Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition.

Contribution of individual histidines to the global stability of human prolactin.

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500-fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.

Analysis of site-specific histidine protonation in human prolactin.

The structural and functional properties of human prolactin (hPRL), a 23 kDa protein hormone and cytokine, are pH-dependent. The dissociation rate constant for binding to the extracellular domain of the hPRL receptor increases nearly 500-fold over the relatively narrow and physiologic range from pH 8 to 6. As the apparent midpoint for this transition occurs around pH 6.

Withanolides from Jaborosa laciniata.

Six new trechonolide type withanolides (compounds 1- 6), together with trechonolide A, jaborotetrol, and 12- O-methyl jaborosotetrol, were isolated from the aerial parts of Jaborosa laciniata. The structures were elucidated on the basis of spectroscopic methods (1D and 2D NMR, MS).

Solution structure and backbone dynamics of an N-terminal ubiquitin-like domain in the GLUT4-regulating protein, TUG.

The GLUT4-regulating protein, TUG, functions to retain GLUT4-containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10-83 of full-length TUG, and confirms a beta-grasp or ubiquitin-like topology.

Induction of quinone reductase by withanolides.

Thirty-seven naturally occurring withanolides (1-37), previously isolated in our laboratories, were evaluated for their potential to induce quinone reductase with cultured murine hepatoma cells (Hepa 1c1c7). Spiranoid (29, 32) and 18-functionalized withanolides (2-5, 7-9, 24) were found to be potent inducers of the enzyme, while 5alpha-substituted derivatives exhibited weak activity. Preliminary studies were performed with compound 29 to evaluate enzyme-inducing capacity in multiple organ sites of BALB/c mice.

Response of Tribolium castaneum (Coleoptera, Tenebrionidae) to Salpichroa origanifolia Withanolides.

Biological effects on Tribolium castaneum larvae were evaluated for three withanolides isolated from Salpichroa origanifolia (Solanaceae), (20S,22R,24S,25S,26R)-5alpha,6alpha:22,26:24,25-triepoxy-26-hydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide A, 1), (20S,22R,24S,25S,26R)-22,26:24,25-diepoxy-5alpha,6beta,26-trihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide C, 2), and (20S,22R,24S,25S,26R)-5alpha,6alpha:22,26:24,25-triepoxy-15,26-dihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide G, 3), and for several chemically modified analogues. The compounds were incorporated into the larval diet at concentrations of 500 and 2000 ppm. Salpichrolide C (2) produced a significant delay in the development of neonate larvae to adults at the highest concentration (2000 ppm); development delays and lethal effects were produced by salpichrolides A (1) and G (3) at both concentrations assayed.

Withanolides from Salpichroa origanifolia.

Five new withanolides, 5 alpha,6 alpha:22,26:24,25-triepoxy-16 alpha,26-dihydroxy-18(13-->17)-abeo-ergosta-2,13-dien-1-one (salpichrolide N, 1), 5 alpha,6 alpha:22,26:24,25-triepoxi-15 alpha,26-dihydroxyergosta-2,16-dien-1-one (salpichrolide L, 2), 5 alpha,6 alpha:22,26-diepoxi-24,25,26-trihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide M, 3a), 5 alpha,6 alpha:22,25:22,26-triepoxy-24-hydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide J, 4), and 5 alpha,6 alpha:22,26-diepoxy-22,24,25-trihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide K, 5), were isolated from the leaves of Salpichroa origanifolia and characterized by a combination of spectroscopic (1D and 2D NMR, MS) and chemical methods.

Antifeedant activity of withanolides from Salpichroa origanifolia on Musca domestica.

The antifeedant effect of several salpichrolides on larvae of Musca domestica was investigated. Three naturally occurring compounds, salpichrolide A (1), salpichrolide C (2), and salpichrolide G (3), previously isolated from Salpichroa origanifolia, and two known (4, 6) and three new (5, 7, 8) synthetic analogues were tested. The maximal effect on development was observed for salpichrolide A (1), while salpichrolide G (3) was the most toxic.

New Hydroxylated Withanolides from Salpichroa origanifolia.

From the leaves of Salpichroa origanifolia three new withanolides, (20S,22R,24S,25S,26R)-5alpha,6alpha:22,26:24,25-triepoxy-15,26-dihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide G, 1), (20S,22R,24S,25R)-5alpha,6alpha:22,26-diepoxy-24,25,26-trihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17-tetraen-1-one (salpichrolide H, 2), and (20S,22R,25S)-5alpha,6alpha:22,26-diepoxy-25,26-dihydroxy-17(13-->18)-abeo-ergosta-2,13,15,17,23-pentaen-1-one (salpichrolide I, 3), were isolated and characterized by spectroscopic methods and with the aid of molecular modeling. The latter two compounds were obtained as an epimeric mixture at C-26.