Permeability factor,cytotoxicity and serotyping ofPseudomonas aeruginosa strains

We analyzed in detail the permeability and cytotoxic activity as well as the serotypes of 127Pseudomonas aeruginosa strains. Sixty-seven strains were isolated from immunocompromised patients (51 from patients with tumors and 16 from patients after transplantation) and 60 strains were isolated from patients’ ears. Culture filtrates of strains isolated from patients after transplantation were responsible for the highest part of permeability reactions corresponding to an intermediate toxin production (68.8%) (categories 2 and 3) and culture filtrates of strains isolated from patients with tumors caused the highest percentage of permeability reactions corresponding to a strong toxin production. Culture filtrates of strains isolated from ears of patients were responsible for the highest percentage of negative permeability reactions (15%). With positive permeability reaction size (categories 2–6) increased also the percentage of cytotoxicity as well as the intensity of morphological changes on Vero cells after 1 and 2 d. We did not observe any relationship between a particular permeability reaction category and the most frequent serotypes (O4, O6) or nontypable strains of the tested groups.     

Mechanisms of α-thrombin,histamine, and bradykinin induced endothelial permeability

α-Thrombin, bradykinin, and histamine are endogenous mediators that increase endothelial permeability. We examined the mechanism by which these three vasoactive mediators could alter permeability to albumin of human umbilical vein endothelial cells (HUVEC). HUVEC were grown to confluence on Transwell membranes and we monitored the flux of fluorescein isothiocyanate-labeled human serum albumin across the membrane from the upper to lower chamber of the Transwell. Addition of α-thrombin, bradykinin, or histamine increased the permeability coefficient of the HUVEC monolayer. At 30 min the permeability coefficient for α-thrombin was 4.92 × 10⁻⁶ cm/sec while histamine was 4.47 × 10⁻⁶ cm/sec. Maximum changes in the permeability coefficient were about three-fold control baseline values (1.59 × 10⁻⁶ cm/sec). There was also a temporal difference in the magnitude of the permeability coefficient. α-Thrombin and bradykinin induced HUVEC permeability was increased for the first 90 min after which it returned to control levels. In contrast, histamine increased the permeability of the HUVEC monolayer throughout the 2 h experiment. To determine a possible intracellular mechanism of the altered permeability coefficients, HUVEC were labeled with FURA-2 and intracellular calcium was monitored by digital fluorescence ratio imaging. Maximum intracellular calcium in HUVEC was increased by α-thrombin (245 ± 20 nM) and histamine (210 ± 22 nM), but not by bradykinin (70 ± 7 nM) as compared to control (69 ± 10). Fluorescent photomicrographs of HUVEC stimulated with the three agonists indicated that α-thrombin and histamine substantially altered HUVEC f-actin arrangement, while bradykinin had no effect on HUVEC f-actin distribution. These data support previous in vitro and in vivo studies demonstrating increased permeability by all three agonists. These data also show, for the first time, that histamine and α-thrombin increased permeability by calcium-dependent intracellular pathways, but bradykinin operates through a calcium-independent mechanism. © 1996 Wiley-Liss, Inc.     

Uterine vascular permeability, blood flow and extracellular fluid space during implantation in rats

Vascular permeability to plasma proteins in uterine implantation and non-implantation sites (i.e. dye sites and non-dye sites) was assessed quantitatively by a method which accounts for steady-state volumes of distribution. Extracellular fluid volume and uterine blood flow were also determined. On both the evening of Day 5 and the morning of Day 6, vascular permeability to 125I-labelled human serum albumin, extracellular fluid volume and blood flow were significantly increased in implantation sites compared to non-implantation sites. Vascular permeability in implantation sites was increased significantly between Days 5 and 6, whereas that in non-implantation sites was unchanged. This increase in vascular permeability between Days 5 and 6 was not accompanied by further increases in extracellular fluid volume and blood flow. This result shows a dissociation between vascular permeability and extracellular fluid volume immediately after the onset of implantation and raises important questions as to whether the rat uterus undergoes a truly oedematous response at implantation as has been generally accepted.     

