William Gaffield

Circular dichroism, optical rotatory dispersion and absolute configuration of flavanones, 3-hydroxyflavanones and their glycosides : Determination of aglycone chirality in flavanone glycosides☆

Abstract Flavanones of 2 S configuration and 3-hydroxyflavanones of 2 R ,3 R configuration, having equatorial 2-aryl substituents in the former or diequatorial 2,3-substituents in the latter, exhibit a positive Cotton effect due to the n → π * transition (∼ 330 nm) and a negative Cotton effect in the n α π * region (∼ 280–290 nm). Flavanone glycosides possessing chiral aglycones show Cotton effects quite similar to their optically active aglycones while flavanone glycosides having racemic aglycones show only weak Cotton effects at 250–350 nm. The π → π * Cotton effect is more suitable for determining aglycone chirality in flavanone glycosides. In addition to the two high wavelength bands, 2 S -flavanones generally showed a positive Cotton effect at 245–270 nm and a negative Cotton effect at 225–240 nm. trans 2 R ,3 R -3-Hydroxyflavanones gave two Cotton effects below 270 nm both of which were positive.

Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation

Wnt signaling defines the colonic epithelial progenitor cell phenotype, and mutations in the gene adenomatous polyposis coli (APC) that activate the Wnt pathway cause the familial adenomatous polyposis coli (FAP) syndrome and most sporadic colon cancers. The mechanisms that regulate the transition of epithelial precursor cells into their differentiated derivatives are poorly characterized. We report that Indian hedgehog (Ihh) is expressed by mature colonocytes and regulates their differentiation in vitro and in vivo. Hedgehog (Hh) signaling restricts the expression of Wnt targets to the base of the colonic crypt in vivo, and transfection of Ihh into colon cancer cells leads to a downregulation of both components of the nuclear TCF4–β-catenin complex and abrogates endogenous Wnt signaling in vitro. In turn, expression of Ihh is downregulated in polyps of individuals with FAP and expression of doxycycline-inducible dominant negative TCF4 (dnTCF4) restores Ihh expression in APC mutant DLD-1 colon cancer cells. These data identify a new Wnt-Hh axis in colonic epithelial renewal.

Hedgehog signalling in colorectal tumour cells: Induction of apoptosis with cyclopamine treatment

Hedgehog (Hh) signalling controls many aspects of development. It also regulates cell growth and differentiation in adult tissues and is activated in a number of human malignancies. Hh and Wnt signalling frequently act together in controlling cell growth and tissue morphogenesis. Despite the fact that the majority of colorectal tumours have a constitutively activated canonical Wnt pathway, few previous studies have investigated the expression of Hh signalling components in colorectal tumours. We describe here epithelial cell lines derived from both nonmalignant colorectal adenomas and colorectal adenocarcinomas that express both Sonic and Indian Hh. Interestingly, these cells also express the Hh receptor Patched and the downstream signalling components Smoothened and Gli1, suggesting autocrine Hh signalling in these cells. To test whether autocrine Hh signalling contributes to cell survival, we treated colorectal tumour cells with cyclopamine, a known inhibitor of Hh signalling. Cyclopamine treatment induced apoptosis in both adenoma‐ and carcinoma‐derived cell lines, which could be partially rescued by further stimulation of Hh signalling. These data suggest that autocrine Hh signalling can increase aberrant cell survival in colorectal tumour cells and may be a novel target for colon cancer therapy using drugs such as cyclopamine.

Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning : fungiform papillae double in number and form in novel locations in dorsal lingual epithelium

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk.

A hedgehog homolog regulates gut formation in leech(Helobdella)

Signaling by the hedgehog (hh)-class gene pathway is essential for embryogenesis in organisms ranging from Drosophila to human. We have isolated a hh homolog (Hro-hh) from a lophotrochozoan species, the glossiphoniid leech, Helobdella robusta, and examined its expression by reverse transcription polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization. The peak of Hro-hh expression occurs during organogenesis (stages 10-11). No patterned expression was detected within the segmented portion of the germinal plate during the early stages of segmentation. In stage 10-11 embryos, Hro-hh is expressed in body wall, foregut, anterior and posterior midgut, reproductive organs and in a subset of ganglionic neurons. Evidence that Hro-hh regulates gut formation was obtained using the steroidal alkaloid cyclopamine, which specifically blocks HH signaling. Cyclopamine induced malformation of both foregut and anterior midgut in Helobdella embryos, and no morphologically recognizable gonads were seen. In contrast, no gross abnormalities were observed in the posterior midgut. Segmental ectoderm developed normally, as did body wall musculature and some other mesodermal derivatives, but the mesenchymal cells that normally come to fill most of the coelomic cavities failed to develop. Taken with data from Drosophila and vertebrates, our data suggest that the role of hh-class genes in gut formation and/or neural differentiation is ancestral to the bilaterians, whereas their role in segmentation evolved secondarily within the Ecdysozoa.

