Luminescent anticancer Ru(II)-arenebipyridine and phenanthroline complexes: Synthesis, characterization, DFT studies, biological interactions and cellular imaging application.

A series of ruthenium(II)-arene complexes of several bipyridine and phenanthroline derivatives have been synthesized by employing a green and efficient protocol involving water as a solvent under sonication. The structures of all the complexes were elucidated by the spectroscopic analysis. The geometry of the chlorido and PTA (1,3,5-Triaza-7-phosphaadamantane) complexes were further confirmed by DFT and single crystal XRD. The stability study in various solvents, specifically in the intracellular one was conducted. Most of the compounds exhibited significant potency and selectivity against MCF7 and HeLa cell lines with respect to normal HEK-293 cells compared to cisplatin and RAPTA-C (Ruthenium(II)-arene PTA complex). Complex [(η6-hexamethylbenzene)RuCl(κ2-N,N-4,4'-di-n-nonyl-2,2'-bpy)]Cl (3e) presented best anticancer profiles against all the human cancer cells. Interestingly, few complexes turned up to be highly fluorescent depicted by the quantum yield values. Remarkably, [(η6-p-cymene)RuCl(κ2-N,N-bpy)]Cl (3i) was identified as most significant anticancer theranostic agent interms of potency, selectivity and fluorescence quantum yield. This complex also represented itself as significant cellular imaging agent in live U-87 MG cells which was monitored by confocal microscope. Absorption and emission spectral studies of bypyridine and phenanthroline complex series revealed that the complexes interacted with calf thymus DNA through groove binding as well as intercalative mode. In addition to this, strong binding efficacy of these scaffolds wih BSA (Bovin Serum Albumin) also enhanced their transportation property inside the cells.

Fusion of Aromatic Ring to Azoarenes: One-Pot Access to 5,6-Phenanthroliniums for Mitochondria-Targeted Far-Red/NIR Fluorescent Probes.

Disclosed herein is a highly efficient strategy to fuse an aromatic ring to azoarenes for one-pot access to 5,6-phenanthrolinium skeletons via tandem ortho-C-H arylation and aryl quaternization. This protocol enables ortho-hindered azobenzenes to solely form 5-aryl-5,6-phenanthroliniums and ortho-unhindered azobenzenes to exclusively generate 5,7-diaryl-5,6-phenanthroliniums. The diarylated products (5k-5r) exhibit far-red to NIR emissions (678-742 nm) with large Stokes shifts, can specifically light up mitochondria in living cells, and, moreover, possess excellent photostability and low cytotoxicity.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits improved transrepression/transactivation dissociation.

Classic glucocorticoids that have outstanding anti-inflammatory effects are still widely prescribed for the treatment of various inflammatory and autoimmune diseases. Conversely, glucocorticoids cause numerous unwanted side effects, particularly systemically dosed glucocorticoids. Therefore, selective glucocorticoid receptor modulator (SGRM), which maintains beneficial anti-inflammatory effects while reducing the occurrence of side effects, is one of the most anticipated drugs. However, there have been no SGRMs marketed to date. The assumption is that there are two major mechanisms of action of glucocorticoids via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, the anti-inflammatory effects of glucocorticoids are mostly mediated through TR, while the side effects associated with glucocorticoids are largely caused by TA. We started to evaluate novel orally available SGRMs that maintain anti-inflammatory effects while minimizing adverse effects by favoring TR over TA. Based on this evaluation, we discovered JTP-117968, (4b'S,7'R,8a'S)-4b'-benzyl-7'-hydroxy-N-(2-methylpyridin-3-yl)-7'-(trifluoromethyl)-4b',6',7',8',8a',10'-hexahydro-5'H-spiro[cyclopropane-1,9'-phenanthrene]-2'-carboxamide, a non-steroidal SGRM. JTP-117968 has partial TR activity, but exhibits extremely low TA activity. The maximum TR efficacy of JTP-117968 was comparable to its structural analogue, PF-802, (4bS,7R,8aR)-4b-Benzyl-7-hydroxy-N-(2-methylpyridin-3-yl)-7-(trifluoromethyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide, which is the active form of Fosdagrocorat that has been developed clinically as a first-in-class orally available SGRM. Remarkably, the TA activity of JTP-117968 was much weaker than PF-802 not only in in vitro assays, but also in in vivo mice experiments. These findings indicate that JTP-117968 exhibits improved TR/TA dissociation because the compound has significantly lower TA activity compared with an already reported SGRM. Therefore, JTP-117968 is expected to be a useful compound for evaluating ideal SGRMs in the future.