Intestinal permeability is reduced and IL-10 levels are increased in septic IL-6 knockout mice

Sepsis is associated with increased intestinal permeability, but mediators and mechanisms are not fully understood. We examined the role of interleukin (IL)-6 and IL-10 in sepsis-induced increase in intestinal permeability. Intestinal permeability was measured in IL-6 knockout (IL-6 -/-) and wild-type (IL-6 +/+) mice 16 h after induction of sepsis by cecal ligation and puncture or sham operation. In other experiments, mice or intestinal segments incubated in Ussing chambers were treated with IL-6 or IL-10. Intestinal permeability was assessed by determining the transmucosal transport of the 4.4-kDa marker fluorescein isothiocyanate conjugated dextran and the 40-kDa horseradish peroxidase. Intestinal permeability for both markers was increased in septic IL-6 +/+ mice but not in septic IL-6 -/- mice. Treatment of nonseptic mice or of intestinal segments in Ussing chambers with IL-6 did not influence intestinal permeability. Plasma IL-10 levels were increased in septic IL-6 -/- mice, and treatment of septic mice with IL-10 resulted in reduced intestinal permeability. Increased intestinal permeability during sepsis may be regulated by an interaction between IL-6 and IL-10. Treatment with IL-10 may prevent the increase in mucosal permeability during sepsis.     

Central Asian cobra venom cytotoxins-induced aggregation, permeability and fusion of liposomes

The processes of membrane aggregation, permeability and fusion induced by cytotoxins from Central Asian cobra venom were investigated by studying optical density of liposome samples, permeability of liposome membranes for ferricyanide anions and exchange of lipid material between the membranes of adjacent liposomes. Cytotoxins Vc5 and Vc1 were found to induce aggregation of PC + CL and PC + PS liposomes. Cytotoxin Vc5 increased also the permeability of the liposomes for K3[Fe(CN)6] and enhanced their fusion. Cytotoxin Vc1 increased membrane permeability and enhanced fusion of PC + CL samples only. The changes in membrane permeability and fusion were found to occur within a single value of cytotoxin concentrations. The fusogenic properties of the cytotoxins studied are supposed to be due to the ability to dehydrate membrane surface and to destabilize the lipid bilayer structure. Fusion probability is largely defined by the phospholipid composition of the membranes. A model of interaction of cytotoxins with cardiolipin-containing membranes is offered.     

射线照射后大鼠骨髓、十二指肠、小脑微血管通透性的改变

本文观察了r线全身照射后,大鼠骨髓、十二指肠和小脑微血管通透性的改变。结果表明:正常时骨髓微血管通透性最高,十二指肠次之,小脑最低。照后骨髓微血管通透亢进发生早、增加快、变化大、恢复慢,主要发生在血窦;照后十二指肠微血管通透亢进发生较早、增加较快、变化较大、恢复较快,主要发生在肠绒毛的毛细血管和细静脉;照后小脑微血管通透性变化较小,在400.0Gy组可见通透亢进,主要发生在小脑皮质的毛细血管。所有这三个脏器微血管通透亢进部位,实质细胞的退变明显加重。说明微血管邇透亢进可明显加重实质细胞的损伤,而实质细胞的再生修复,就发生在微血管通透恢复,基底背景较清净地方。说明:照后骨髓、十二指肠、小脑微血管通透性的改变,在造血型、胃肠型和脑型急性放射病的发病机理上有重要意义。     

Effects of leukotriene B4 on permeability, prostacyclin and thromboxane release by normal and oxygen-preexposed isolated, perfused rat lungs

This study assessed the hemodynamic and permeability effects of exogenous, synthetic leukotriene B4 (LTB4) on normal rat lungs and lungs from rats preexposed to oxygen for 48 h, which were isolated and perfused at constant flow in vitro. Adult, Sprague-Dawley rats were exposed to air or greater than 97% O2 for 48 h. After exposure, their lungs were removed from the thorax, ventilated with normoxic gas, and perfused at 12 ml/min with Krebs-Ringer bicarbonate buffer which contained 5 mM glucose and 3 mg/ml albumin. A total of 5.55 micrograms of synthetic LTB4 was infused in three separate boluses over 15 minutes. Perfusion and airway pressures were monitored, and the lungs release of 6-ketoprostaglandin F1 alpha and thromboxane B2 (TXB2) into the effluent from the pulmonary vasculature was measured by radioimmunoassay. The LTB4 had no measureable effects on pulmonary vascular pressures. LTB4 infusion caused a pronounced increase in permeability, indicated by increased albumin concentrations in alveolar lavage fluid from O2-preexposed lungs. Release of TXB2 from both air- and O2-preexposed lungs was increased after LTB4 infusion, while the change in 6-ketoprostaglandin F1 alpha release was not statistically significant. Both the increase in permeability enhanced TXB2 released after LTB4 infusion were inhibited by 10 microM indomethacin in the perfusate. These data indicate that exogenous LTB4 increases microvascular permeability in O2-exposed lungs in association with increased release of TXB2 into the pulmonary vascular effluent.     

