Concise Synthesis of Pyrrolidine and Indolizidine Alkaloids by a Highly Convergent Three-Component Reaction

The synthesis of pyrrolidine and indolizidine derivatives through radical carboazidation of alkenes with alpha-iodoketones, followed by reductive amination, is described. When properly substituted, further lactamization afforded pyrrolizidinones in good yield. This carboazidation/reductive amination sequence was efficiently applied to the total synthesis of three different simple alkaloids, including (+/-)-monomorine I.

A MICROFLUIDIC METHOD TO MEASURE DIFFUSION IN HYDROGELS

A novel microfluidic method is proposed for studying diffusion of small molecules in a hydrogel. Microfluidic devices were prepared with semi-permeable microchannels defined by crosslinked poly(ethylene glycol) (PEG). Uptake of dye molecules from aqueous solutions flowing through the microchannels was observedoptically and diffusion of the dye into the hydrogel was quantified. To complement the diffusion measurements from the microfluidic studies, nuclear magnetic resonance(NMR) characterization of the diffusion of dye in the PEG hydrogels was performed. The diffusion of small molecules in a hydrogel is relevant to applications such asdrug delivery and modeling transport for tissue-engineering applications. The diffusion of small molecules in a hydrogel is dependent on the extent of crosslinking within the gel, gel structure, and interactions between the diffusive species and the hydrogel network. These effects were studied in a model environment (semi-infinite slab) at the hydrogelfluid boundary in a microfluidic device. The microfluidic devices containing PEG microchannels were fabricated using photolithography. The unsteady diffusion of small molecules (dyes) within the microfluidic device was monitored and recorded using a digital microscope. The information was analyzed with techniques drawn from digital microscopy and image analysis to obtain concentration profiles with time. Using a diffusion model to fit this concentration vs. position data, a diffusion coefficient was obtained. This diffusion coefficient was compared to those from complementary NMR analysis. A pulsed field gradient (PFG) method was used to investigate and quantify small molecule diffusion in gradient (PFG) method was used to investigate and quantify small molecule diffusion in hydrogels. There is good agreement between the diffusion coefficients obtained from the microfluidic methods and those found from the NMR studies. The microfluidic approachused in this research enables the study of diffusion at length scales that approach those of vasculature, facilitating models for studying drug elution from hydrogels in blood-contacting applications.

Quantifying The Diffusion And Erosion Of Degradable Hydrogels Using A Microfluidic Technique

Hydrogels are composed of cross-linked networks of hydrophilic polymers that are biocompatible due to their high water content. Mass transfer through hydrogels has been suggested as an effective method of drug delivery, specifically in degradable polymers to minimize lasting effects within the body. Diffusion of small molecules in poly (ethylene glycol) diacrylate (PEG-DA) and dextran methacrylate (dex-MA) hydrogels was characterized in a microfluidic device and by complementary techniques. Microfluidic devices were prepared by crosslinking a formulation of hydrogel and photo-initiator, with and without visible dye, using photolithography to define a central microchannel. Channel sizes within the devices were approximately 600 ¿m to simulate vessels within the body. The microfluidic technique allows for both image and effluent analyses. To visualize the diffusive behavior within the dextran hydrogel, methylene blue and sulforhodamine 101 dyes were used in both elution and uptake experiments. Three analysis techniques for measuring diffusion coefficients were used to quantify the diffusion of solute in the hydrogel, including optical microscopy, characterization of device effluent, and NMR analyses. The optical microscopy technique analyzes images of the dye diffusion captured by a stereomicroscope to generate dye concentration v. position profiles. The data was fit to a diffusion model to determine diffusion coefficients and the dye release profile. In a typical elution experiment, aqueous solution is pumped through the microchannel and dye diffuses out of the hydrogel and into the aqueous phase. During elution, images are taken at regular time intervals and the effluent was collected. Analysis of the device effluent was performed using ultraviolet-visible (UV/Vis) spectroscopy to determine the effluent dye concentration and thus a short-time diffusion coefficient. Nuclear magnetic resonance (NMR) was used to determine a free diffusion coefficient of molecules in hydrogel without the effect of a concentration gradient. Diffusion coefficients for methylene blue and sulforhodamine 101 dyes in dex-MA hydrogel calculated using the three analysis methods all agree well. It was determined that utilizing a combination of the three techniques offers greater insight into molecular diffusion in hydrogels than employing each technique individually. The use of the same microfluidic devices used to measure diffusion is explored in the use of studying the degradation of dex-MA hydrogels. By combining what is known about the degradation rate in regards to the effect of pH and crosslinking and the ability to use a dye solution in contrast to establish the hydrogel boundaries could be a novel approach to studying hydrogel degradation.

