Latarcins, Antimicrobial Peptides from Spider Venom: a Variety of the Mechanisms of Action DUBOVSKII PV Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
Background: The latarcins (Ltc) peptides are present in the venom of L. tarabaevi spider and found to possess antibacterial activity, most likely due to their ability to deteriorate plasmatic bacterial membranes. Aim: to determine the mechanism of antibacterial activity of these peptides, the two representatives, the 26-residue peptides, Ltc1 and Ltc2a have been investigated in model membranes of the lipid composition mimicking one of Gram-negative microorganisms (mixture of phosphatidylethanolamine and phosphatidylglycerol, 7:3 molar ratio).
Methods: Using a number of biophysical techniques we have performed: (i) structural study of the both peptides by CD spectroscopy in phospholipid liposomes and by 1H NMR in detergent micelles; (ii) determination of the peptide’s effect on the liposomes and planar membranes by a dye leakage fluorescent assay, 31P NMR spectroscopy, and voltage-clamp technique.
Results: The Ltc2a molecule was found to consist of two helical regions (residues 3–9 and 13–21) connected via a poorly ordered fragment. The effect of the peptide on the liposomes suggests the carpet mechanism of the membrane deterioration. In contrast, Ltc1 molecule is featured by an uninterrupted helix within residues 8-23. This peptide affects liposomes weakly, but induces erratic current fluctuations in planar membranes, causing lesions in them.
Conclusions: Ltc family is represented by the peptides exploiting the diverse mechanisms of the membrane perturbation: (i) the carpet mechanism (Ltc2a); (ii) a voltage-dependent pore-forming mechanism (Ltc1). The spider venom, a natural mixture of these peptides, possesses a broad-spectrum antibacterial activity. This implies that mixing the peptides with different mechanisms of activity, is of potential use in the design of effective anti-infectives.
Authors’ disclosure statement: This work was supported by the Russian Foundation for Basic Research, project no. 07-04-00910a.
Pathophysiology of Enteropathogenic E. coli-induced diarrhea: Potential Therapeutic Role of Probiotics DUDEJA PK, GILL R, HODGES K, BORTHAKUR A, RAMASWAMY K, TURNER JR, HECHT GA
Univ. of Illinois at Chicago, Univ. of Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612
Background: Enteropathogenic Escherichia coli (EPEC) is a gram-negative food-borne pathogen primarily associated with infantile diarrhea. However, the pathophysiology underlying EPEC induced early diarrhea is not fully understood. Diarrhea results from either an increased ion secretion or decreased absorption, or both. Our aims were to test the hypothesis that EPEC infection decreases NaCl absorption (via a coupled operation of Na+-H+ exchanger (NHE) and Cl--HCO3-(OH-) exchangers) in human intestinal epithelial cells (IECs) to cause diarrhea; whereas probiotics act as pro-absorptive agents by increasing NaCl absorption.
Methods: Caco2 cells in culture and mouse models were utilized for assessing the effects of EPEC infection and probiotics treatment. The effect of bacteria at 30 min to 3h on ethylisopropyl amiloride (EIPA) sensitive 22Na uptake and DIDS sensitive 36Cl- uptake was determined. Cell surface biotinylation and confocal microscopy were utilized to assess expression of ion transporters. Real time QPCR was utilized to measure mRNA expression.
Results: EPEC infection markedly inhibited the activities of NHE3 (the predominant sodium absorbing isoform by ~ 50%) and Cl-/OH- exchange activity (50-75%) in Caco-2 cells. The effects of EPEC were dependent upon type 3 secretion system (TTSS) of the bacterium and occurred via EPEC effector molecules: EspF and EspG, respectively. EPEC infection reduced surface expression of apical anion exchanger, SLC26A3 on the plasma membrane (~70%) in Caco-2 cells and in the mouse colon. Treatment of cells with Lactobacillus acidophilus/rhamnosus (LR) stimulated NHE and Cl-/OH- exchange activities and an increase in NHE3 and SLC26A3 mRNA expression in a differential manner.
Conclusion: Our data demonstrate that EPEC infection to human intestinal epithelial cells inhibits NaCl absorption, which may underlie the pathophysiology of EPEC-induced early diarrhea. Our findings also highlight the potential therapeutic roles of probiotics in prevention of diarrhea associated with enteric infections or inflammatory bowel diseases.