Phase 1 study of APTO-253 HCl, an inducer of KLF4, in patients with advanced or metastatic solid tumors.

INTRODUCTION: This phase I, multicenter, open-label, single-arm, dose-escalation study evaluated the safety, pharmacokinetics and antitumor activity of APTO-253, an inducer of the transcription factor KLF4, in adults with advanced solid tumors. METHODS: APTO-253 was administered IV on days 1 and 2, and 15 and 16 of each 28 day cycle; the dose were escalated from 20 to 387 mg/m(2) in 9 cohorts until DLT was observed. RESULTS: Thirty-two patients were treated on this trial (50 % colon cancer, 22 % other gastrointenstinal malignancies and 18 % non-small cell lung cancer). Fatigue was the only drug-related treatment-emergent adverse event to occur in >10 % of patients. Dose-limiting toxicities of hypersensitivity reaction and transient hypotension despite prophylaxis occurred at 387 mg/m(2) which led to identification of 298 mg/m(2) as the MTD. Only 1 patient had any drug-related treatment-emergent grade 3 adverse event at or below 229 mg/m(2). A total of 21 patients underwent at least one restaging after 2 cycles; 11 patients discontinued prior to the end of cycle 2 due to adverse events (9) or disease progression (2). The best overall response was stable disease (SD) in 5 of these 21 (23.8 %) with durations ranging from 3.6 to 8.4 months. CONCLUSION: APTO-253 was well tolerated at the Phase 2 recommended dose and produced evidence of antitumor activity in the form of stable disease in patients with advanced solid tumors. Based on the drug levels achieved and the lower frequency of treatment-emergent adverse events encountered, 229 mg/m(2) was selected as the recommended Phase 2 dose. Overall APTO-253 was found to be well tolerated and to have favorable pharmacokinetics, and treatment was associated with stable disease in 5 of 21 (24 %) of patients with far advanced solid tumors.

Rhenium(I) polypyridine diamine complexes as intracellular phosphorogenic sensors: synthesis, characterization, emissive behavior, biological properties, and nitric oxide sensing.

We report the development of a series of rhenium(I) polypyridine complexes appended with an electron-rich diaminoaromatic moiety as phosphorogenic sensors for nitric oxide (NO). The diamine complexes [Re(N^N)(CO)3 (py-DA)][PF6 ] (py-DA=3-(N-(2-amino-5-methoxyphenyl)aminomethyl)pyridine; N^N=1,10-phenanthroline (phen) (1 a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4 -phen) (2 a), 4,7-diphenyl-1,10-phenanthroline (Ph2 -phen) (3 a)) have been synthesized and characterized. In contrast to common rhenium(I) diimines, these diamine complexes were very weakly emissive due to quenching of the triplet metal-to-ligand charge-transfer ((3) MLCT) emission by the diaminoaromatic moiety through photoinduced electron transfer (PET). Upon treatment with NO, the complexes were converted into the triazole derivatives [Re(N^N)(CO)3 (py-triazole)][PF6 ] (py-triazole=3-((6-methoxybenzotriazol-1-yl)methyl)pyridine; N^N=phen (1 b), Me4 -phen (2 b), Ph2 -phen (3 b)), resulting in significant emission enhancement (I/I0 ≈60). The diamine complexes exhibited high reaction selectivity to NO, and their emission intensity was found to be independent on pH. Also, these complexes were effectively internalized by HeLa cells and RAW264.7 macrophages with negligible cytotoxicity. Additionally, the use of complex 3 a as an intracellular phosphorogenic sensor for NO has been demonstrated.

