Determination of Serotonin and Dopamine Metabolites in Human Brain Microdialysis and Cerebrospinal Fluid Samples by UPLC-MS/MS: Discovery of Intact Glucuronide and Sulfate Conjugates

An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.     

Microwave-assisted synthesis of quinoline, isoquinoline, quinoxaline and quinazoline derivatives as CB2 receptor agonists

Quinoline, isoquinoline, quinoxaline, and quinazoline derivatives were synthesized using microwave-assisted synthesis and their CB1/CB2 receptor activities were determined using the [3?S]GTPγS binding assay. Most of the prepared quinoline, isoquinoline, and quinoxalinyl phenyl amines showed low-potency partial CB2 receptor agonists activity. The most potent CB2 ligand was the 4-morpholinylmethanone derivative (compound 40e) (-log EC?? = 7.8; E(max) = 75%). The isoquinolin-1-yl(3-trifluoromethyl-phenyl)amine (compound 26c) was a high efficacy CB2 agonist (-log EC?? = 5.8; E(max) = 128%). No significant CB1 receptor activation or inactivation was shown in these studies, except 40e, which showed weak CB1 agonist activity (CB1 -log EC?? = 5.0). These ligands serve as novel templates for the development of selective CB2 receptor agonist.     

Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma

Background

Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs.

Methodology/Principal Findings

We studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections.

Conclusions/Significance

Intraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.     

Kinetics of PKCε activating and inhibiting llama single chain antibodies and their effect on PKCε translocation in HeLa cells

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing V(max) and/or decreasing K(m) values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.     

Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.     

LPS,Oleuropein and Blueberry extracts affect the survival,morphology and Phosphoinositide signalling in stimulated human endothelial cells

Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes’ expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.     

Natural extracellular nanovesicles and photodynamic molecules: is there a future for drug delivery?

Photodynamic molecules represent an alternative approach for cancer therapy for their property (i) to be photo-reactive; (ii) to be not-toxic for target cells in absence of light; (iii) to accumulate specifically into tumour tissues; (iv) to be activable by a light beam only at the tumour site and (v) to exert cytotoxic activity against tumour cells. However, to date their clinical use is limited by the side effects elicited by systemic administration. Extracellular vesicles are endogenous nanosized-carriers that have been recently introduced as a natural delivery system for therapeutic molecules. We have recently shown the ability of human exosomes to deliver photodynamic molecules. Therefore, this review focussed on extracellular vesicles as a novel strategy for the delivery of photodynamic molecules at cancer sites. This completely new approach may enhance the delivery and decrease the toxicity of photodynamic molecules, therefore, represent the future for photodynamic therapy for cancer treatment.     

Role of ω-Conotoxin GVIA-Sensitive Ca2+ Entry in Angiotensin II-Stimulated [3H]Phorbol 12, 13-Dibutyrate Binding in Bovine Adrenal Medullary Cells

Abstract: The relative contributions of Ca²⁺ influx and intracellular Ca²⁺ mobilization were examined for angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding, which reflects the level of activated protein kinase C in bovine chromaffin cells. Angiotensin II receptors activate phospholipase C in chromaffin cells, leading to a shortlived mobilization of intracellular Ca²⁺. Angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding was largely blocked in Ca²⁺-free buffer and by pretreatment with the Ca²⁺-channel blocker ω-conotoxin GVIA. The [³H]phorbol 12, 13-dibutyrate binding response to [Sar¹]angiotensin II also appeared to be voltage sensitive, as no additivity was observed with the response to the depolarizing agent 4-aminopyridine (3 m M ). Threshold sensitivities of the extra-and intracellular Ca²⁺-mobilizing pathways to angiotensin II were similar, and all examined effects of angiotensin II in these cells were apparently mediated by losartan-sensitive (AT₁-Iike) receptors. The dependence of angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding on extracellular Ca²⁺ entry, in contrast to stimulation by other phospholipase C-linked receptor agonists (bradykinin and methacholine), suggests that angiotensin II preferentially stimulates protein kinase C translocation to the plasma membrane, rather than to internal membranes, in bovine adrenal medullary cells.     

Production of pectin methylesterase from Erwinia chrysanthemi B374 in Bacillus subtilis

Summary The gene coding for pectin methylesterase (PME) of Erwinia chrysanthemi B374 (pme) was cloned by a polymerase chain reaction. The pme gene was expressed in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the -amylase gene from B. amyloliquefaciens. The cultivation of B. subtilis cells carrying the cloned pme resulted in efficient secretion of PME into the culture medium based on enzymatic and sodium dodecyl sulphate-polyacrylamide gel electrophoresis characterizations. The NH₂-terminal sequence analysis of the secreted PME revealed two different NH₂-termini. Heterologous processing was probably due to a second putative signal peptidase cleavage site at the joint region between the PME and -amylase signal peptide. Offprint requests to: R. Heikinheimo