Beneficial Effects of Anti-Interleukin-6 Antibodies on Impaired Gastrointestinal Motility,Inflammation and Increased Colonic Permeability in a Murine Model of Sepsis Are Most Pronounced When Administered in a Preventive Setup

MethodsOF-1 mice were randomized to either the preventive or curative protocol, in which they received 1 mg/kg of antibodies to interleukin-6, or its IgG isotype control solution. They subsequently underwent either the caecal ligation and puncture procedure, or sham-surgery. GI motility was assessed 48h following the procedure, as well as colonic permeability, serum and colon cytokines, colonic tight junction proteins at the mRNA level; cultures of blood and mesenteric lymph nodes were performed.ResultsPreventive administration of anti-interleukin-6 antibodies successfully counteracted the gastrointestinal motility disturbances and impaired colonic barrier function that could be observed in vehicle-treated septic animals. Serum and colonic levels of proinflammatory cytokines were significantly lower when animals were preventively treated with anti-interleukin-6 antibodies. A repetitive injection 24h later resulted in the most pronounced effects. Curative treatment significantly lowered systemic and colonic inflammation markers while the effects on transit and permeability were unfortunately no longer significant.ConclusionsCaecal ligation and puncture resulted in septic ileus with an increased colonic permeability. Antibodies to interleukin-6 were able to ameliorate gastro-intestinal motility, suppress inflammation and normalize the permeability of the colonic wall, with the preventive administration combined with a repeat injection being far more efficacious than the sole preventive or curative one.     

Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model

A series of novel lipophilic vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids possessing a branched-acyl chain of varying length from C(8) to C(16) (6-bAcyl-AA-2G), were evaluated as topical prodrugs of ascorbic acid (AA) with transdermal activity in a human living skin equivalent model. The permeability of 6-bAcyl-AA-2G was compared with those of the derivatives having a straight-acyl chain (6-sAcyl-AA-2G). Out of 10 derivatives of 6-sAcyl-AA-2G and 6-bAcyl-AA-2G, 6-sDode-AA-2G and 6-bDode-AA-2G exhibited most excellent permeability in this model. Measurement of the metabolites permeated from the skin model suggested that 6-bDode-AA-2G was mainly hydrolyzed via 6-O-acyl AA to AA by tissue enzymes, while 6-sDode-AA-2G was hydrolyzed via 2-O-alpha-D-glucopyranosyl-L-ascorbic acid to AA. The former metabolic pathway seems to be advantageous for a readily available source of AA, because 6-O-acyl AA, as well as AA, is able to show vitamin C activity.     

Mosquito (Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes.

Previous results showed that mRNA encoding a putative aquaporin (AQP) (GenBank accession number AF218314) is present in the tracheolar cells associated with female Aedes aegypti Malpighian tubules. In this study, immunohistochemistry detected the protein, AeaAQP, also in tracheolar cells, suggesting its involvement in water movement in the respiratory system. When expressed in Xenopus oocytes, AeaAQP increased the osmotic water permeability from 15 x 10(-6) to 150 x 10(-6) m x s-1, which was inhibited by mercury ions. No permeability to glycerol or other solute was observed. AeaAQP expressed in oocytes was solubilized as a homotetramer in nondenaturing detergent as deduced from velocity centrifugation on density gradients. Phylogenetic analysis of MIP (major intrinsic protein) family sequences shows that AeaAQP clusters with other native orthogonal array forming proteins. Specific orthogonal arrays were detected by freeze-fracture analysis of AeaAQP oocyte membranes. We conclude that, in tracheolar cells of A. aegypti, AeaAQP is probably a highly water-permeable homotetrameric MIP which natively can form 2D crystals.     

