BacMam recombinant baculovirus in transporter expression: a study of BCRP and OATP1B1

Human BCRP and OATP1B1 have recently been identified as important transporters in the absorption, distribution, and elimination of clinically significant drugs. In this report, we illustrate the use of modified baculoviruses, termed BacMam viruses for the expression of functional BCRP and OATP1B1 in mammalian cells. We show a variety of host cells efficiently transduced to express BCRP including HEK 293, LLC-PK, and U-2 OS, where protein levels on the cell-surface were modulated by titrating different amounts of viral inoculum. In addition, using the BODIPY-prazosin efflux assay and the BacMam reagent we illustrate inhibition of BCRP activity with GF120918 or Fumitremorgin C. Furthermore, we present data demonstrating simultaneous expression of BCRP and OATP1B1 in BacMam transduced mammalian cells by simply adding viral inoculum of each transporter. Thus these results indicate that BacMam mediated gene delivery provides a novel and efficient research tool for the investigation of single or multiple transporters in vitro.     

Quantitative immunogold study of increased expression of metallothionein-I/II in the brain perivascular areas of diabetic scrapie-infected mice

Quantitative immunogold procedure was used to study the distribution of metallothionein I/II (MT-I/II) at the ultrastructural level in the perivascular areas, including microvascular endothelial cells (ECs) and astrocytes with their perivascular end-feet, in brains of scrapie-infected hyperglycemic (diabetic) and normoglycemic (non-diabetic) mice. Samples of the fronto-parietal cortex obtained from diabetic and non-diabetic scrapie-infected, as well as from non-infected (control) SJL/J mice, were processed for immunocytochemical examination. In control mice, the labelling of the ECs was of low intensity, restricted to few immunogold particles in the cytoplasm. More intense labelling was present in the cytoplasm of astrocytic perivascular processes and perikarya, where it was associated with endoplasmic reticulum and fibrils. A few immunosignals were also present inside the nuclei of astrocytes. In diabetic mice the labelling of the EC cytoplasm was slightly increased, whereas in the cytoplasm of perivascular processes and pericarya of astrocytes, including their nuclei, there was significant enhancement of labelling. In these cells the density of immunosignals was highest in the areas of cytoplasm containing bundles of fibrils. In non-diabetic, scrapie-infected mice the intensity of immunolabelling was higher than in control mice but slightly lower than in diabetic mice. These results are similar to those in Alzheimer’s disease reported by other authors, and suggest that neurodegenerative diseases as well as metabolic stress enhance the metallothionein expression in perivascular regions of brain cerebral cortex, predominantly in astrocytes.     

Synthesis and antiprotozoal activity of 2,5-bis[amidinoaryl]thiazoles

Seven novel diamidino 2,5-bis(aryl)thiazoles (5a–g) were synthesized and evaluated against Trypanosoma brucei rhodensiense (T. b. r.) and Plasmodium falciparum (P. f.). The diamidines were obtained directly from the corresponding bis-nitriles (4a–g) by the action of lithium bis(trimethylsilyl)amide. The bis-nitriles 4a–f were synthesized in four steps starting with the Stille coupling of 2-tributyltinthiazole with the appropriate cyanoaryl halide. The bis-nitrile 5g was obtained by the palladium facilitated coupling of the mixed tin-silyl reagent 2-trimethylsilyl-5-trimethyltinthiazole with 2-bromo-5-cyanopyridine. The amidoxime potential prodrugs 6a–e, 6g were obtained by the reaction of hydroxylamine with the bis-nitriles. O-Methylation of the amidoximes gave the corresponding N-methoxyamidines 7a–c, 7e, 7g. The diamidines showed strong DNA binding affinity as reflected by ΔT_m measurements. Four of the diamidines 5a, 5b, 5d and 5e were highly active in vitro against P. f. giving IC₅₀ values between 1.1 and 2.5 nM. The same four diamidines showed IC₅₀ values between 4 and 6 nM against T. b. r. The selectivity indices ranged from 233 to 9175. One diamidine 5a produced one of four cures at an ip dose of 4 × 5 mg/kg in the STIB900 mouse model for acute African trypanosomiasis. The amidoxime and N-methoxyamidine of 5a were the only produgs to provide cures (1/4 cures) in the same mouse model on oral dosage at 4 × 25 mg/kg.     

Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats

The aim of this study was to examine the effect of an infection with Hymenolepis diminuta on ion transport in an isolated colon and blood picture of rats. Fifty rats were orally infected with five cysticercoids of H. diminuta. The experimental groups of rats were assigned to four groups: group I - 8 days post-infection (dpi), group II - 16 dpi, group III - 40 dpi and group IV- 60 dpi. The control group comprised non-infected rats. The experiments consisted of measuring the transepithelial electrical potential difference (PD) and the transepithelial electrical resistance (R) of the rat colon under controlled conditions as well as during mechanical stimulation (MS) using a modified Ussing chamber. Ion transport was modified using inhibitors of the epithelial sodium channel (amiloride - AMI) and the epithelial chloride channel (bumetanide - BUME), and also using capsaicin (CAPSA), a substance which activates C-fibres. The experimental data presented in this study indicates that experimental hymenolepidosis inhibits sodium and chloride ion transport in the epithelium of the rat colon, with preserved tight junction continuity (except at 40 dpi) and a decreased mechanical sensitivity. The effect of capsaicin on ion transport in the rat colon was varied. In control rats it increased ionic current, and in H. diminuta-infected rats it did not cause any changes in PD.Blood picture in this study showed a statistically significantly lower red blood cells (RBC) count and haemoglobin (HGB) concentration in infected rats in comparison to non-infected. Red cell distribution width (RDW) values and platelet (PLT) count were negatively correlated with the duration of infection, whereas mean corpuscular volume (MCV) value was positively correlated. We did not observe leukocytosis during infection, and amongst the differential leukocyte counts eosinophils and basophils showed statistically significant lower values in infected rats in comparison to non-infected.Our results indicate that hymenolepidosis is associated with the activation of inflammatory mediators and stimulation of nervous fibres, which significantly affects the function of ion channels in the epithelium of the colon in the host. At the same time, a significant decrease in eosinophil count during infection suggests that such an infection did not trigger a strong immunological reaction in rats.     

The influence of the two different light intensities duringthe daytime on the colon motility in patients with irritable bowel syndrome (IBS): a preliminary report

Irritable bowel syndrome (IBS) is the most frequent functional disorder of the gastrointestinal tract, with great economical impact. The etiology and pathogenesis of the disease are unclear. Among patients seeking medical attention for IBS, 70 - 90% have psychiatric co-morbidity, most commonly major depression. In this study we test the influence of bright light on colon motility and subjectively felt symptoms. Eight IBS patients participated in the experiment. The passage rate was evaluated twice for every person: (1) after three days of exposure to bright light -3000 lux (from 8:00 to 18:00), (2) after three days exposure to dim light -100 lux (from 8:00 to 18:00). The comparison of colonic time in IBS patients point to differences in the elimination of markers, depending on experimental light conditions. After bright light conditions a tendency is observed to slower elimination of markers in the IBS-diarrhoea patients, and to a quicker elimination in the IBS-constipated patients. According to subjective feeling, all IBS patients reported an improvement in bowel function and relief of pain after bright but not after dim light application. Results of this preliminary study suggest that phototherapy might be a valuable addition to the conventional treatment of IBS.     

Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env(121-129) epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.     

In vitro study of AMP-deaminase from fish (Cyprinus carpio) treated with hydrolyzable tannins

AMP-deaminase (EC 3.5.4.6) is an enzyme responsible for stabilising adenylate energy changes. The properties of this enzyme are controlled by various ligands of hydrophobic nature. An investigation of enzyme activity alterations under the influence of natural phenolic acids (tannic, ellagic and gallic) which are soluble in water, could evidence the biological toxicity of these compounds. In our study purified AMP-deaminase isolated from white muscle of Cyprinus carpio was exposed to phenolic acids in the concentration range of 1 to 50 microM as well as to tannic acid in the presence of Cu2+ ions (5 microM). On the basis of the obtained results we can conclude that among the tested acids, gallic acid did not contribute to the change in AMP-deaminase activity, whereas ellagic acid diminished its activity at the highest concentration (50 microM). Tannic acid caused a significant decrease in the enzyme activity in comparison to control for all used concentrations. Cu2+ ions alone reduced the activity of AMP-deaminase for all studied concentrations. A combined action of a chosen Cu2+ ions concentration (5 microM) with tannic acid at the concentration higher than 2 microM resulted in a decrease in the enzyme activity, but for lower tannic acid concentration of 1 microM the activity of AMP-deaminase was stimulated. These experiments showed that tannic acid may stop free radical chain reactions only at low concentrations (1 microM) in the presence of Cu2+ ions (5 microM).