Structural characterization of MAO and related aluminum complexes. 1. Solid-state (27)Al NMR with comparison to EFG tensors from ab initio molecular orbital calculations.

Experimental and ab initio molecular orbital techniques are developed for study of aluminum species with large quadrupole coupling constants to test structural models for methylaluminoxanes (MAO). The techniques are applied to nitrogen- and oxygen-containing complexes of aluminum and to solid MAO isolated from active commercial MAO preparations. (Aminato)- and (propanolato)aluminum clusters with 3-, 4-, and 6-coordinate aluminum sites are studied with three (27)Al NMR techniques optimized for large (27)Al quadrupole coupling constants: field-swept, frequency-stepped, and high-field MAS NMR. Four-membered (aminato)aluminum complexes with AlN(4) coordination yield slightly smaller C(q) values than similar AlN(2)C(2) sites: 12.2 vs 15.8 MHz. Planar 3-coordinate AlN(2)C sites have the largest C(q) values, 37 MHz. In all cases, molecular orbital calculations of the electric field gradient tensors yields C(q) and eta values that match with experiment, even for a large hexameric (aminato)aluminum cage. A D(3d) symmetry hexaaluminum oxane cluster, postulated as a model for MAO, yields a calculated C(q) of -23.7 MHz, eta = 0.7474, and predicts a spectrum that is too broad to match the field-swept NMR of methylaluminoxane, which shows at least three sites, all with C(q) values greater than 15 MHz but less than 21 MHz. Thus, the proposed hexaaluminum cluster, with its strained four-membered rings, is not a major component of MAO. However, calculations for dimers of the cage complex, either edge-bridged or face-bridged, show a much closer match to experiment. Also, MAO preparations differ, with a gel form of MAO having significantly larger (27)Al C(q) values than a nongel form, a conclusion reached on the basis of (27)Al NMR line widths in field-swept NMR spectra acquired from 13 to 24 T.

Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the expression of EGF and/or TGF-alpha.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure produces hydronephrosis and cleft palate in mice. These responses are correlated with disruption of expression of epidermal growth factor (EGF) receptor ligands, primarily EGF and transforming growth factor-alpha (TGF-alpha), and altered epithelial cell proliferation and differentiation. This research examined the role of these growth factors in TCDD-induced teratogenicity by using wild type (WT) and knockout (-/-) mice that do not express EGF, TGF-alpha, or both EGF and TGF-alpha. Pregnant females were weighed on GD 12 and dosed by gavage with either corn oil or TCDD at 24 microg/kg, 5 ml/kg. On GD 17.5, the maternal parameters evaluated included body weight, body weight gain, liver weight (absolute and adjusted for body weight). The number of implantations, live and dead fetuses, early or late resorptions, the proportion of males, fetal body weight, fetal absolute and relative liver weight, placenta weight, incidence of cleft palate, and the severity and incidence of hydronephrosis were recorded. TCDD did not affect maternal weight gain, fetal weight, or survival, but maternal and fetal liver weights and liver-to-body weight ratios were increased in all genotypes. The WT and TGF-alpha (-/-), but not the EGF (-/-) and EGF + TGF-alpha (-/-) fetuses, developed cleft palate after exposure to 24 microg TCDD/kg. Hydronephrosis was induced by TCDD in all genotypes, with the incidence in EGF + TGF-alpha (-/-) fetuses comparable to that of the WT. The incidence and severity of this defect was substantially increased in EGF (-/-) and TGF-alpha (-/-). In conclusion, this study demonstrated that expression of EGF influences the induction of cleft palate by TCDD. Also, EGF and TGF-alpha are not required for the induction of hydronephrosis, but when either is absent the response of the fetal urinary tract to TCDD is enhanced.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fetal mouse urinary tract epithelium in vitro.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis by altering the differentiation and proliferation of ureteric epithelial cells in the fetal C57BL/6N mouse urinary tract. This study tests the hypothesis that the late fetal urinary tract epithelial cells respond to TCDD with increased proliferation and that the responses do not require contributions from other maternal or fetal tissues. This was achieved by exposing late gestation fetal urinary tract cells to TCDD in an in vitro model. Isolated ureteric cells from gestation day (GD) 18 fetal ureters were plated in medium supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. Both epithelial and mesenchymal cells remain viable under these conditions. The cultures were exposed to 0.1% dimethylsulfoxide (DMSO), 1x10(-8), 1x10(-9) or 1x10(-10) M TCDD. Exposure to 1x10(-10) M TCDD did not affect the cultures, while 1x10(-8) and 1x10(-9) M TCDD supported epithelial, but not mesenchymal, cell survival and stimulated epithelial cell proliferation and differentiation. The TCDD-exposed cells expressed high levels of keratin and little or no vimentin, confirming that the cells, which survive and differentiate are epithelial. However, after continuous exposure to epidermal growth factor (EGF), the TCDD-induced stimulation of ureteric epithelial growth could not be detected. In conclusion, this study demonstrates that late gestational ureteric cells respond to TCDD in vitro with the stimulation of epithelial cell growth and differentiation.