A Microfluidic Method to Measure Small Molecule Diffusion in Hydrogels

Drug release from a fluid-contacting biomaterial is simulated using a microfluidic device with a channel defined by solute-loaded hydrogel; as water is pumped through the channel, solute transfers from the hydrogel into the water. Optical analysis of in-situ hydrogels, characterization of the microfluidic device effluent, and NMR methods were used to find diffusion coefficients of several dyes (model drugs) in poly( ethylene glycol) diacrylate (PEG-DA) hydrogels. Diffusion coefficients for methylene blue and sulforhodamine 101 in PEG-DA calculated using the three methods are in good agreement; both dyes are mobile in the hydrogel and elute from the hydrogel at the aqueous channel interface. However, the dye acid blue 22 deviates from typical diffusion behavior and does not release as expected from the hydrogel. Importantly, only the microfluidic method is capable of detecting this behavior. Characterizing solute diffusion with a combination of NMR, optical and effluent methods offer greater insight into molecular diffusion in hydrogels than employing each technique individually. The NMR method made precise measurements for solute diffusion in all cases. The microfluidic optical method was effective for visualizing diffusion of the optically active solutes. The optical and effluent methods show potential to be used to screen solutes to determine if they elute from a hydrogel in contact with flowing fluid. Our data suggest that when designing a drug delivery device, analyzing the diffusion from the molecular level to the device level is important to establish a complete picture of drug elution, and microfluidic methods to study such diffusion can play a key role. (C) 2013 Elsevier B.V. All rights reserved.

Effect on infection resistance of a local antiseptic and antibiotic coating on osteosynthesis implants: an in vitro and in vivo study

The purpose of this study was to acquire information about the effect of an antibacterial and biodegradable poly-L-lactide (PLLA) coated titanium plate osteosynthesis on local infection resistance. For our in vitro and in vivo experiments, we used six-hole AO DC minifragment titanium plates. The implants were coated with biodegradable, semiamorphous PLLA (coating about 30 microm thick). This acted as a carrier substance to which either antibiotics or antiseptics were added. The antibiotic we applied was a combination of Rifampicin and fusidic acid; the antiseptic was a combination of Octenidin and Irgasan. This produced the following groups: Group I: six-hole AO DC minifragment titanium plate without PLLA; Group II: six-hole AO DC minifragment titanium plate with PLLA without antibiotics/antiseptics; Group III: six-hole AO DC minifragment titanium plate with PLLA + 3% Rifampicin and 7% fusidic acid; Group IV: six-hole AO DC minifragment titanium plate with PLLA + 2% Octenidin and 8% Irgasan. In vitro, we investigated the degradation and the release of the PLLA coating over a period of 6 weeks, the bactericidal efficacy of antibiotics/antiseptics after their release from the coating and the bacterial adhesion of Staphylococcus aureus to the implants. In vivo, we compared the infection rates in white New Zealand rabbits after titanium plate osteosynthesis of the tibia with or without antibacterial coating after local percutaneous bacterial inoculations at different concentrations (2 x 10(5)-2 x 10(8)): The plate, the contaminated soft tissues and the underlying bone were removed under sterile conditions after 28 days and quantitatively evaluated for bacterial growth. A stepwise experimental design with an "up-and-down" dosage technique was used to adjust the bacterial challenge in the area of the ID50 (50% infection dose). Statistical evaluation of the differences between the infection rates of both groups was performed using the two-sided Fisher exact test (p < 0.05). Over a period of 6 weeks, a continuous degradation of the PLLA coating of 13%, on average, was seen in vitro in 0.9% NaCl solution. The elution tests on titanium implants with antibiotic or antiseptic coatings produced average release values of 60% of the incorporated antibiotic or 62% of the incorporated antiseptic within the first 60 min. This was followed by a much slower, but nevertheless continuous, release of the incorporated antibiotic and antiseptic over days and weeks. At the end of the test period of 42 days, 20% of the incorporated antibiotic and 15% of the incorporated antiseptic had not yet been released from the coating. The antibacterial effect of the antibiotic/antiseptic is not lost by integrating it into the PLLA coating. The overall infection rate in the in vivo investigation was 50%. For Groups I and II the infection rate was both 83% (10 of 12 animals). In Groups III and IV with antibacterial coating, the infection rate was both 17% (2 of 12 animals). The ID50 in the antibacterial coated Groups III and IV was recorded as 1 x 10(8) CFU, whereas the ID50 values in the Groups I and II without antibacterial coating were a hundred times lower at 1 x 10(6) CFU, respectively. The difference between the groups with and without antibacterial coating was statistically significant (p = 0.033). Using an antibacterial biodegradable PLLA coating on titanium plates, a significant reduction of infection rate in an in vitro and in vivo investigation could be demonstrated. For the first time, to our knowledge, we were able to show, under standardized and reproducible conditions, that an antiseptic coating leads to the same reduction in infection rate as an antibiotic coating. Taking the problem of antibiotic-induced bacterial resistance into consideration, we thus regard the antiseptic coating, which shows the same level of effectiveness, as advantageous.

Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC

Gemcitabine (2'2'-difluorodeoxycytidine) is a pyrimidine analog used in the treatment of a variety of solid tumors. After intravenous (i.v.) administration, it is rapidly inactivated to 2'-deoxy-2',2'-difluorouridine (dFdU). A sensitive analytical method for the quantitation of gemcitabine is required for the assessment of alternative dosage and treatment schemes. A rapid and robust RP-HPLC assay for analysis of gemcitabine in human and animal plasma and serum was developed and validated using 2'-deoxyuridine (dU) and 5-fluoro-2'-deoxyuridine (5FdU) as internal standards. It is based on protein precipitation, the use of an Atlantis dC18 column of 100 mm length (inner diameter, 4.6 mm; particle size, 3 microm) and isocratic elution using a 10 mM phosphate buffer, pH 3.0, followed by isocratic elution with the same buffer containing 3% of ACN. For gemcitabine, RSD values for intraday and interday precision were < 4.4 and 5.3%, respectively, the LOQ was 20 ng/mL, and the assay was linear in the range of 0.020-20 microg/mL with an accuracy of > or =89%. The recovery for gemcitabine, dU and 5FdU was 86-98%. The assay was applied to determine gemcitabine levels in plasma samples of patients collected during and shortly after conventional infusion of 25-30 mg/kg body mass (levels: 2.0-18.9 microg/mL) and rats that received lower doses (1.5 mg/kg) via i.v., subcutaneous and oral drug administration (levels: 0.20-2.60 microg/mL). It could also be applied to estimate dFdU levels in human plasma.

Temporary scaffolding of coronary arteries with bioabsorbable magnesium stents: a prospective, non-randomised multicentre trial

BACKGROUND: Coronary stents improve immediate and late results of balloon angioplasty by tacking up dissections and preventing wall recoil. These goals are achieved within weeks after angioplasty, but with current technology stents permanently remain in the artery, with many limitations including the need for long-term antiplatelet treatment to avoid thrombosis. We report a prospective multicentre clinical trial of coronary implantations of absorbable magnesium stents. METHODS: We enrolled 63 patients (44 men; mean age 61.3 [SD 9.5 years]) in eight centres with single de novo lesions in a native coronary artery in a multicentre, non-randomised prospective study. Follow-up included coronary angiography and intravascular ultrasound at 4 months and clinical assessment at 6 months and 12 months. The primary endpoint was cardiac death, non-fatal myocardial infarction, or clinically driven target lesion revascularisation at 4 months FINDINGS: 71 stents, 10-15 mm in length and 3.0-3.5 mm in diameter, were successfully implanted after pre-dilatation in 63 patients. Diameter stenosis was reduced from 61.5 (SD 13.1%) to 12.6 (5.6%) with an acute gain of 1.41 mm (0.46 mm) and in-stent late loss of 1.08 mm (0.49 mm). The ischaemia-driven target lesion revascularisation rate was 23.8% after 4 months, and the overall target lesion revascularisation rate was 45% after 1 year. No myocardial infarction, subacute or late thrombosis, or death occurred. Angiography at 4 months showed an increased diameter stenosis of 48.4 (17.0%). After serial intravascular ultrasound examinations, only small remnants of the original struts were visible, well embedded into the intima. Neointimal growth and negative remodelling were the main operating mechanisms of restenosis. INTERPRETATION: This study shows that biodegradable magnesium stents can achieve an immediate angiographic result similar to the result of other metal stents and can be safely degraded after 4 months. Modifications of stent characteristics with prolonged degradation and drug elution are currently in development.

