Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.     

Modification of lipid phase behavior with membrane-bound cryoprotectants

Several derivatives of cholesterol containing oxyethylene headgroups with and without a terminal galactose have been synthesized in order to examine the effects of immobilizing a cryoprotectant at a membrane surface. In this work, we have studied the ability of the triethoxycholesterol (TEC) and triethoxycholesterol galactose (TEC-Gal) derivatives to modulate the phase behavior of phosphatidylcholine and phosphatidylethanolamine membranes. Methods of fluorescence polarization, 31P-NMR and freeze-fracture electron microscopy were employed to monitor these changes in lipid phase behavior. Fluorescence polarization data demonstrated the ability of the derivatives to fluidize gel state and rigidify liquid-crystalline state phosphatidylcholines in a manner similar to that observed for cholesterol. Unlike cholesterol, however, the Tm of dipalmitoylphosphatidylcholine (DPPC) was reduced in a concentration-dependent manner with each of the derivatives. Freeze-fracture electron microscopy and 31P-NMR of DOPE dispersions indicate an increase in the lamellar to hexagonal phase-transition temperature on the order of 10-20 C degrees above room temperature for mixtures with 20 mol% of the derivatives. These results are discussed in terms of the properties exhibited by compounds such as carbohydrates, which are known to serve as cryoprotectants for synthetic and biological membranes.     

N,N-expeditious dialkylation of cyclen (1,4,7,10-tetraazacyclododecane). An astonishing reactivity of cyclen tricarbonyl molybdenum and chromium complexes

After reaction with alkyl iodides and subsequent oxidative removal of the M(CO)₃ triprotection, molybdenum and chromium fac-LM(CO)₃ complexes of cyclen (L) unexpectedly lead to N¹,N⁷-dialkylated cyclen derivatives.     

Thynnine wasps discriminate among heights when seeking mates: tests with a sexually deceptive orchid

The flower of a sexually deceptive orchid, Chiloglottis reflexa, mimics both the sex pheromone and the appearance of a female thynnine wasp (Neozeloboria nr. proxima). The flower is pollinated when visited by male wasps, who attempt mating with the flower. We have used these mimetic flowers to investigate mating behavior of the male wasps. In field choice experiments, males strongly prefer to visit flowers that are very low in the habitat, 15 cm, vs. flowers that are placed at 55 or 105 cm. These studies suggest that male precopulatory response is strongly dependent on the microlocation of the female (or female mimic). Other insect-mimicking orchids, which together attract several groups of Hymenoptera, may be useful in analogous experiments on mating behavior. Additionally, these experiments help elucidate features of the mimetic flowers, particularly stature, that act to efficiently attract potential pollinators.     

THE IMPACT OF A FLOWER-COLOR POLYMORPHISM ON MATING PATTERNS IN EXPERIMENTAL POPULATIONS OF WILD RADISH (RAPHANUS RAPHANISTRUM L.)

We conducted field experiments to determine how a naturally occurring petal-color polymorphism influences mating patterns in wild radish (Raphanus raphanistrum). The polymorphism is controlled at a single genetic locus, with white petal color being completely dominant to yellow. In experimental populations with equal numbers of yellow- and white-flowered homozygous individuals, insect visitors strongly discriminated against white flowers. Pieris rapae, the most frequent pollinator, was almost 50% more likely to visit yellow than white flowers. Maternal fecundity did not differ between the morphs and was not significantly influenced by a plant's compatibility with potential donors, suggesting that seed production was not limited by receipt of compatible pollen. In contrast, the yellow-flowered morph sired approximately 75% of all seeds produced during the study. This paternity proportion was consistently greater than that expected on the basis of postpollination compatibility measures and was indistinguishable from that expected on the basis of pollinator-visitation frequency. We conclude that the male-fitness advantage of the yellow morph resulted from enhanced pollen export due to the greater attractiveness of its flowers to insect pollinators. With color morphs evenly distributed in experimental arrays, insects did not move assortatively on the basis of petal color, and we found no evidence for assortative pollen flow due to the floral polymorphism. Once postpollination compatibility relationships within populations were taken into account, paternal success of yellow donors did not differ between yellow- and white-flowered maternal plants.     

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease

-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly¹⁵N- and ¹³C/¹⁵N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed.     

De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

The transport of morphologically abnormal sperm in the female reproductive tract of mice

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.