Design,synthesis and biological evaluation of phosphopeptides as Polo-like kinase 1 Polo-box domain inhibitors

Polo-like kinase 1 (Plk1) is an anti-cancer target due to its critical role in mitotic progression. A growing body of evidence has documented that Peptide-Plk1 inhibitors showed high Plk1 binding affinity. However, phosphopeptides-Plk1 inhibitors showed poor cell membranes permeability, which limits their clinical applications. In current study, nine candidate phosphopeptides consisting of non-natural amino acids were rationally designed and then successfully synthesized using an Fmoc-solid phase peptide synthesis (SPPS) strategy. Moreover, the binding affinities and selectivity were evaluated via fluorescence polarization (FP) assay. The results confirmed that the most promising phosphopeptide 6 bound to Plk1 PBD with the IC₅₀ of 38.99?nM, which was approximately 600-fold selectivity over Plk3 PBD (IC₅₀?=?25.44?μM) and nearly no binding to Plk2 PBD. Furthermore the intracellular activities and the cell membrane permeability of phosphopeptide 6 were evalutated. Phosphopeptide 6 demonstrated appropriate cell membrane permeability and arrested HeLa cells cycle in G2/M phase by regulating CyclinB1-CDK1. Further, phosphopeptide 6 showed typical apoptotic morphology and induced caspase-dependent apoptosis. In conclusion, we expect our discovery can provide new insights into the further optimization of Plk1 PBD inhibitors.     

The anti-cancer drug, doxorubicin, causes oxidant stress-induced endothelial dysfunction

The anticancer drug doxorubicin (DOX) is toxic to target cells, but also causes endothelial dysfunction and edema, secondary to oxidative stress in the vascular wall. Thus, the mechanism of action of this drug may involve chemotoxicity to both cancer cells and to the endothelium. Indeed, we found that the permeability of monolayers of bovine pulmonary artery endothelial cells (BPAEC) to albumin was increased by approximately 10-fold above control, following 24-h exposure to clinically relevant concentrations of DOX (up to 1 microM). DOX also caused >4-fold increases in lactate dehydrogenase leakage and large decreases in ATP and reduced glutathione (GSH) in BPAECs, which paralleled the increases in endothelial permeability. A large part of the ATP loss could be attributed to DOX-induced hydrogen peroxide production which inhibited key thiol-enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PDH). Depletion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) appeared to be a major factor leading to DOX-induced GSH depletion. At low concentrations, the sulfhydryl reagent, iodoacetate (IA), inhibited GAPDH, caused a decrease in ATP and increased permeability, without inhibiting G6PDH or decreasing GSH. These results, coupled with those of previous work on a related quinone, menadione, suggest that depletion of either GSH or ATP may lead independently to endothelial dysfunction during chemotherapy, contributing to the cardiotoxicity and other systemic side-effects of the drug.     

Gramicidin, valinomycin, and cation permeability of Streptococcus faecalis

Gramicidin and valinomycin in concentrations of 10(-7) and 10(-6)m, respectively, inhibited the growth of Streptococcus faecalis. Inhibition of growth was associated with loss of Rb(+) and K(+) from the cells, and could be reversed by addition of excess K(+). Cells treated with these antibiotics exhibited greatly increased permeability to certain cations; no effect was observed on the penetration of other small molecules. Unlike normal cells, cells treated with gramicidin rapidly lost internal Rb(+) by passive exchange with external cations, including H(+), all monovalent alkali metals, NH(4) (+), Mg(++), and tris(hydroxymethyl)aminomethane. Exchange was rapid even at 0 C and was independent of energy metabolism. The effect of valinomycin was more selective. Cellular Rb(+) was rapidly displaced by external H(+), K(+), Rb(+), and Cs(+); other cations were less effective. The exchange was independent of metabolism but strongly affected by temperature. Under certain conditions, polyvalent cations inhibited exchange between (86)Rb and Rb(+) induced by valinomycin. The antibiotic apparently neither stimulates nor inhibits the energy-dependent K(+) pump of S. faecalis, but exerts its effect on the passive permeability of the membrane to cations. The increased permeability to specific cations induced by gramicidin and valinomycin is a sufficient explanation for the inhibition of growth, glycolysis, and other processes.     

