A stereotypic,transplantable liver tissue-culture system

A method of coculturing adult rat hepatic parenchymal cells (PC) and stromal cells in a three-dimensional framework of nylon filtration screens or biodegradable polymer meshes was developed in our laboratory. Rat liver stroma, which includes vascular and bile duct endothelial cells, fat-storing cells, fibroblasts, and Kupffer cells, were isolated by gradient centrifugation afterin situ liver perfusion and expanded in monolayer culture prior to seeding onto nylon screens or bioresorbable polyglycolic acid (PGA) polymers oriented into a felt-like construct. A second inoculum of freshly isolated PC was applied after the stromal cells became established. Histological analyses revealed that PC proliferation occurred until all available space for expansion within the template was exhausted. These cells retained their rounded morphology, and after 4–5 wk 7–9 “layers” of PC filled the 140-μm deep template. Dioxin-inducible cytochrome P450 activity was detected for up to 58 d in culture, and albumin, fibrinogen, transferrin, and soluble fibronectin were detected in the medium by enzyme-linked immunosorbent assay (ELISA) for 48 d in vitro. Immunohistochemical analysis of sections through the cultures confirmed the presence of these proteins as well as cytokeratin at the cellular level; the extracellular matrix stained for both collagen type III and laminin. Long-term PC proliferation and function were enhanced by the presence of stromal cells as well as by a meshwork template whose geometry allows the interaction of PC with stroma and matrix on several different planes. To permit transplantation, co-cultures of hepatic PC and stromal cells were established on PGA felt constructs instead of nylon screens. After 24 d in vitro, these constructs were grafted into sites in the mesentery, omentum, and sub-cutaneous tissues of adult Long-Evans rats. The growth of hepatocytes after 30 din situ was evident by histological analysis; grafts of co-cultures regenerated a liver-like architecture consisting of sinusoids and putative biliary structures. In addition, PC at these extrahepatic graft sites were positive for albumin, transferrin, and fibrinogen synthesis by immunohistochemistry. Graft survival was enhanced when recipients were subjected to 40% hepatectomy. Hepatic PC:stromal cell cocultures may prove useful in the restoration of liver function either by direct transplantation using PGA or similar templates, or as extracorporeal devices, using nylon screens.     

Development and validation of a method for the determination of trace alkylphenols and phthalates in the atmosphere

An analytical method has been developed for the simultaneous extraction and determination of trace tertiary octylphenol (t-OP), technical nonylphenol isomers (NP), nonylphenol monoethoxylate isomers (NP1EO) and seven phthalates in the atmosphere using gas chromatography-mass spectrometry (GC-MS). High volume samples were collected using a high-volume pump equipped with a PUF/XAD-2 column for air and glass fiber filter for particles. The detection limits of the method for alkylphenols (APs) and the phthalates ranged from 0.0006 to 0.034 ng m⁻³ in air. The recoveries of t-OP, NP, NP1EO and the phthalates for the entire procedure were satisfactory (>69%). The method was successfully applied to the determination of the analytes in the atmosphere samples collected over land and the ocean. The concentrations of t-OP, NP, NP1EO showed decline trends from land to the open sea, and the phthalates present over land and the North Sea were comparable. It is suggested that the atmosphere is a significant pathway for the transport of alkylphenols and the phthalates in the environment.     

Heterocyclic Rearrangement of 4,5,6,7-Tetrahydro-6,6-dimethyl-benzo[c][1,2,5]oxadiazol-4-one (Z)-Arylhydrazones into Corresponding 2-Aryl-4,5,6,7-tetrahydro-2H-benzo[d][1,2,3]triazol-4-one Oximes

The thermal base-catalysed and photochemical transformation (Boulton-Katritzky rearrangement) of the title tetrahydrobenzo[c][1,2,5]oxadiazoles to tetrahydro-2H-benzo[d][1,2,3]triazoles is studied. Attempts to induce analogous rearrangement in tetrahydro-2H-benzo[d][1,2,3]triazol-4-one arylhydrazones or oximes failed. Some CNDO/2 calculation are also carried out.     

An efficient closed-shell singles and doubles coupled-cluster method

A reformulated set of equations for the closed-shell singles and doubles coupled-cluster (CCSD) method is presented. A computational cost of n_v⁴n₀²+7n_v³n₀³+1n_v²n₀⁴ for the n⁶ steps is obtained, where n_v is the number of virtual molecular orbitals included in the CCSD procedure, n₀ is the number of doubly occupied molecular orbitals and n=n₀+n_v. Test calculations for the cis and trans isomers of FNNF and planar and pyramidal CH₃⁻ are presented. Equilibrium structures determined with large Gaussian basis sets at the second-order Møller-Plesset (MP2) perturbation level of theory are reported and used for the other electron correlation methods. With the largest one-particle basis set (144 contracted Gaussian functions), the equilibrium geometries of cis- and trans-FNNF agree with experiment. Based on analyses of planar and pyramidal CH₃⁻ wavefunctions and the calculated inversion barrier, it is suggested that the molecular anion may not exist in a planar configuration but that autodetachment of an electron occurs before the transition state is reached. Comparisons of our new CCSD procedure demonstrate that coupled-cluster methods are not significantly more expensive than similar electron correlation techniques.     