Thrombosis and drug-eluting stents: an objective appraisal

Stent thrombosis (ST) after percutaneous coronary intervention has been the focus of intense interest because of its attendant morbidity and mortality. There is controversy about several facets of the problem. These include the frequency of ST with drug-eluting stents (DES) versus bare-metal stents (BMS), the timing of the event, clinical consequences, risk factors, adjunctive therapy, and new preventive approaches. Information has accrued rapidly from several sources, including randomized controlled clinical trials of DES versus BMS in carefully selected subsets of patients and registry experiences in larger patient groups, which provide a more universal real-world picture. The results from these different data sets are not completely concordant. However, several general conclusions can be made: 1) ST is an infrequent but very severe complication of both BMS and DES; 2) at the present time, during 4 years of follow-up from randomized controlled trials that compared DES and BMS, there is no apparent difference in overall ST frequency, although the time course for occurrence appears to differ, with a relative numeric excess of ST late after DES implant; 3) despite this relative imbalance, no differences in the end points of death or death and infarction between DES and BMS are observed; 4) longer-term follow-up of these patients as well as larger angiographic and clinical subsets of patients who receive this technology outside of randomized trials are required to fully study this issue; and 5) advances in stent platforms for drug elution as well as adjunctive pharmacologic therapy are being evaluated to enhance long-term safety.

Syntheses and characterization of monomeric Mo(VI) complexes with bidentate phosphine oxide ligands and dimeric and tetrameric Mo(V) clusters with benzoic acid and phosphinic acid derivatives, containing MoO2, Mo2O2(μ-O)2 and Mo4O4(μ3-O)4

Mo(VI) oxo complexes have been persistently sought after as epoxidation catalysts. Further, Mo(V) oxo clusters of the form M4(µ3-X)4 (M = transition metal, X = O, S) have been rigorously studied due to their remarkable structures and also their usefulness as models for electronic studies. The syntheses and characterizations of new Mo(VI) and Mo(V) oxo complexes have been described in this dissertation. Two new complexes MoO2Cl2Ph2P(O)CH2COOH and MoO2Cl2Ph2P(O)C6H4tBuS(O) were synthesized from reactions of “MoO2Cl2” with ligands Ph2P(O)CH2COOH and Ph2P(O)C6H4tBuS(O). Tetrameric packing arrangements comprised of hydrogen bonds were obtained for the complex MoO2Cl2Ph2P(O)CH2COOH and the ligand Ph2P(O)CH2COOH. Further the stability of an Mo-O bond was preferred over the Mo-S bond even though this resulted in the formation of a more strained seven membered ring. Tetranuclear Mo(V) complexes of the form [Mo4(µ3-O)4(µ-O2PR2)4O4], (PR2 = PPh2, PMe2) were synthesized using reactions of MoO2(acac)2 with diphenyl and dimethyl phosphinic acids, in ethanol. In the crystal structure of these complexes four Mo=O units are interconnected by four triply bridging oxygen atoms and bridging phosphinate ligands. The complex exhibited fourfold symmetry as evidenced by a single 31P NMR peak for the P atoms in the coordinated ligands. Reaction of WO2(acac)2 with Ph2POOH in methanol resulted in a dimeric W(VI) complex [(CH3O)2(O)W(µ-O)( µ-O2PPh2)2W(O)(CH3O)2] which contained a packing disorder in its crystal structure. Similar reactions of MoO2(acac)2 with benzoic acid derivatives resulted in dimeric complexes of the form [Mo2O2(acac)2(µ-O)(µ-OC2H5)(µ-O2CR)] (R = C6H5, (o-OH)C6H4, (p-Cl)C6H4, (2,4-(OH)2)C6H3, (o-I)C6H4) and one tetrameric complex [Mo2O2(acac)2(µ-O)(µ-OC2H5)(µ-O2C)C6H4(p-µ-O2C)Mo2O2(acac)2(µ-O)(µ-OC2H5)] with terephthalic acid. 1H NMR proved very useful in the prediction of the formation of dimers with the substituted benzoic acids, which were also confirmed by elemental analyses. The reductive capability of ethanol proved instrumental in the syntheses of Mo(V) tetrameric and dimeric clusters. Synthetic details, IR, 1H and 31P NMR spectroscopy and elemental analyses are reported for all new complexes. Further, single crystal X-ray structures of MoO2Cl2Ph2P(O)CH2COOH, MoO2Cl2Ph2P(O)C6H4tBuS(O), [Mo4(µ3-O)4(µ-O2PR2)4O4], (PR2 = PPh2, PMe2), [(CH3O)2(O)W(µ-O)( µ-O2PPh2)2W(O)(CH3O)2] and [Mo2O2(acac)2(µ-O)(µ-OC2H5)(µ-O2CR)] (R = C6H5, (o-OH)C6H4) are also presented.