In vivo measurement of tissue oxygenation by time-resolved luminescence spectroscopy: advantageous properties of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate.

Measuring tissue oxygenation in vivo is of interest in fundamental biological as well as medical applications. One minimally invasive approach to assess the oxygen partial pressure in tissue (pO2) is to measure the oxygen-dependent luminescence lifetime of molecular probes. The relation between tissue pO2 and the probes' luminescence lifetime is governed by the Stern-Volmer equation. Unfortunately, virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity. For that reason, we studied the oxygen sensitivity and phototoxicity of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate [Ru(Phen)] using a dedicated optical fiber­based, time-resolved spectrometer in the chicken embryo chorioallantoic membrane. We demonstrated that, after intravenous injection, Ru(Phen)'s luminescence lifetime presents an easily detectable pO2 dependence at a low drug dose (1 mg∕kg) and low fluence (120 mJ∕cm2 at 470 nm). The phototoxic threshold was found to be at 10 J∕cm2 with the same wavelength and drug dose, i.e., about two orders of magnitude larger than the fluence necessary to perform a pO2 measurement. Finally, an illustrative application of this pO2 measurement approach in a hypoxic tumor environment is presented.

Danshen diversity defeating dementia.

Salvia miltiorrhiza (danshen) is widely used for the clinical treatment of cerebral ischemia and cardiovascular diseases. Its diverse molecular makeup of simple and poly hydroxycinnamic acids and diterpenoid quinones are also associated with its beneficial health effects such as improved cognitive deficits in mice, protection of neuronal cells, prevention of amyloid fibril formation and preformed amyloid fibril disaggregation related to Alzheimer's disease. Whilst the in vitro studies have therapeutic promise, the anti-dementia effect/impact of danshen however depends on its absorbed constituents and pharmacokinetic properties. Both the water and lipid danshen fractions have been shown to have low oral bioavailability and at physiological pH, the polyphenolic carboxylate anions are not brain permeable. To tap into the many neuroprotective and other biological benefits of danshen, the key challenge resides in developing danshen nanopharmaceuticals, semi-synthetic pro-drug forms of its constituents to improve its biocompatability, that is, absorption, circulation in bloodstream and optimization of BBB permeability.

Investigation of the absorbed and metabolized components of Danshen from Fuzheng Huayu recipe and study on the anti-hepatic fibrosis effects of these components.

ETHNOPHARMACOLOGY: Fuzheng Huayu recipe (FZHY) was formulated on the basis of Chinese medicine theory in treating liver fibrosis. It has a significant efficacy against liver fibrosis caused by chronic hepatitis B, with the action mechanisms of inhibition of hepatic stellate cell activation, protection of hepatocyte oxidative injury and regulations of hepatic matrix remodeling etc. AIM OF THE STUDY: To identify the absorbed components and metabolites of Danshen in FZHY in rat serum, and find their active components for anti-liver fibrosis. MATERIAL AND METHODS: A valid high performance liquid chromatography-electrospray ionization ion trap mass spectrometry (HPLC-ESI/MS(n)) method was established to investigate the absorbed and metabolized compounds of Danshen in FZHY in rat serum after oral administration. Mass spectra were acquired in both negative and positive modes. Otherwise, to evaluate the anti-hepatic fibrosis efficacies of absorbed and metabolized compounds, the LX-2 cell line of hepatic stellate cell (HSC), which was crucial cellular basis of fibrogenesis, was cultured and incubated with absorbed compounds, the cytotoxicity was determined with the cellomics Multiparameter Cytotoxicity Kit 1 by High Content Screening (HCS), the cell proliferation was assayed with EdU-DNA incorporation, and the cell activation was analyzed through α-smooth muscle actin (α-SMA) expression with high content screening technology. RESULTS: More than 11 compounds and 2 metabolites from Danshen were identified in the serum after oral administration of FZHY by comparing their mass spectra and retention behavior with reference compounds or literature data. Among these compounds, there were no obvious changes in nuclear morphology, membrane permeability with blow 96 µM of six polar compounds treatment in comparison with control cells, respectively. And the salvianolic acid B (6 µM, 48 µM), caffeic acid (6 µM, 48 µM) and rosmarinic acid (48 µM) could obviously inhibit LX-2 cells proliferation, down-regulate α-SMA expression. CONCLUSION: The results proved that the established method could be applied to analyze the absorbed into blood compounds of Danshen after oral administration FZHY. These absorbed compounds included 11 compounds and 2 metabolites of Danshen. Among them, the salvianolic acid B, caffeic acid and rosmarinic acid were the effective components of FZHY to anti-hepatic fibrosis effects.

