Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Immunohistochemical demonstration of hyaluronan and its possible involvement in axolotl neural crest cell migration

Hyaluronan (HA), an extracellular matrix component, is involved mainly in the control of cell proliferation, neural crest and tumor cell migration, and wound repair. We investigated the effect of hyaluronan on neural crest (NC) cell migration and its ultrastructural localization in dark (wild-type) and white mutant embryos of the Mexican axolotl (Ambystoma mexicanum, Amphibia). The axolotl system is an accepted model for studying mechanisms of NC cell migration. Using a biotinylated hyaluronan binding protein (HABP), major extracellular matrix (ECM) spaces, including those of NC cell migration, reacted equally positive on cryosections through dark and white embryos. Since neural crest-derived pigment cells migrate only in subepidermal spaces of dark embryos, HA does not seem to influence crest cell migration in vivo. However, when tested on different alternating substrates in vitro, migrating NC cells in dark and white embryos prefer HA to fibronectin. In vivo, such an HA migration stimulating effect might exist as well, but be counteracted to differing degrees in dark and white embryos. The ultrastructural localization of HA was studied by means of transmission electron microscopic immunohistochemistry using HABP and different protocols of standard chemical fixation, cryofixation, embedding, and immunolabeling. The binding reaction of HA to HABP was strong and showed an equal distribution throughout ECM spaces after both standard chemical fixation/freeze substitution and cryofixation. A preference for the somite or subepidermal side was not observed. Following standard fixation/freeze substitution HABP-labeled "honeycomb"-like networks reminiscent of fixation artifacts were more prominent than labeled fibrillar or irregular net-like structures. The latter predominated in adequately frozen specimens following high-pressure freezing/freeze substitution. For this reason fibrillar or irregular net-like structures very likely represent hyaluronan in the complex subepidermal matrix of the axolotl embryo in its native arrangement.     

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3⁺ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4⁺ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr^−/−Pd1^−/−) displayed striking increases in systemic CD4⁺ and CD8⁺ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3⁺ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr^−/−Pd1^−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr^−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.     

Bone morphogenetic protein-4 and Noggin signaling regulates pigment cell distribution in the axolotl trunk

Abstract Wild-type (dark) and white mutant axolotl ( Ambystoma mexicanum ) embryos were used to investigate the role of the secreted growth factor bone morphogenetic protein-4 (BMP-4) and its antagonist, Noggin, in dorso-lateral trunk neural crest (NC) migration. Implantation of a BMP-4-coated microbead caused a melanophore-free zone around the bead, reduction of the dorsal fin above the bead, and disappearance of myotome tissue. We established a novel method that allows controlled induction of protein synthesis and release. Xenopus animal cap (XAC) cells injected with heat shock-inducible constructs for BMP-4 and Noggin were implanted into axolotl embryos and protein expression was induced at defined time points. With this approach, we could demonstrate for the first time that Noggin can stimulate melanophore migration in the white mutant. We further showed that implantation of BMP-4 expressing XAC cells alters pigment cell distribution without affecting muscle and dorsal fin development.     

Transient expression of a lysine-rich vicilin gene ofVicia faba in barley endosperm detected by immunological tissue printing after particle bombardment

Summary Using immunological tissue printing we detected transient expression of a faba bean vicilin gene with or without introns driven by the B1 hordein promoter in barley endosperm after particle bombardment. The described method generally allows the analysis of transient expression of genes without depending on reporter gene constructs and specifically suggests correct splicing of dicot introns by a monocot splicing machinery.Abbreviations GUS -glucuronidase - NPTII neomycin phosphotransferase II - ELISA enzyme-linked immunosorbent assay     

CO2-Fixierung und Stofftransport in benthischen marinen Algen

Summary After local assimilation of NaH¹⁴CO₃ by an old frond of Laminaria hyperborea, radioactive photosynthate is translocated to the growing region of the thallus. The pathway of this long-distance transport was studied by histoautoradiography. Cellular localization of the conducting channels was accomplished by new autoradiographic methods including freeze-substitution and 1 m-cuttings of epoxy resin embedded tissue. In the autoradiographs, patches of silver grains overlying single trumpet-filaments (=Trompetenzellen) indicated that downward translocation occurs in these cells only. It could be shown that predominantly young trumpetfilaments contain the bulk of labeling. It is concluded that the young filaments rather than the older ones are particularly active in translocation. A lateral movement of labelled material was not observed except in the growing region.     

Applications of an optimized monocot expression vector in studying transient gene expression and stable transformation of barley

Protoplasts of a barley ( Hordeum vulgare L. cv. Golden Promise) suspension cell line were used for PEG-mediated gene transfer. Transient gene expression in barley protoplasts was studied using a chimeric CaMV 35S cat construct, which was only poorly expressed in barley cells. However, insertion of exon 1 and intron 1 of the maize Shrunken-1 (Sh1) gene in the 5'-untranslated leader of the construct strongly stimulated gene expression. By using the optimized chimeric cat construction the amount of CAT protein that was reached 19 hours after DNA uptake was 0.5% of total protein, which was calculated from western blot data.
As an alternative marker gene for expression studies, we also tested the firefly luciferase gene in barley protoplasts. Low level expression of chimeric CaMV 35S luciferase genes could be highly stimulated when Sh1 exon1 and intron1 were inserted in the 5'-untranslated leader of the constructs. Enhanced luciferase gene expression by Shrunken-1 intronic sequences enabled us to monitor gene integration events early after DNA uptake using a promoterless luciferase marker gene, which could only be expressed after integration behind an endogenous promoter.