Method for Aqueous Gold Thiosulfate Extraction Using Copper-Cyanide Pretreated Carbon Asdortion

A gold thiosulfate leaching process uses carbon to remove gold from the leach liquor. The activated carbon is pretreated with copper cyanide. A copper (on the carbon) to gold (in solution) ration of at least 1.5 optimizes gold recovery from solution. To recover the gold from the carbon, conventional elution technology works but is dependent on the copper to gold ratio on the carbon.

Combinatorial synthesis and biological evaluation of isoxazole-based libraries as antithrombotic agents

The 3-substitutedphenyl-5-isoxazolecarboxaldehydes have been identified as activated aldehydes for the generation of isoxazole-based combinatorial libraries on solid phase through automation. Three highly functionalized isoxazole-based libraries comprising of 32, 96 and 45 compounds each have been synthesized in parallel format using Baylis Hillman reaction, Michael addition, reductive amination and alkylation reactions. With an objective of lead generation all the three libraries were evaluated for their antithrombin activity in vivo.

Proceedings of the Third Biochemical Engineering Symposium (April 28, 1973)

This report presents the proceedings of the Biochemical Engineering Symposium held at Kansas State University, April 28, 1973. Since a number of the contributions will be published in detail elsewhere, only brief summaries of each contribution are included here. Requests for additional information on projects conducted at The University of Nebraska should be directed to Dr. Peter J. Reilly, and those at Kansas State University to the editors. ContentsKenneth J. Jacobson, Andrew H.C. Chan, and Raymond C. Eliason, "Properties and Utilization of Small Particulates in Cattle Manure" Cady R. Engler and James S. Yohn, "Protein from Manure" Robert J. Williams, "Kinetics of Sucrose Inversion Using Invertase Immobilized on Hollow Fibers of Cellulose Acetate" David F. Aldis and Thomas A. Carlisle, "Study of a Triiodide-Resin Complex Disinfection System" John C. Heydweiller, "Modeling and Analysis of Symbiotic Growth" Kenneth J. Jacobson, "Synchronized Growth of the Blue Green Alga Microcystis aeruginosa" Clarence C. Y. Ron arui Lincoln L. S. Yang, "Computer Modeling of the Reductive Pentose Phosphate Cycle" Ming-ching T. Kuo, "Application of a Parallel Biochemical Oxidation Kinetic Model to the Design of an Activated Sludge System Including a Primary Clarifier" Prakash N. Mishra, "Optimal Synthesis of Water Renovation Systems"

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Experimental dissection and characterization of the functional domains in cardiac troponin C