Intracellular surface-enhanced Raman scattering (SERS) with thermally stable gold nanoflowers grown from Pt and Pd seeds.

SERS provides great sensitivity at low concentrations of analytes. SERS combined with near infrared (NIR)-resonant gold nanomaterials are important candidates for theranostic agents due to their combined extinction properties and sensing abilities stemming from the deep penetration of laser light in the NIR region. Here, highly branched gold nanoflowers (GNFs) grown from Pd and Pt seeds are prepared and their SERS properties are studied. The growth was performed at 80 °C without stirring, and this high temperature growth method is assumed to provide great shape stability of sharp tips in GNFs. We found that seed size must be large enough (>30 nm in diameter) to induce the growth of those SERS-active and thermally stable GNFs. We also found that the addition of silver nitrate (AgNO3) is important to induce sharp tip growth and shape stability. Incubation with Hela cells indicates that GNFs are taken up and reside in the cytoplasm. SERS was observed in those cells incubated with 1,10-phenanthroline (Phen)-loaded GNFs.

Air flow-assisted ionization imaging mass spectrometry method for easy whole-body molecular imaging under ambient conditions.

Whole-body molecular imaging is able to directly map spatial distribution of molecules and monitor its biotransformation in intact biological tissue sections. Imaging mass spectrometry (IMS), a label-free molecular imaging method, can be used to image multiple molecules in a single measurement with high specificity. Herein, a novel easy-to-implement, whole-body IMS method was developed with air flow-assisted ionization in a desorption electrospray ionization mode. The developed IMS method can effectively image molecules in a large whole-body section in open air without sample pretreatment, such as chemical labeling, section division, or matrix deposition. Moreover, the signal levels were improved, and the spatial assignment errors were eliminated; thus, high-quality whole-body images were obtained. With this novel IMS method, in situ mapping analysis of molecules was performed in adult rat sections with picomolar sensitivity under ambient conditions, and the dynamic information of molecule distribution and its biotransformation was provided to uncover molecular events at the whole-animal level. A global view of the differential distribution of an anticancer agent and its metabolites was simultaneously acquired in whole-body rat and model mouse bearing neuroglioma along the administration time. The obtained drug distribution provided rich information for identifying the targeted organs and predicting possible tumor spectrum, pharmacological activity, and potential toxicity of drug candidates.

Identification of phenanthroindolizines and phenanthroquinolizidines as novel potent anti-coronaviral agents for porcine enteropathogenic coronavirus transmissible gastroenteritis virus and human severe acute respiratory syndrome coronavirus.

