The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC50), assessed by parasite incorporation of [3H]hypoxanthine, were between 0.2 and 1.1 μM. CLT concentrations of 2 μM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (≈48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 μM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

The infected cell protein 0 (ICP0) of herpes simplex virus 1, a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection, interacts with numerous cellular proteins and has been linked to the disruption of ND10 and degradation of several proteins. ICP0 contains a RING finger domain characteristic of a class of E3 ubiquitin ligases. We report that: (i) in infected cells, ICP0 interacts dynamically with proteasomes and is bound to proteasomes in the presence of the proteasome inhibitor MG132. Also in infected cells, cdc34, a polyubiquitinated E2 ubiquitin-conjugating enzyme, exhibits increased ICP0-dependent dynamic interaction with proteasomes. (ii) In an in vitro substrate-independent ubiquitination system, the RING finger domain encoded by exon 2 of ICP0 binds cdc34, whereas the carboxyl-terminal domain of ICP0 functions as an E3 ligase independent of the RING finger domain. The results indicate that ICP0 can act as a unimolecular E3 ubiquitin ligase and that it promotes ubiquitin-protein ligation and binds the E2 cdc34. It differs from other unimolecular E3 ligases in that the domain containing the RING finger binds E2, whereas the ligase activity maps to a different domain of the protein. The results also suggest that ICP0 shuttles between nucleus and cytoplasm as a function of its dynamic interactions with proteasomes.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.

Amino ozonides exhibit in vitro activity against Echinococcus multilocularis metacestodes.

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20μM, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20μM, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11μM to 14μM. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection.

Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous urotensin-converting enzyme (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 muM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37degreesC) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37degreesC), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTF-ProhU-II to hU-II, and this was inhibited with 35 muM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37degreesC), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.

AP-2 transcription factor family member expression, activity, and regulation in human epidermal keratinocytes in vitro

The AP-2 transcription factor family is presumed to play an important role in the regulation of the keratinocyte squamous differentiation program; however, limited functional data are available to support this. In the present study, the activity and regulation of AP-2 were examined in differentiating human epidermal keratinocytes. We report that (1) AP-2 transcriptional activity decreases in differentiated keratinocytes but remains unchanged in differentiation-insensitive squamous cell carcinoma cell lines, (2) diminished AP-2 transcriptional activity is associated with a loss of specific DNA-bound AP-2 complexes, and (3) there is an increase in the ability of cytoplasmic extracts, derived from differentiated keratinocytes, to phosphorylate AP-2alpha and AP-2beta when cells differentiate. In contrast, extracts from differentiation-insensitive squamous cell carcinoma cells are unable to phosphorylate AP-2 proteins. Finally, the phosphorylation of recombinant AP-2alpha by cytosolic extracts from differentiated keratinocytes is associated with decreased AP-2 DNA-binding activity. Combined, these data indicate that AP-2 trans-activation and DNA-binding activity decrease as keratinocytes differentiate, and that this decreased activity is associated with an enhanced ability to phosphorylate AP-2alpha and beta.

The C1q binding activity of IgG is modified in vitro by reactive oxygen species: implications for rheumatoid arthritis

IgG can be denatured in vitro by reactive oxygen species (ROS). Native IgG activates the complement cascade through C1q. Using a modified ELISA, C1q binding activity of rheumatoid IgG has been compared to IgG denatured by neutrophil-derived ROS. The C1q binding activity of rheumatoid synovial fluid IgG is greater than the corresponding serum IgG (P < 0.01). Denaturation of IgG by activated polymorphs or the Fenton reaction decreased its C1q binding activity (P < 0.01). In vitro exposure of IgG to OH. and ROO. increased its interaction with C1q (P < 0.01). Hypochlorous acid had no effect. ROS-induced alteration to IgG-C1q binding activity may promote the inflammatory response in rheumatoid arthritis.

Pharmacologically induced and stimulus evoked rhythmic neuronal oscillatory activity in the primary motor cortex in vitro

Parkinson's disease (PD) is associated with enhanced synchronization of neuronal network activity in the beta (15-30 Hz) frequency band across several nuclei of the basal ganglia (BG). Deep brain stimulation of the subthalamic nucleus (STN) appears to reduce this pathological oscillation, thereby alleviating PD symptoms. However, direct stimulation of primary motor cortex (M1) has recently been shown to be effective in reducing symptoms in PD, suggesting a role for cortex in patterning pathological rhythms. Here, we examine the properties of M1 network oscillations in coronal slices taken from rat brain. Oscillations in the high beta frequency range (layer 5, 27.8 +/- 1.1 Hz, n=6) were elicited by co-application of the glutamate receptor agonist kainic acid (400 nM) and muscarinic receptor agonist carbachol (50 mu M). Dual extracellular recordings, local application of tetrodotoxin and recordings in M1 micro-sections indicate that the activity originates within deep layers V/VI. Beta oscillations were unaffected by specific AMPA receptor blockade, abolished by the GABA type A receptor (GABAAR) antagonist picrotoxin and the gap-junction blocker carbenoxolone, and modulated by pentobarbital and zolpidem indicating dependence on networks of GABAergic interneurons and electrical coupling. High frequency stimulation (HFS) at 125 Hz in superficial layers, designed to mimic transdural/transcranial stimulation, generated gamma oscillations in layers 11 and V (incidence 95%, 69.2 +/- 7.3 Hz, n=17) with very fast oscillatory components (VFO; 100-250 Hz). Stimulation at 4 Hz, however, preferentially promoted theta activity (incidence 62.5%, 5.1 +/- 0.6 Hz, n=15) that effected strong amplitude modulation of ongoing beta activity. Stimulation at 20 Hz evoked mixed theta and gamma responses. These data suggest that within M1, evoked theta, gamma and fast oscillations may coexist with and in some cases modulate pharmacologically induced beta oscillations.

Celiac disease IgA modulates vascular permeability in vitro through the activity of transglutaminase 2 and RhoA

Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients' serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a "gatekeeper" in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).