Support: Work supported by awards from NIH and Dept. of Veterans Affairs
Ganciclovir treatment of infants with cytomegalovirus infection and
central nervous system involvement. DUNIN-WASOWICZ D1, JÓŹWIAK S1, KASZPRZYK-OBARA J1 The Children’s Memorial Health Institute, Warsaw, Poland
Background: Congenital and acquired human cytomegalovirus (HCMV) infection is very frequent and dangerous especially in the central nervous system involvement. So far vaccination against cytomegalovirus is not possible ,and the search for effective and safe antiviral drug is on. Ganciclovir (GVC) is one of the “oldest “antiviral drugs against HCMV. In The Children’s Memorial Health Institute in Warsaw ganciclovir treatment of newborns and infants has been used for several years after the endorsement of the Bioethics Committee and the informed consent of the parents. Aim: Estimation of the efficacy and tolerability of ganciclovir in infants with cytomegalovirus neuroinfection.
Methods: .66 infants at the age from 2 to 12 months with detection DNA HCMV in cerebrospinal fluid by qualitative PCR method were treated with intravenous infusions of ganciclovir. The dose of GCV was established individually after the pharmacokinetic examinations. The longest time of treatment was 12 weeks ( 3 courses with 1 months interval).The analysis of the blood cell count as well as chemistry were regularly performed. The infants had also neuroimaging and electroencephalographic examinations , were taken into multispecialistic care and followed-up.
Results: Epileptic seizures, hypertonia, chorioretinitis, sensorineural hearing loss, central nervous system malformations, calcifications, hepatosplenomegaly, hepatitis, thrombocytopenia, anemia were clinical symptoms and signs of the cytomegalovirus infection. In all infants, after antiviral treatment cerebrospinal fluid DNA HCMV wasn’t found in control. GCV treatment was particularly effective in chorioretinitis, hepatosplenomegaly and thrombocytopenia. After combined antiviral and antiepileptic treatment infants were long term seizures free and even withdrawal of the antiepileptic drugs was possible in 8 cases .Only transient neutropenia was observed in 5 out of 66 infants (7.57%). No other side effects of antiviral treatment were stated during the twelve year follow up (mean 7 years).
Conclusions: 1)Cytomegalovirus neuroinfection treatment with ganciclovir was effective and well tolerated. 2) Side effects of the GCV treatment were mild and transient.
Phenotypization of Cyclosporine A in Stable Renal Transplant Patients
DURICOVA J, MARSALKOVA P, KOMZAKOVA I, BROZMANOVA H, GRUNDMANN M
Univ. Hospital and Medico Social Faculty Ostrava Univ., Ostrava, Czech Republic
Background: The immunosuppressive drug cyclosporine A (CsA) shows broad interindividual pharmacokinetic variability due to different intestinal absorption and metabolism. CsA is metabolized extensively in the liver and intestine by the cytochrome P450 3A (CYP3A) to 3 primary metabolites with the AM1 being the predominant. The CYP3A isoenzymes (CYP3A4 and CYP3A5) show function variability between individuals ranging from 7-fold to 10-fold in vivo. Different probe drugs have been proposed for the phenotypization of CYP3A metabolic activity.
The aim of this study was to compare the values of blood CsA levels and its metabolite AM1 during the 12 h interval with the AUC0-12 for the phenotypization of CsA.
Methods: 39 stable renal transplant patients on CsA therapy in steady state were included. The mean age was 49 years (49.1 ± 6.3), the mean weight was 76 kg (76.2 ± 7.4), the mean CsA dose was 198 mg (198.4 ± 56.1). CsA dose was adjusted for individual patient so as to reach the therapeutic range measured by trough CsA concentrations. Totally 9 venous blood samples were drawn in each patient – at 0, 0.5, 1, 1.5, 2, 3, 5, 8, 12 h after CsA intake. Blood concentrations of CsA and its metabolite AM1 were measured by means of HPLC with UV detection. Metabolic ratios (MR) of blood concentration CsA/AM1 and the AUC0-12 CsA/AM1 were calculated. Spearman´s rank correlation test was used.
Results: There were large intra- and interindividual differences in the MR CsA/AM1 at different blood sample times. In only 10 out of 39 patients (26%) the MR CsA/AM1indicated the same metabolic group throughout the 0-12 h blood sample time interval. However there were significant correlations between MR CsA/AM1and MR AUC0-12 CsA/AM1 at the particular blood sample time, the strongest correlation was found with the MR CsA/AM1 at 5 h after CsA intake (r=0.9501, P<0.0001). The MR AUC0-12 CsA/AM1 was then divided into 3 metabolic groups with 16 patients having the lowest MR (0.6-1.6) and thus presenting the highest metabolic activity, with 15 patients having MR 1.7-3.1 and 8 patients having the highest MR (3.2-5.3) and presenting the lowest metabolic activity.
Conclusion: Our results show that the MR AUC0-12 CsA/AM1 correlated with all the MR CsA/AM1 throughout the 0-12 h blood sample time interval, however the strongest correlation was found with the MR CsA/AM1 at 5 h after CsA intake.