Organic synthesis with sulphones—XVII: The anti-markownikoff halosulphonylation of olefins via an ionic pathway,and a new method of preparing benzenesulphonyl iodide

Iodine and bromine in the presence of sodium benzenesulphinate react with olefins in acetone solution to give halosulphones resulting from an apparent steric direction of attack at the intermediate halonium ion. A straightforward preparation of benzenesulphonyl iodide from benzenesulphonyl chloride and sodium iodide is also described.     

Adsorption of<Emphasis Type="Italic"> N</Emphasis>-dodecyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethyl-3-ammonio-1-propanesulfonate (DDAPS), a model zwitterionic surfactant,on the Au(111) electrode surface

Electrochemical measurements including cyclic voltammetry, differential capacity, and chronocoulometry have been used to characterize the adsorption behaviors of the zwitterionic surfactant N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (DDAPS) on the Au(111) electrode surface. The thermodynamics of the ideally polarized electrode have been employed to determine the Gibbs excess and the Gibbs energy of adsorption. The results show that the adsorption of DDAPS has a multistate character. The first two states are observed at potentials close to zero charge. At low bulk DDAPS concentrations, it corresponds to the formation of a film of nearly flat adsorbed molecules. At higher concentrations it is converted into a hemimicellar state. The second state is formed at negative potentials and charge densities close to 0 C cm^–2. It corresponds to a film of tilted molecules oriented with the hydrocarbon tail towards the metal and the polar head toward the solution.Dedicated to Professor Zbigniew Galus on the occasion of his 70^th birthday and in recognition of his many contributions to electrochemistry.     

STRESS PROTEIN EXPRESSION IN MURINE TUMOR CELLS FOLLOWING PHOTODYNAMIC THERAPY WITH BENZOPORPHYRIN DERIVATIVE

Abstract— Photodynamic therapy (PDT) has been proven as a method of tumor eradication and is currently being used clinically to treat a wide variety of malignancies. Although it is understood that the interaction of light and sensitizer results in the production of potentially damaging oxygen species, the mechanism by which tumors are destroyed has yet to be defined fully. Using a new porphyrin sensitizer, benzoporphyrin derivative(BPD), we examined protein expression in murine tumor cells following treatment as an indication of molecular changes to target tissue concurrent with PDT-mediated damage. In order to assess the relevance of the results obtained using an in vitro PDT model, metabolic labeling of proteins synthesized subsequent to PDT was performed both in tumor cells grown and treated in tissue culture dishes and in cells explanted from PDT-treated solid tumors. We observed that the oxidative stress associated with PDT-resulted in the induction of a number or proteins corresponding to a set of heat-shock or stress proteins, and that the pattern of expression was similar when tumor cells were treated in vitro and in vivo . These results support the use of in vitro models in the dissection of the molecular erects of PDT and provide the foundation for future experiments that will examine the role of the immune system in tumor eradication by PDT.     

Solid state separated-local-field NMR spectroscopy on half-integer quadrupolar nuclei: principles and applications to borane analysis

New multidimensional NMR methods correlating the quadrupolar and heteronuclear dipolar interactions affecting a half-integer quadrupolar spin in the solid state are introduced and exemplified. The methods extend separated-local-field magic-angle spinning (SLF MAS) NMR techniques that have been used successfully in spin-(1)/(2) spectroscopy to the study of S >/= (3)/(2) nuclei. In our implementation, these techniques avoid homonuclear proton decoupling requirements by relying on moderately fast MAS rates (6-15 kHz) and use rotor-synchronized constant-time pulse sequences to achieve nearly arbitrary amplifications of the apparent dipolar coupling strengths. The result is a suite of simple 2D NMR experiments, whose line shapes carry valuable information about the structure and dynamics of solids containing quadrupolar and proton nuclei. The potential of these sequences was exploited to gather new insight into the structure and dynamics of a variety of boron-containing samples. These experimental SLF schemes were also extended to 3D NMR experiments that incorporate multiple-quantum MAS, thus enabling the resolution needed to study multiple chemical sites in a solid and providing a useful tool for the assignment of inequivalent sites.     

The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V³⁺, 3Fe³⁺, 4Fe²⁺]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined ⁵¹V (I = 7/2) hyperfine splitting, a_iso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V³⁺, 4Fe³⁺, 3Fe²⁺]. Our findings suggest that the V³⁺ does not change valency throughout the catalytic cycle.

Active site FeV-cofactor of the V-nitrogenase and the EPR spectrum of the reduced cofactor showing ⁵¹V-hyperfine coupling.