Hauptman-Woodward Medical Research Institute

About: Hauptman-Woodward Medical Research Institute is a nonprofit organization based out in Buffalo, New York, United States. It is known for research contribution in the topics: Dihydrofolate reductase & Protein structure. The organization has 284 authors who have published 537 publications receiving 16412 citations.

Structural basis for androgen specificity and oestrogen synthesis in human aromatase

Abstract: Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer. In a three-step process, each step requiring 1 mol of O(2), 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16alpha-hydroxytestosterone to oestrone, 17beta-oestradiol and 17beta,16alpha-oestriol, respectively. The first two steps are C19-methyl hydroxylation steps, and the third involves the aromatization of the steroid A-ring, unique to aromatase. Whereas most P450s are not highly substrate selective, it is the hallmark androgenic specificity that sets aromatase apart. The structure of this enzyme of the endoplasmic reticulum membrane has remained unknown for decades, hindering elucidation of the biochemical mechanism. Here we present the crystal structure of human placental aromatase, the only natural mammalian, full-length P450 and P450 in hormone biosynthetic pathways to be crystallized so far. Unlike the active sites of many microsomal P450s that metabolize drugs and xenobiotics, aromatase has an androgen-specific cleft that binds the androstenedione molecule snugly. Hydrophobic and polar residues exquisitely complement the steroid backbone. The locations of catalytically important residues shed light on the reaction mechanism. The relative juxtaposition of the hydrophobic amino-terminal region and the opening to the catalytic cleft shows why membrane anchoring is necessary for the lipophilic substrates to gain access to the active site. The molecular basis for the enzyme's androgenic specificity and unique catalytic mechanism can be used for developing next-generation aromatase inhibitors.

The design and implementation of SnB version 2.0

Abstract: SnB is a direct-methods program based on the Shake-and-Bake methodology. It has been used to solve difficult or large structures that could not be solved by traditional reciprocal-space routines based on the tangent formula. Recently, it has also been used to determine the Se sites in large selenomethionyl-substituted proteins. SnB version 1.5 has been available for several years and is being used regularly in many laboratories. In this paper, we introduce SnB version 2.0, which incorporates a graphical user interface written in Java, a dynamic histogram display, and an interactive Java/VRML-based visualization facility. In addition, it provides the user with several utility routines and a variety of new algorithmic options.

Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz‐F1 disrupted mice

Abstract: Ad4BP (or SF-1) has been iden- tified as a transcription factor which regulates all the steroidogenic P450 genes in the peripheral or- gans, and is encoded by the mammalian homo- logue of Drosophila FTZ-F1 gene. mRNA coding for Ad4BP was detected in the hypothalamus and pituitary of rats by RT-PCR. Immunohistochemi- cal analyses using an antiserum to Ad4BP in the brain and pituitary revealed that the transcrip- tion factor is expressed in nuclei of the dorso- medial part of the ventromedial hypothalamus (dmVMH) and in some subpopulation of the ade- nohypophysial cells. Double immunostaining of the pituitary for Ad4BP and trophic peptide hor- mones, FSH, TSH, and ACTH, indicated a re- stricted localization of Ad4BP to the gonadotroph. Disruption of the mouse Ftz-FI gene was clarified to induce severe defects in the organization of the dmVMH and the function of the pituitary gona- dotroph. However, some of the dm VMH neurons and pituitary gonadotrophs persisted, which pro- vided a sharp contrast to complete agenesis of the peripheral steroidogenic tissues (adrenal and go- nads) in the mutant mouse. Additional abnormal- ities were seen in the ventrolateral part of VMH and dorsomedial hypothalamic nucleus, both of which do not express Ad4BP but have strong reciprocal fiber-connections with the dmVMH. Aromatase P450-containing cells in the medial preoptico-amygdaloid region, which were devoid of Ad4BP, persisted even in the brain of the gene disrupted mice. The present results clearly showed that the hypothalamic and pituitary Ad4BPs are essential to normal development of the functional VMH and gonadotroph through some mechanism distinct from that in the periph-

Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium.

Abstract: To determine whether local estrogen production takes place in endometriotic or adenomyotic tissues, in eutopic endometrium from patients with endometriosis or adenomyosis, and in normal endometrium, tissue specimens were examined by immunohistochemistry, catalytic activity, and mRNA expression for aromatase cytochrome P450 (P450arom). P450arom was immunohistochemically localized in the cytoplasm of glandular cells of endometriotic and adenomyotic tissues, and of eutopic endometrium from patients with the respective diseases, whereas estrogen receptors and progesterone receptors were localized in the nuclei of the glandular cells and stroma. Aromatase activity in the microsomal fraction of adenomyotic tissues was inhibited by the addition of danazol, aromatase inhibitors, and anti-human placental P450arom monoclonal antibody (mAb3-2C2) in a manner similar to such inhibition in other human tissues. Reverse transcription polymerase chain reaction and Southern blot analysis also revealed P450arom mRNA in these tissues. However, neither P450arom protein activity nor mRNA was detected in endometrial specimens obtained from normal menstruating women with cervical carcinoma in situ but without any other gynecological disease. These results suggest that at a local level, endometriotic and adenomyotic tissues produce estrogens, which may be involved in the tissue growth through interacting with the estrogen receptor.

The K1 Capsular Polysaccharide of Acinetobacter baumannii Strain 307-0294 Is a Major Virulence Factor

Abstract: Acinetobacter baumannii is a pathogen of increasing medical importance with a propensity to be multidrug resistant, thereby making treatment challenging. Little is known of virulence traits in A. baumannii. To identify virulence factors and potential drug targets, random transposon (Tn) mutants derived from the A. baumannii strain AB307-0294 were screened to identify genes essential for growth in human ascites fluid in vitro, an inflammatory exudative fluid. These studies led to the identification of two genes that were predicted to be required for capsule polymerization and assembly. The first, ptk, encodes a putative protein tyrosine kinase (PTK), and the second, epsA, encodes a putative polysaccharide export outer membrane protein (EpsA). Monoclonal antibodies used in flow cytometric and Western analyses confirmed that these genes are required for a capsule-positive phenotype. A capsule-positive phenotype significantly optimized growth in human ascites fluid, survival in human serum, and survival in a rat soft tissue infection model. Importantly, the clearance of the capsule-minus mutants AB307.30 (ptk mutant, capsule minus) and AB307.45 (epsA mutant, capsule minus) was complete and durable. These data demonstrated that the K1 capsule from AB307-0294 was an important protectin. Further, these data suggested that conserved proteins, which contribute to the capsule-positive phenotype, are potential antivirulence drug targets. Therefore, the results from this study have important biologic and translational implications and, to the best of our knowledge, are the first to address the role of capsule in the pathogenesis of A. baumannii infection.