Circular dichroism and absolute configuration of α-chloro and α-bromo alkyl carboxylic acids: Effect of α-alkyl substitution on the → π* cotton effect.

Abstract CD, ORD and UV properties are reported for seventeen α-chloro and α-bromo alkyl carboxylic acids, with alkyl groups varying from Me to t-Bu. Two CD bands are present in the n → π* absorption region, for S compounds a positive maximum at 195–222 nm and a negative maximum at 233–270 nm. The former CD band is the most useful for assigning absolute configuration. The CD properties of these compounds are interpreted on the basis of a conformational equilibrium and a quadrant rule is proposed to correlate the CD results with the preferred conformations. The preferred conformation of all halo acids is probably the rotamer which has the halogen eclipsed with the CO group. While 2-bromopropanoic acid shows a much stronger CD band for the other conformer this is probably due to the greater rotational strength of a halogen atom in comparison to an alkyl group. 2-Bromopropanoic acid was found to be an exception to the rule of configurational correlation using Co (III) complexes of asymmetric carboxylic acids.

C-2 stereochemistry of naringin and its relation to taste and biosynthesis in maturing grapefruit

Abstract Circular dichroism and proton magnetic resonance spectroscopy can be used to determine the C-2 chirality of naringin, the main bitter principle of grapefruit. Thus, the aglycone chirality of naringin has been studied as a function of grapefruit maturity. The amount of (2 S ) isomer is 85–92% in immature grapefruit but only 55–60% in mature grapefruit. These results are discussed in relation to the current postulates relating to flavanone biosynthesis. A naringin sample predominant in the (2 R ) isomer has been prepared and found by some tasters to be more bitter than the (2 S ) naringin. It is concluded that the naringin isomerization in ripening grapefruit is not responsible for debittering.

Steroidal Alkaloid Teratogens: Molecular Probes for Investigation of Craniofacial Malformations

AbstractHoloprosencephaly, a malformation sequence that results from impaired midline cleavage of the embryonic forebrain, is expressed as a spectrum of craniofacial anomalies of which cyclopia is the most severe. The Veratrum alkaloids are the most prominent of the teratogenic agents known to induce holoprosencephaly in mammals. Jervine and 11-deoxojervine (cyclopamine) are potent steroidal alkaloid teratogens from Veratrum californicum that are responsible for inducing cyclopic malformations in sheep.Extensive structure-terata investigations of jervanes, solanidanes, and spirosolanes have shown that teratogenicity induced upon oral administration of all three structural types is significantly higher if the C-5, C-6 bond is unsaturated. Research in progress on the pathogenesis of holoprosencephalic malformations in both hamsters and humans offers the potential to provide information on the receptors involved in the expressions of these craniofacial syndromes. A clearer understanding of steroidal alkaloid...

Spirosolane-containing Solanum species and induction of congenital craniofacial malformations

Comparison by GC analysis of purified alkaloid extracts of Solanum species revealed no measurable free solasodine, other spirosolanes, or any non-spirosolane steroidal alkaloid aglycones in unhydrolyzed total alkaloid fractions of fruit of Solanum elaeagnifolium Cav. (silverleaf nightshade), Solanum sarrachoides (S. villosum Lam.--hairy nightshade), Solanum dulcamara L. (European bittersweet nightshade) or Solanum melongena L. (eggplant). All alkaloidal material was apparently present as glycoside. Conversely, sprouts of Solanum tuberosum L. (potato) contained 67% of its alkaloids as glycosides, which was freed only upon hydrolysis with the remaining 33% present as free solanidine. GC/MS analysis of hydrolysates of purified extracts of the test Solanum species revealed that solasodine was a principal or sole aglycone of the alkaloid glycosides in each of the test species except Solanum tuberosum. In the latter, solanidine was the sole aglycone. Among the test species, exclusive of S. tuberosum, only S. dulcamara contained aglycones other than solasodine. In addition to solasodine, S. dulcamara contained appreciable amounts of an unknown spirosolane, an aglycone provisionally identified as soladulcidine. The induction of congenital craniofacial malformations in hamsters by high oral doses of the four Solanum species that contained mainly solasodine glycosides--S. elaeagnifolium, S. dulcamara, S. sarrachoides and S. melongena was compared to inductions of malformations by Solanum tuberosum, that contained mainly solanidane glycosides. Compared to controls, Solanum elaeagnifolium and Solanum dulcamara fruit both induced a high percentage incidence of deformed litters (20.4 and 16.3, respectively) that was statistically significant (P less than 0.001 level) while percentage incidence of deformed litters induced by Solanum sarrachoides and Solanum melongena fruit (9.5 and 7.6 respectively) were both higher than controls (3.4%), in neither case was the incidence statistically significant (P less than .05). Deformed litter incidence induced by sprouts of Solanum tuberosum was 24.0%, (P less than 0.001).