 protein was analyzed. Wnt8 protein was predominantly found in the endoplasmic reticulum (ER) and resided at the periphery of the cells; however, this protein was restricted to the mRNA‐injected cellular region as shown by lineage tracing. A mutant Wnt8 that contained an ER retention signal (Wnt8‐KDEL) could dorsalize Xenopus embryos. Finally, Wnt8‐induced dorsalization occurred only in cells injected with Wnt8 mRNA. These experiments suggest that the Wnt8 protein acts within the cell, likely in the ER or on the cell surface in an autocrine manner for dorsalization.     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Induction of Normal Cardiovascular Development in the Vitamin A-Deprived Quail Embryo by Natural Retinoids

The biological activity of various natural retinoids and the time "window" when vitamin A activity is required for normal cardiovascular development were examined in vitamin A-deprived Japanese quail embryos. The administration of 1 μg of retinol at the beginning of incubation resuited in normal cardiovascular development in 97% of embryos; retinoic acid was toxic at this dose level. Treatment of embryos with 0.1 μg of all-trans-retinol or 13-cis-retinoic acid at the beginning of incubation resulted in normal cardiovascular development in 47 and 12% of embryos, respectively; administration of these retinoids at other time points attenuated the percentage of embryos with normal cardiovascular development. Didehydroretinol, 0.1 μg, and 9-cis-retinoic acid, 0.1 μg, were inactive at all time points examined; 9-cis-retinoic acid did not enhance the biological activity of all-trans-retinoic acid. All-trans-retinoic acid, 0.1 μg, administered during 22-28 hr of incubation induced normal cardiovascular development in 20-34% of embryos; biological activity was optimal when it was administered at 24 hr. All retinoids tested were inactive in establishing normal cardiovascular development when administered at 36 hr of incubation or later. The studies suggest that all-trans-retinoic acid is the biologically active form of vitamin A required for normal cardiovascular development in the avian embryo. There is a critical time point within the first 22-28 hr of quail embryogenesis when all-trans-retinoic acid initiates events that lead to normal cardiovascular development.     

High Diversity of Indigenous Populations of Dalmatian Sage (Salvia officinalis L.) in Essential‐Oil Composition

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential‐oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis‐thujone, camphor, trans‐thujone, 1,8‐cineole, β‐pinene, camphene, borneol, and bornyl acetate) formed 78.13–87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and β‐pinene, β‐pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans‐thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis‐thujone from those rich in trans‐thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis‐thujone; trans‐tujone, and camphor/β‐pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.     

Enantiopurity and absolute configuration determination of arene cis‐dihydrodiol metabolites and derivatives using chiral boronic acids

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis‐1,2‐diol metabolites, including boronate formation, using racemic or enantiopure (+) and (?)‐2‐(1‐methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono‐ and poly‐cyclic arene cis‐dihydrodiol, cyclohex‐2‐en‐1‐one cis‐diol, heteroarene cis/trans‐2,3‐diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2‐[1‐methoxy‐2,2‐dimethylpropyl]phenyl boronic acid (MDPBA) and 2‐[1‐methoxy‐1‐phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis‐diol metabolites, are presented.     

Epithelial‐to‐mesenchymal transition in epicardium is independent of snail1

The epicardium is the outer epithelial covering the heart. This tissue undergoes an epithelial‐to‐mesenchymal transition (EMT) to generate mesenchymal epicardial‐derived cells (EPDCs) that populate the extracellular matrix of the subepicardium and contribute to the development of the coronary vessels and cardiac interstitial cells. Although epicardial EMT plays a crucial role in heart development, the molecular regulation of this process is incompletely understood. Here we examined the possible role of the EMT regulator Snail1 in this process. Snail1 is expressed in the epicardium and EPDCs during mouse cardiac development. To determine the function of Snail1 in epicardial EMT, we deleted Snail1 in the epicardium using Wt1‐ and Tbx18‐Cre drivers. Unexpectedly, epicardial‐specific Snail1 mutants are viable and fertile and do not display any obvious morphological or functional cardiac abnormalities. Molecular analysis of these mice reveals that epicardial EMT occurs normally, and epicardial derivatives are established in these mutants. We conclude that Snail1 is not required for the initiation and progression of embryonic epicardial EMT. genesis 51:32–40, 2013. © 2012 Wiley Periodicals, Inc.     

Retinoic acid is a negative physiological regulator of N‐cadherin during early avian heart morphogenesis

The vitamin A‐deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A‐active form, retinoic acid (RA). Here we examine the role of N‐cadherin (N‐cad) in RA‐regulated early cardiovascular morphogenesis. N‐cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N‐cad overproducing VAD embryos reveal N‐cad involvement in the RA‐regulated cardiovascular development and suggest that N‐cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N‐cad. We hypothesize that a critical endogenous level of N‐cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage‐specific, developmentally regulated RA signaling.