Biochemical and biological evaluation of novel potent coumarin inhibitor of 17ß-HSD type 1.

Human 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 is an enzyme that acts at the pre-receptor level. It catalyzes the NADPH-dependent reduction of the weak estrogen estrone into the most potent estrogen 17ß-estradiol, which exerts proliferative effects via estrogen receptors. Overexpression of 17ß-HSD type 1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis. 17ß-HSD type 1 thus represents an attractive target for development of new drugs. Recently, we discovered that substituted coumarin derivatives potently and selectively inhibit 17ß-HSD type 1. In the present study, we have performed additional biochemical and biological evaluation of the most promising coumarin derivative. First, we used an efficient method for isolation and purification of the active, soluble recombinant human 17ß-HSD type 1 from Escherichia coli. This 17ß-HSD type 1 showed a specific activity of 0.64±0.08 µmol min(-1) mg(-1) for estrone reduction in the presence of NADPH at pH 6.5, and a K(m) of 62 nM for estrone. Next, we evaluated the best of the coumarin-derivative inhibitors, showing its reversible and competitive inhibition of 17ß-HSD type 1 reductive activity with a K(i) of 53 nM. We confirmed that this coumarin inhibitor acts not only in a cell-free assay, but also decreases endogenous 17ß-HSD type 1 activity in human T-47D breast cancer cells. This inhibitor also reduced estrone dependent growth of T-47D cells after 48 h of incubation.

Optimization of UDP-N-acetylmuramic acid synthesis.

UDP-N-acetylmuramic acid (UDP-MurNAc) is a substrate of MurC, an important enzyme in the intracellular pathway of bacterial peptidoglycan biosynthesis. Various approaches towards preparation of UDP-MurNAc have been published but these synthetic preparations were shown to include many problematic steps. An optimization study with the focus on muramyl phosphate and UMP-morpholidate coupling was performed, resulting in a synthetic procedure enabling robust and easily reproducible production on a multi-gram scale.

Kepone (chlordecone) disrupts adherens junctions in human breast epithelial cells cultured on matrigel.

Perturbations in cell-extracellular matrix (ECM) interactions are a consistent feature of mammary tumors and cells in culture. We have utilized MCF-10ATG3B human breast epithelial cells to examine whether the organochlorine Kepone induces alterations in cell adhesion molecules important to cell-cell and cell-ECM interactions. Kepone effects on the levels and association of proteins involved in adherens junctions or desmosomes were examined using immunoblot analysis and immunoprecipitation. MCF-10ATG3B cells cultured on an ECM of Matrigel form lattice-like structures that are disrupted with 0.1 and 1 microM Kepone. E-cadherin protein levels decreased significantly by approximately 23% and approximately 69% following treatment with 0.1 and 1.0 microM Kepone, respectively, relative to solvent-treated cells. Desmoglein and alpha- and gamma-catenin levels did not vary significantly with Kepone. Beta-catenin protein levels decreased significantly by approximately 37%, 36% and 53% at 0.01, 0.1 and 1.0 microM Kepone, respectively. E-cadherin-gamma-catenin association was disrupted with 0.1 and 1.0 microM Kepone. Thus, Kepone disrupts cellular architecture, specifically E-cadherin-gamma-catenin containing adherens junctions, which may ultimately affect cellular phenotype.

Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease.

BACKGROUND: Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice. METHODS: The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided. RESULTS: Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series. CONCLUSION: Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.

[Amputation of the lower extremities in angiopathies and reamputation as a sequelae of postoperative complications]. / Amputacije donjih ekstremiteta kod angiopatija i reamputacije kao posledice komplikacija u postoperativnom toku.

In the period from January 1985 till January 1995 in the Clinic of Traumatology and Orthopedics of Military Medical Academy were treated 305 patients with 317 primary amputations of the lower extremities that were performed as the sequelae of ischemia and necrosis on the basis of occlusive changes, arteriosclerosis and diabetes. Out of the total number of operated patients, 210 were with diabetes mellitus. The amputations were performed at the level of: foot in 102 (32.17%), lower leg in 145 (45.74%) and upper leg in 70 patients (22.09%). Reamputations were performed in 52 patients (16.4%): at the level of foot in 22 (6.94%), lower leg in 17 (5.36%) and upper leg in 13 patients (4.10%). Tourniquet was used during the surgery of primary amputation in 56 patients.