The discovery and development of new, highly potent anti-coronavirus agents and effective approaches for controlling the potential emergence of epidemic coronaviruses still remains an important mission. Here, we identified tylophorine compounds, including naturally occurring and synthetic phenanthroindolizidines and phenanthroquinolizidines, as potent in vitro inhibitors of enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV). The potent compounds showed 50% maximal effective concentration (EC50) values ranging from 8 to 1468 nM as determined by immunofluorescent assay of the expression of TGEV N and S proteins and by real time-quantitative PCR analysis of viral yields. Furthermore, the potent tylophorine compounds exerted profound anti-TGEV replication activity and thereby blocked the TGEV-induced apoptosis and subsequent cytopathic effect in ST cells. Analysis of the structure-activity relations indicated that the most active tylophorine analogues were compounds with a hydroxyl group at the C14 position of the indolizidine moiety or at the C3 position of the phenanthrene moiety and that the quinolizidine counterparts were more potent than indolizidines. In addition, tylophorine compounds strongly reduced cytopathic effect in Vero 76 cells induced by human severe acute respiratory syndrome coronavirus (SARS CoV), with EC50 values ranging from less than 5 to 340 nM. Moreover, a pharmacokinetic study demonstrated high and comparable oral bioavailabilities of 7-methoxycryptopleurine (52.7%) and the naturally occurring tylophorine (65.7%) in rats. Thus, our results suggest that tylophorine compounds are novel and potent anti-coronavirus agents that may be developed into therapeutic agents for treating TGEV or SARS CoV infection.

Pharmacokinetic interactions induced by content variation of major water-soluble components of Danshen preparation in rats.

AIM: To investigate the pharmacokinetic interactions induced by content variation of the main water-soluble components of Danshen injection in rats. METHODS: Intravenous Danshen injection (control) or Danshen injection with danshensu (DSS), protocatechuic aldehyde (PAL), salvianolic acid A (Sal A) or salvianolic acid B (Sal B) were administered to female Sprague Dawley rats. Plasma concentrations of DSS, Sal A, PAL and its oxidative metabolite protocatechuic acid (PA) were analyzed simultaneously with LC-MS/MS; concentrations of Sal B were determined by the LC-MS method. Non-compartmental pharmacokinetic parameters were calculated and compared for identifying the pharmacokinetic interactions among these components. RESULTS: Compared with the control group, the DSS, Sal A, and Sal B groups had significant increases in AUC(0-infinity) in response to elevated concentrations of PAL (by 78.1%, 51.0%, and 82.9%, respectively), while the clearances (CL) were markedly reduced (by 42.5%, 32.9%, and 46.8%, respectively). Similarly, Sal A increased the AUC(0-infinity) of DSS and Sal B (26.7% and 82.4%, respectively) and substantially decreased their clearances (21.4% and 45.6%, respectively). In addition, the pharmacokinetics of DSS and Sal B were significantly affected by the content variation of the other major components; the AUC(0-infinity) increased by 45.1% and 52.1%, respectively, the CL dropped by 29.6% and 27.1%, respectively, and the T(1/2) was decreased by 22.0% and 19.6%, respectively. CONCLUSION: Complex, extensive pharmacokinetic interactions were observed among the major water-soluble constituents in the Danshen injection. The content variation of PAL had the most significant effect on the pharmacokinetic behaviors of other major constituents. Furthermore, the pharmacokinetics of DSS and Sal B were the most susceptible to the content change of other components.

Synthesis of a novel tricyclic 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system and CXCR4 antagonists with potent activity against HIV-1.

Stereorandom and diastereoselective syntheses of a novel 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system are described. Derivatives of all four diastereomers were prepared and isolated in >98% ee. The pure enantiomers were compared in order to determine the preferred absolute and relative configuration required for optimal anti-HIV activity. Anti-HIV potency and pharmacokinetic properties of the newly synthesized tricyclic octahydrophenanthroline inhibitors are presented and comparisons are made to previously reported bicyclic (8S)-N-methyl-5,6,7,8-tetrahydro-8-quinolinamine analogs.

Preparation and quality control of ¹77Lu-[tris(1,10-phenanthroline) lutetium(III)] complex for therapy.