27Al field-swept and frequency-stepped NMR for sites with large quadrupole coupling constants.

Spectra of nonspinning samples with large quadrupole coupling constants, 16-32 MHz, are acquired by frequency-stepping. A series of spin-echoes are acquired at arbitrary frequency increments, frequency-shifted in the time domain, and co-added as magnitude spectra. This procedure is derived from a method in use for field-swept NMR. The two methods are compared.

Modeling of parenteral container headspace pressure.

When containers and related closure systems holding fluids are heated, internal pressures are generated. Depending upon conditions, surprisingly high pressures can be developed. These pressures are often sufficient to break system integrity. Numerous factors combine to determine the pressure generated within the container and closure system. Each of these factors can vary during the manufacture and service life of the product. Prudent pharmaceutical design seeks to set and control all the factors in such a manner that container and related closure integrity are maintained from the time of assembly to the time of use. Answers can be obtained by pure experimentation ("cut, try, recut and retry until it comes out right") or by predicting performance with a good mathematical model and testing once to verify the model. Pure experimentation requires extensive prototype parts and testing thereof. The mathematical model approach is more exact and produces a better product quicker and at lower cost. This paper uses a simple tubing vial to demonstrate the two approaches to controlling internal pressures from assembly to use. A mathematical model with experimental verification produces high confidence that the system integrity will be maintained as desired. The model used to predict the headspace pressure of the tubing vial is of significance in estimating performance of similar containers and related closures. It can rapidly produce product with predictable behavior. As such, the model is an excellent tool for designers of pharmaceutical products.

Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces hydronephrosis in C57BL/6N mice. The etiology of this response involves TCDD-induced hyperplasia of ureteric epithelium, which occludes the ureteric lumen, blocking the flow of urine. The present study localizes and examines the effects of TCDD on the expression of the Ah receptor (AhR), the Ah receptor nuclear translocator (ARNT), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) in the epithelial cells of the developing urinary tract, particularly the ureteric bud derivatives (ureter and tubules). Pregnant C57BL/6N mice were dosed on gestation day (GD) 10 with either corn oil or TCDD at 12 micrograms/kg; a dose of 24 micrograms/kg is expected to induce 100% hydronephrosis. The metanephric urinary tract is morphologically detectable as early as GD 12; thus, embryos were removed on GD 12, 13, and 14, and the lower dorsal torso was prepared for immunohistochemistry or in situ hybridization. Regardless of treatment, the expression of both AhR and ARNT increased in epithelial cells of the ureter and AhR increased in the metanephric tubules from GD 12-14. In situ hybridization localized the expression of AhR and ARNT mRNAs to these derivatives of the ureteric bud and levels of mRNA increased throughout the developmental period examined. There were no significant effects of TCDD treatment on expression of AhR, while TCDD significantly decreased levels of ARNT in tubules on GD 14. The epithelial cells of the ureter and tubules expressed TGF-alpha and EGF. EGF increased from GD 12 to 13 in the tubules and ureter, but there was no difference from GD 13 to 14. Treatment with TCDD reduced TGF-alpha significantly only in tubules on GD 13. TCDD exposure significantly decreased EGF in ureter and tubule cells on both GD 13 and 14. In summary, the epithelial cells of the embryonic mouse urinary tract expressed AhR, ARNT, EGF, and TGF-alpha in developmentally dependent patterns. These proteins are involved in the regulation of embryonic cell proliferation during normal urinary tract development and are probably involved in the hyperplastic response to TCDD.

Cancer rehabilitation: assessment of need, development, and evaluation of a model of care.

A sample of 805 cancer patients, comparable to but not identical with a national study, was screened to identify: rehabilitation problems encountered at different cancer sites; the need for rehabilitation services; and gaps in the delivery of rehabilitation care. Significant numbers of rehabilitation problems were found that could be improved by rehabilitation care. Psychologic problems were commonly encountered and seemed more severe in patients with physical disabilities; these patients have to make adjustments to both life-threatening disease and to a disabling condition. The findings suggest a need for psychosocial support services on any oncology service; where cancer is associated with significant physical disability, a comprehensive rehabilitation team with psychologic management capability is often needed. Primary barriers to optimal delivery of rehabilitation care are the lack of identification of patient problems and/or lack of appropriate referral by physicians unfamiliar with the concept of rehabilitation. Health care financial support for the patients in the sample came primarily from private insurance, Medicare and Medicaid; financial support from the patient's family held up well even in the advanced stages of the disease. A model of rehabilitation care delivery was established and implemented, with the result being that gaps and barriers to rehabilitation service delivery disappeared rapidly.