Contraction of vertebrate cardiac muscle is regulated by the binding of Ca$\sp{2+}$ to the troponin C (cTnC) subunit of the troponin complex. In this study, we have used site-directed mutagenesis and a variety of assay techniques to explore the functional roles of regions in cTnC, including Ca$\sp{2+}$/Mg$\sp{2+}$-binding sites III and IV, the functionally inactive site I, the N-terminal helix, the N-terminal hydrophobic pocket and the two cysteine residues with regard to their ability to form disulfide bonds. Conversion of the first Ca$\sp{2+}$ ligand from Asp to Ala inactivated sites III and IV and decreased the apparent affinity of cTnC for the thin filament. Conversion of the second ligand from Asn to Ala also inactivated these sites in the free protein but Ca$\sp{2+}$-binding was recovered upon association with troponin I and troponin T. The Ca$\sp{2+}$-concentrations required for tight thin filament-binding by proteins containing second-ligand mutations were significantly greater than that required for the wild-type protein. Mutation of site I such that the primary sequence was that of an active site with the first Ca$\sp{2+}$ ligand changed from Asp to Ala resulted in a 70% decrease in maximal Ca$\sp{2\sp+}$ dependent ATPase activity in both cardiac and fast skeletal myofibrils. Thus, the primary sequence of the inactive site I in cTnC is functionally important. Major changes in the sequence of the N-terminus had little effect on the ability of cTnC to recover maximal activity but deletion of the first nine residues resulted in a 60 to 80% decrease in maximal activity with only a minor decrease in the pCa$\sb{50}$ of activation, suggesting that the N-terminal helix must be present but that a specific sequence is not required. The formation of an inter- or intramolecular disulfide bonds caused the exposure of hydrophobic surfaces on cTnC and rendered the protein Ca$\sp{2+}$ independent. Finally, elution patterns from a hydrophobic interactions column suggest that cTnC undergoes a significant change in hydrophobicity upon Ca$\sp{2+}$ binding, the majority of which is caused by site II. These latter data show an interesting correlation between exposure of hydrophobic surfaces on and activation of cTnC. Overall, these results represent significant progress toward the elucidation of the functional roles of a variety of structural regions in cTnC. ^

AN ANALYSIS OF THE DNA DAMAGE INDUCED BY NICKEL COMPOUNDS IN CHINESE HAMSTER OVARY CELLS (CARCINOGENESIS, HETEROCHROMATIN, CYTOTOXICITY, REPLICATION, PROTEIN CROSSLINKING)

The carcinogenic activity of water-insoluble crystalline nickel sulfide requires phagocytosis and lysosome-mediated intracellular dissolution of the particles to yield Ni('2+). This study investigated the extent and nature of the DNA damage in Chinese hamster ovary cells treated with various nickel compounds using the technique of alkaline elution. Crystalline NiS and water-soluble NiCl(,2) induced single strand breaks that were repaired quickly and DNA-protein crosslinks that persisted up to 24 hr after exposure to nickel. The induction of single strand breaks was concentration dependent at both noncytotoxic and lethal amounts of nickel. The induction of DNA-protein crosslinks was concentration dependent but was absent at lethal amounts of nickel. The cytoplasmic and nuclear uptake of nickel was concentration dependent even at the toxic level of nickel. However, the induction of DNA-protein crosslinks by nickel required active cell cycling and occurred predominantly in mid-late S phase of the cell cycle, suggesting that the lethal amounts of nickel inhibited DNA-protein crosslinking by inhibiting active cell cycling. Since the DNA-protein crosslinking induced by nickel was resistant to DNA repair, the nature of this lesion was investigated using various methods of DNA isolation and chromatin fractionation in combination with SDS-polyacrylamide gel electrophoresis. High molecular weight, non-histone chromosomal proteins and possibly histone 1 were preferentially crosslinked to DNA by nickel. The crosslinked proteins were concentrated in a magnesium-insoluble fraction of sonicated chromatin (5% of the total) that was similar to heterochromatin in solubility and protein composition. Alterations in DNA structure and function, brought about by the effect of nickel on protein-DNA interactions, may be related to the carcinogenicity of nickel compounds. ^