Glutathione and glutathione S-transferase in the rainbow trout olfactory mucosa during retrograde degeneration and regeneration of the olfactory nerve.

In the peripheral olfactory organ, continual olfactory receptor neuron (ORN) turnover exposes neighboring cells to potentially damaging cellular debris such as free radicals. These, in turn, may be inactivated by binding directly onto glutathione (GSH) or by enzymatic conjugation with glutathione S-transferase (GST). In this study, we have investigated GSH and GST during retrograde degeneration and regeneration of the olfactory nerve in rainbow trout. In these fish, prolonged ORN physiological activity and structural integrity following transection of the olfactory nerve may be mediated by GSH and GST. In the olfactory mucosa, early changes following nerve lesion and prior to ORN degeneration included a shift of intense GSH labeling from the dendrites and perikarya of a subpopulation of ORN, and from melanophores, to olfactory nerve fascicles. GSH levels were unchanged, but GST activity decreased by 33% and GST-immunoreactivity (GST-IR) in nerve fascicles diminished slightly. When the process of massive degeneration terminated and ORN were largely absent, GSH levels and GST activity decreased further, GSH labeling was confined to melanophores, and GST-IR was absent. As ORN repopulated the olfactory mucosa, GST-IR was widespread. The combination of increased GST activity (92% of preoperative values) and low GSH levels suggests GSH utilization for GST conjugation reactions. These changes imply that GSH provides protection from cellular debris associated with ORN degeneration. Recovery of GST activity and widespread GST-IR during regeneration indicates modulation of neuroprotective, developmental, and/or physiological processes by GST.

Issues in detecting abuse of xenobiotic anabolic steroids and testosterone by analysis of athletes' urine.

Over the last decade the number of laboratories accredited by the International Olympic Committee (IOC) has grown to 25. Nearly half of the approximately 90,000 samples tested annually are collected on short notice-the most effective means to deter the use of anabolic androgenic steroids (AAS). The major urinary metabolites of AAS have been characterized and are identified by their chromatographic retention times and full or partial mass spectra. The process of determining if an athlete has used testosterone (T) begins with finding a T to epitestosterone (E) ratio > 6 and continues with a review of the T/E-time profile. For the user who discontinues taking T, the T/E reverts to baseline (typically approximately 1.0). For the extremely rare athlete with a naturally increased T/E ratio, the T/E remains chronically increased. Short-acting formulations of T transiently increase T/E, and E administration lowers it. Among ancillary tests to help discriminate between naturally increased T/E values and those reflecting T use, the most promising is determination of the carbon isotope ratio.

Immunohistochemical localization of glutathione S-transferase pi in rainbow trout olfactory receptor neurons.

In the fish olfactory system, glutathione S-transferases (GST) which detoxify electrophilic substances and participate in reactions of lipophilic compounds, may be active in the biotransformation of odorants and xenobiotics. In this study GST activity in the rainbow trout olfactory mucosa was high (477.6 +/- 218 nmol/min per mg protein). The GST pi class was demonstrated by Western immunoblot analysis and localized by immunofluorescence to the dendritic and perinuclear regions of olfactory receptor neurons; areas previously shown to contain elevated glutathione. The presence of GST and glutathione in fish olfactory receptor neurons suggests that these cells utilize the glutathione pathway.

[Surgical treatment of habitual luxation of the shoulder joint using the Dickson-O'Dell method. Our modification]. / Operativno lijecenje habitualne luksacije ramenog zgloba po metodi Dickson-O'Dell. Nasa modifikacija.

The authors have presented their experience in the treatment of 40 patients with habitual dislocation of the shoulder joints using the method of Dickson-O'Dell during a period of 10 years. They have compared their results with results obtained by other methods and consider their modification as the method of choice on the basis of excellent results obtained in their patients.