The interaction of 3,3',4',5-tetrachlorosalicylanilide with phosphatidylcholine bilayers.

1. The interaction of the germicide 3,3',4',5-tetrachlorosalicylanilide (T4CS) with vesicles and dispersions of egg phosphatidylcholine has been studied by gel permeation chromatography, electron microscopy, electron spin resonance spin labelling and ion permeability measurements. 2. Incorporation of T4CS into vesicles of egg phosphatidylcholine gives rise to a large increase in the permeability rate of the paramagnetic cation N,N-dimethyl-N-(1'-oxyl-2',2',6',6'-tetramethyl-4'-piperidyl)-2-hydroxyethylammonium chloride through the lipid bilayer but has no significant effect on the vesicle sizes as measured by gel permeation chromatography or electron microscopy. 3. ESR studies using a spin-labelled fatty acid have demonstrated the presence of two different environments for the spin label when T4CS is incorporated into phosphatidylcholine bilayers. These two environments are identified as (a) highly ordered areas of the bilayer, rich in T4CS and (b) areas with very similar ordering to that in pure egg phosphatidylcholine. 4. The effectiveness of very low concentrations of the germicide in increasing vesicle permeability is explained in terms of its clustering to give rigid patches, rich in T4CS, rather than being evenly distributed throughout the bilayer. It is proposed that the increased ion permeability arises from leakage at the interfaces between the rigid and flexible regions of the lipid bilayer. 5. Comparisons between the effective levels of T4CS in phosphatidylcholine vesicles and its minimum inhibitory concentration with a Gram-positive bacterium confirm the validity of phospholipid vesicles as a model for studies of germicidal activity.     

Ionic permeability of K, Na, and Cl in crayfish nerve. Regulation by membrane fixed charges and pH.

Teorell's fixed charge theory for membrane ion permeability was utilized to calculate specific ionic permeabilities from measurements of membrane potential, conductance, and specific ionic transference numbers. The results were compared with the passive ionic conductances calculated from the branched equivalent circuit membrane model of Hodgkin Huxley. Ionic permeabilities for potassium, sodium, and chloride of crayfish (Procambarus clarkii) medial giant axons were examined over an external pH range from 3.8 to 11.4. Action potentials were obtained over this pH range. Failures occurred below pH 3.8 during protonation of membrane phospholipid phosphate and carboxyl, and above pH 11.4 from calcium precipitation. In general, chloride permeability increases with membrane protonation, while cation permeability decreases. At pH 7.0, PK = 1.33 X 10(-5), PCl = 1.49 X 10(-6), PNa = 1.92 X 10(-8) cm/s. PK: PCl: PNa = 693:78:1. PCl is zero above pH 10.6 and is opened predominately by protonation of epsilon-amino, and partially by tyrosine and sulfhydryl groups from pH 10.6 to 9. PK is activated in part by ionization of phospholipid phosphate and carboxyl around pH 4, then further by imidazole from pH 5 to 7, and then predominately from pH 7 to 9 by most probably phosphatidic acid. PNa permeability parallels that of potassium from pH 5 to 9.4. Below pH 5 and above pH 9.4, PNa increases while PK decreases. Evidence was obtained that these ions possibly share common passive permeable channels. The data best support the theory of Teorell, that membrane fixed charges regulate permiability and that essentially every membrane ionizable group appears involved in various amounts in ionic permeability control.     

The Interactions of P-Glycoprotein with Antimalarial Drugs,Including Substrate Affinity,Inhibition and Regulation

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10⁻⁶ cm/sec, followed by amodiaquine around 20 x 10⁻⁶ cm/sec; both mefloquine and artesunate were around 10 x 10⁻⁶ cm/sec. Methylene blue was between 2 and 6 x 10⁻⁶ cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.     