The ¹77Lu-[tris(1,10-phenanthroline)lutetium(III)] complex (¹77Lu-PQ3) was prepared successfully with high radiochemical purity (> 99%). Lu-177 chloride was obtained by thermal neutron flux (4 × 10¹³ n.cm⁻².s⁻¹) of natural Lu2(NO3)3 sample, dissolved in acidic media. The radiochemical yield was checked by measuring the radiochemical purity of the (177)Lu-PQ complex by ITLC (10 mM DTPA, pH = 5, as mobile phase). The final complex solution was injected intravenously into wild-type male rats and bio-distribution of the complex was checked for up to 48 hours. The dose limiting organs were shown to be the reticulu-endothelial system. The bio-distribution of the labelled compounds in tumour-bearing animals is under investigation.

[Evaluation of different combinations of components of Chinese formulation shuangshentongguan by using AUC values].

A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was applied for the quantification of each component: tetrahydropalmatine (THP), dehydrocorydaline (DHC), salvianolic acid B (SAB), ginsenoside Rg1, Re, Rb1 and Rd in the Chinese herbal component SSTG (Shuangshentongguan) with different combinations. The pharmacokinetic data were analyzed with WinNonlin 5.2 software. The results showed that combination can increase the THP AUC value while the AUC values of SAB, ginsenoside Rg1, Re, Rb1 and Rd were reduced. These results showed significant differences. The AUC value of ginsenoside Rb1 was increased when combined with Danshen or Yanhusuo, but reduced when combined with Danshen and Yanhusuo. The DHC concentration in serum was too low to be determined.

Effect of danshen extract on pharmacokinetics of theophylline in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Danshen extract is widely used for the treatment and prevention of coronary heart disease and other diseases of senility in Asia. Danshen extract and theophylline may be prescribed together to treat patients with asthma. In human, theophylline with low therapeutic index is mainly metabolized by CYP1A2. In vitro findings have shown that human CYP1A2 is inhibited by the ethyl acetate extract of danshen and danshen pharmaceutical product. There may be drug interactions between danshen extract and theophylline (CYP1A2 substrate). WHAT THIS STUDY ADDS: This study concerned drug interactions between danshen extract and theophylline in Chinese volunteers. Long-term oral intake of danshen extract does not change the basic pharmacokinetic parameters of theophylline. Dose adjustment of theophylline thus may not be necessary in patients receiving concomitant therapy with danshen extract. AIMS: To examine the potential effect of danshen extract on the pharmacokinetics of theophylline. METHODS: In a sequential cross-over study with two phases, 12 volunteers took 100 mg theophylline on day 1 and day 15. From day 2 to day 15, volunteers received danshen extract tablets three times daily, four tablets each time for 14 days. On day 15, they received four danshen extract tablets with 100 mg theophylline. Plasma concentrations of theophylline were measured on days 1 and 15 periodically for 24 h. RESULTS: The 90% confidence interval of C(max), t(1/2) and CL/F of theophylline with 14-day danshen extract tablets vs. without comedication were (101.42, 121.36) (84.57, 106.72) and (88.82, 105.72), respectively. The time to peak plasma theophylline concentration was unchanged by danshen (P > 0.05). The pharmacokinetics parameter of theophylline was unaffected by danshen extract. CONCLUSIONS: Danshen extract does not influence the metabolism of theophylline in healthy volunteers. Dose adjustment of theophylline thus may not be necessary in patients receiving concurrent therapy with danshen extract tablets.

Simultaneous determination of magnesium lithospermate B, rosmarinic acid, and lithospermic acid in beagle dog serum by liquid chromatography/tandem mass spectrometry.

A rapid, sensitive and specific isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantitative determination of magnesium lithospermate B (MLB), rosmarinic acid (RA), and lithospermic acid (LA) in beagle dog serum, with silibinin as internal standard. The serum samples were treated by liquid-liquid extraction and analyzed using LC/MS/MS with a TurboIonSpray source. A short run-time (3 min) fulfilled the need for monitoring serum levels of MLB, RA, and LA in large-scale studies. The calibration curves for MLB, RA, and LA were linear over the ranges 8-2048, 4-1024, and 4-1024 ng/mL, respectively, with coefficients of correlation >0.999. The intra- and inter-day precision (CV) of analysis was <10%, and accuracy ranged from 90-104%. This quantitation method was successfully applied to a pharmacokinetic study of salvianolate administrated by intravenous infusion with dosage of 6 mg/kg in beagle dogs.

