Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

Seed Coat Microsculpturing in Brassica and Allied Genera (Subtribes Brassicinae, Raphaninae, Moricandiinae)

To obtain new information on the taxonomy of Brassica and alliedgenera, seed surfaces of 44 species (78 accessions) belongingto 11 genera of subtribes Brassicinae, Raphaninae and Moricandiinaewere examined using a scanning electron microscope. Ten typesof basic ornamentation patterns were recognized. While fourtypes were represented by only one species each, six types hadmore than one species either representing one genus (two types)or more (four types). The different species in each of the sixtypes could generally be distinguished from each other on thebasis of differences in microsculpturing features. This analysisprovides evidence for the close relationship among the variousgenera within the subtribe Brassicinae, and also the closenessof Raphanus, Enarthrocarpus and Moricandia of subtribe Raphaninaeand Moricandiinae, respectively, with the Brassicinae. Copyright2000 Annals of Botany Company Brassicinae, Moricandiinae, Raphaninae, SEM, seed coat microsculpturing, taxonomic implications     

The phylogeny of Patrinieae sensu Graebner (Valerianaceae) revisited: additional evidence from ndhF sequence data

 DNA sequences of both 5′ and 3′ regions of the plastid ndhF gene were generated in order to study the position of Patrinia and Nardostachys, to check the potential paraphyletic nature of Patrinieae, and to evaluate the possible link between the tribe and Linnaeaceae. Parsimony analysis showed very strong support for Patrinia as sister to all members of Valerianaceae (including Nardostachys) and indicated the paraphyletic nature of the tribe Patrinieae. Additionally, trees were constructed from available rbcL data separately and supplemented with ndhF sequences. Topologies of these combined cladograms are in agreement with the ndhF phylogeny, suggesting that the traditionally circumscribed Patrinieae can no longer be recognized but must be considered as part of a basal grade in Valerianaceae. Parsimony analysis based on a morphological data set supported a monophyletic Patrinieae; combination with the molecular data showed a paraphyletic Patrinieae. Furthermore, the possible link between Patrinieae and Linnaeaceae is evaluated. Received July 12, 2001 Accepted February 25, 2002     

Obesity-inducing lesions of the central nervous system alter leptin uptake by the blood-brain barrier.

Leptin regulates body adiposity by decreasing feeding and increasing thermogenesis. Obese humans and some obese rodents are resistant to peripherally administered leptin, suggesting a defect in the transport of leptin across the blood-brain barrier (BBB). Defective transport of exogenous leptin occurs in some models of obesity, but in other models transport is normal. This shows that factors other than obesity are associated with impairment of leptin transport across the BBB. In order to further investigate these factors, we determined leptin transport in rats made obese by lesioning of the ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), or posterodorsal amygdala (PDA). These regions all contain leptin receptors and lesions there induce obesity and hyperleptinemia and alter the levels of many feeding hormones which might participate in leptin transporter regulation. We measured the uptake of radioactively labeled leptin by the BBB by multiple-time regression analysis which divides uptake into a reversible phase (Vi, e.g., receptor/transporter binding to the brain endothelial cell) and an irreversible phase (Ki, complete transport across the BBB). Leptin uptake was not affected in rats with VMH lesions. No significant change occurred in the entry rate (Ki) for any group, although Ki declined by over 35% in rats with PVN lesions. Decreased uptake was observed in rats with PVN lesions and with PDA lesions. This was primarily due to a reduced Vi (about 21% for the PDA). This decreased uptake is most likely explained by decreased binding of leptin to the brain endothelial cell, which could be because of decreased binding by either receptors or transporters. This suggests that some of the feeding hormones controlled by the PVN and PDA may participate in regulating leptin uptake by the BBB.