Absorption of antisense oligonucleotides in rat intestine: effect of chemistry and length

An in situ single-pass perfusion model was used to assess the effect of chemical modification and length on permeability and absorption of various oligonucleotides in rat intestine. Phosphorothioate oligodeoxynucleotides (PS-ODN) were compared with oligoribonucleotides with 2'-methoxyethyl (MOE) or 2'-O-methyl (OMe) modifications. A 25-mer PS-OMe-modified oligonucleotide showed relatively poor permeability in this model, as did unmodified 20-mer PS-ODN (permeability coefficient [P(eff)] = 2-8 X 10(-6)cm/sec). Modifying some or all of the oligonucleotides with 2'-MOE groups on deoxyribose and 5'-methylation of the cytosines substantially increased intestinal permeability of oligonucleotides. Both partially and fully modified PS-MOE oligonucleotides showed a (2-4)-fold increase in permeability as compared with unmodified PS-ODN. The presence of a phosphodiester backbone in MOE-modified compounds led to further increases in intestinal permeability. PS-MOE composed of 6, 8, 10, 12, 14, 16, 18, 20, and 22 nucleotides were also examined. It was found that the permeability of these oligonucleotides increased linearly with decreasing length.     

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.     

The permeability and cytotoxicity of insulin-mimetic vanadium (III,IV,V)-dipicolinate complexes

Vanadium (III,IV,V)-dipicolinate complexes with different redox properties were selected to investigate the structure-property relationship of insulin-mimetic vanadium complexes for membrane permeability and gastrointestinal (GI) stress-related toxicity using the Caco-2 cell monolayer model. The cytotoxicity of the vanadium complexes was assayed with 3-(4,5-dimethylthiazoyl-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assays and the effect on monolayer integrity was measured by the trans-epithelial electric resistance (TEER). The three vanadium complexes exhibited intermediate membrane permeability (P(app) = 1.4-3.6x10(-6) cm/s) with low cellular accumulation level (<1%). The permeability of all compounds was independent of the concentration of vanadium complexes and excess picolinate ligands. Both V(III) and V(V)-dipicolinate complexes induced 3-4-fold greater reactive oxygen and nitrogen species (RONS) production than the V(IV)-dipicolinate complex; while the vanadium (III)-dipicolinate was 3-fold less damaging to tight junction of the Caco-2 cell monolayer. Despite the differences in apparent permeability, cellular accumulation, and capacity to induce reactive oxygen and nitrogen species (RONS) levels, the three vanadium complexes exhibited similar cytotoxicity (IC50 = 1.7-1.9 mM). An ion pair reagent, tetrabutylammonium, increased the membrane apparent permeability by 4-fold for vanadium (III and IV)-dipicolinate complexes and 16-fold for vanadium (V)-dipicolinate as measured by decrease in TEER values. In addition, the ion pair reagent prevented damage to monolayer integrity. The three vanadium (III,IV,V)-dipicolinate complexes may pass through caco-2 monolayer via a passive diffusion mechanism. Our results suggest that formation of ion pairs may influence compound permeation and significantly reduce the required dose, and hence the GI toxicity of vanadium-dipicolinate complexes.     

Isolation, biological and chemical characterization, and synthesis of a neurotensin-related hexapeptide from chicken intestine

A new biologically active peptide of the neurotensin (NT) family, shown previously to cross-react in a COOH-terminal-directed radioimmunoassay for bovine NT, has been isolated from extracts of chicken intestine and identified as H-Lys-Asn-Pro-Tyr-Ile-Leu-OH, which is identical with the biologically active COOH-terminal half of NT except for the amino acid substitutions Lys/Arg and Asn/Arg. It is proposed that this peptide be referred to as Lys8, Asn9, NT8-13 (LANT-6). Synthetic material prepared with this amino acid sequence using the Merrifield technique was immunochemically, chromatographically, and biologically indistinguishable from the native peptide. In contrast to chicken NT which induced hypotension, hyperglycemia, increased vascular permeability, and cyanosis when injected intravenously into anesthetized rats, synthetic LANT-6 brought about primarily a hypertensive response and had little ability to promote hyperglycemia, increased vascular permeability, and cyanosis. In rats pretreated with the alpha-blocker phentolamine and in adrenalectomized rats, the hypertensive response to LANT-6 was blocked, suggesting that adrenal catecholamines mediated this effect. These findings suggest that LANT-6, a natural variant of NT with a different spectrum of biologic activity, may be a NT-related messenger peptide with a different function(s).