Acridine conjugates of 3-clip-phen: influence of the linker on the synthesis and the DNA cleavage activity of their copper complexes.

To increase the DNA cleavage activity and the cell delivery of the bis(phenanthroline) DNA cleaver "3-Clip-Phen", conjugates between 3-Clip-Phen and the intercalators acridine and 6-chloro-2-methoxyacridine, through amino acid linkers of various length, were prepared. After complexation with CuCl(2), the ability of these conjugates to cleave phiX 174 DNA in the presence of a reductant and air was compared. The results indicated that (i) the coupling of 3-Clip-Phen to an acridine derivative increased the DNA cleavage efficiency of the copper complexes, (ii) the acridine derivatives were more active than 6-chloro-2-methoxyacridine derivatives, (iii) the linker length influenced cleavage efficiency, the highest DNA cleavage activity being obtained for an aminocaproic spacer.

Development of a digital fluorescence sensing technique to monitor the response of macrophages to external hypoxia.

Oxygen plays a very important role in living cells. The intracellular level of oxygen is under tight control, as even a small deviation from normal oxygen level affects major cellular metabolic processes and is likely to result in cellular damage or cell death. This paper describes the use of the oxygen sensitive fluorescent dye tris (1,10-phenanthroline) ruthenium chloride [Ru(phen)(3)] as an intracellular oxygen probe. Ru(phen)(3) exhibits high photostability, a relatively high excitation coefficient at 450 nm (18 000 M(-1) cm(-1)), high emission quantum yield ( approximately 0.5), and a large Stoke shift (peak emission at 604 nm). It is effectively quenched by molecular oxygen due to its long excited state lifetime of around 1 micros. The luminescence of Ru(phen)(3) decreases with increasing oxygen concentrations and the oxygen levels are determined using the Stern-Volmer equation. In our studies, J774 Murine Macrophages are loaded with Ru(phen)(3), which passively permeates into the cells. Fluorescence spectroscopy and digital fluorescence imaging microscopy are used to observe the cells and monitor their response to changing oxygen levels. The luminescence intensity of the cells decreases when exposed to hypoxia and recovers once normal oxygen conditions are restored. The analytical properties of the probe and its application in monitoring the cellular response to hypoxia are described.

A novel system to study the impact of epithelial barriers on cellular metabolism.

Medications introduced into the systematic circulation must be transported across biological barriers such as skin, gastrointestinal, or bronchial epithelia, which can alter their kinetic and metabolic profiles. It is, therefore, important to understand diffusion kinetics across barrier membranes when choosing a dosing regime that will elicit the greatest cellular response. An in vitro system that combines membrane transport studies with a downstream cell culture chamber has been developed. The system has been tested with skin and a small intestine model (Caco-2 cell monolayers) as barriers, the peroxovanadium compound [VO(O2)2 1, 10 phenanthroline] bpV(phen), as the test chemical, Hep-G2 (liver) as the test cells, and glucose consumption as the test assay. Peroxovanadium has insulin mimetic properties and has been previously demonstrated to effectively lower blood glucose levels in diabetic rats when administered transdermally. A dose of 10 mM bpV(phen) placed on the skin epidermis with a continuous iontophoretic current of 0.5 mA/cm2 for 4.5 h led to a net 22% increase in glucose consumption by Hep-G2 cells. The same dose of bpV(phen) passively diffusing across a Caco-2 cell monolayer led to an increase in glucose consumption by Hep-G2 cells of 23%. This system is highly versatile and can be used to study many other processes, involving a variety of biological membranes, cell types, chemicals and assays, making it a valuable research tool.