Probing DMPG vesicle surface with a cationic aqueous soluble spin label

A small, highly aqueous soluble, deuterated, cationic spin label, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (dCAT1), was used to directly monitor the negatively charged DMPG vesicle surface in order to test a recent suggestion (Riske et al., Chem. Phys. Lipids, 89 (1997) 31-44) that alterations in the surface potential accompanied apparent phase transitions observed by light scattering. The temperature dependence of the label partition between the lipid surface and the aqueous medium indicated an increase in the surface potential at the gel to liquid-crystal transition, supporting the previous suggestion. Results at the phase transition occurring at a higher temperature were less definitive. Although some change in the dCAT1 ESR spectra was observed, the interpretation of the phenomena is still rather unclear. DMPG surface potentials were estimated from the dCAT1 partition ratios (surface label moles/total label moles), using a simple two-sites model, where the electrostatic potential is zero everywhere but at the vesicle surface, and the interaction between the spin label and the membrane surface is chiefly electrostatic. The Gouy-Chapman-Stern model predicts surface potentials similar to those observed, although the measured decrease in the surface potential with ionic strength is somewhat steeper than that predicted by the model.     

Neoparamoeba Page, 1987: light and electron microscopic observations on six strains of different origin

Although amoebic gill disease (AGD) has emerged as one of the most severe health problems in the fish industry, proof of the identity of AGD agents from various localities is still missing. Six strains of amoebae designated until recently as Paramoeba species (the agents of AGD) were studied in cultures by light and electron microscopy. Although they were isolated from gills of different hosts (Dicentrarchus labrax and Scophthalmus maximus) and from distant localities, their morphology was identical. The strains differed from Paramoeba eilhardi, the type species of the genus, in that they lacked the boat-shaped microscales on the cell surface but could be safely identified as belonging to the genus Neoparamoeba Page, 1987. Transmission electron microscopy revealed the presence of a symbiotic organism, Perkinsiella amoebae Hollande, 1980, in all strains under study. The only difference among the strains examined was found in the size of trophozoites, which could be attributed to the different origins of the strains, but until more refined diagnostic methods are available, in addition to N. pemaquidensis, the closely related species N. aestuarina also has to be taken into consideration as the agent of AGD.     

In vivo spin-label murine pharmacodynamics using low-frequency electron paramagnetic resonance imaging.

A novel, very-low-frequency electron paramagnetic resonance (EPR) technique is used to image the distribution of several nitroxides with distinct pharmacologic compartment affinities in the abdomens of living mice. Image acquisition is sufficiently rapid to allow a time sequence of the distribution for each compound. The spectra and concentrations of these nitroxides are imaged with the use of spectral-spatial imaging to distinguish a single spatial dimension. Liver and bladder of the mouse anatomy are distinguished by this technique. After an intraperitoneal injection of the spin-label probes, a shift in the distribution of the compounds from the upper abdomen (primarily liver) to the lower abdomen (primarily bladder) is observed. The time dependence of the shift in regional distribution depends on the structural properties of the side chain attached to the spin label. These results indicate that this application of in vivo electron paramagnetic resonance imaging will provide a new method of magnetic resonance imaging for determination of pharmacodynamics in the body of an intact animal.     

MAGE-A3/4 and NY-ESO-1 antigens expression in metastatic esophageal squamous cell carcinoma

In the present study we analyzed immunohistochemical expression of MAGE-A 3/4 and NY-ESO-1 in 55 samples of esophageal squamous cell carcinomas (ESCC) and their respective lymph node metastases. To our knowledge this is the first study to assess and compare the expression of these antigens in ESCC lymph node metastases. Fifty (90.9%) primary ESCC were positive for MAGE-A 3/4 and 53 (96.6%) were positive for NY-ESO-1. MAGE-A 3/4 was expressed in all lymph node metastases and the intensity of expression was high in a majority of cases. NY-ESO-1 was negative in 2 (7.1%) lymph nodes metastases, while the reaction was predominantly moderate in the positive group. In primary tumors MAGE-A 3/4 showed a significantly higher intensity of expression compared to NY-ESO-1 (P=0.047), while in lymph node metastases the intensity of expression was not significantly different (P=0.387). Primary tumors with and without lymph node metastases showed no significant differences in MAGE-A 3/4 (P=0.672) and NY-ESO-1 (P=0.444) expression. Intensity of MAGE-A 3/4 (P=0.461) and NY-ESO-1 (P=0.414) expression in primary tumors was not significantly different compared to the expression in their respective lymph nodes metastases. Expression of MAGE-A 3/4 in primary tumors showed significant positive correlation with primary tumor expression of NY-ESO-1 (P=0.021) but no significant correlation with the expression of MAGE-A 3/4 in lymph node metastases (P=0.056). Expression of NY-ESO-1 in primary tumors showed significant positive correlation with the expression of NY-ESO-1 in lymph node metastases (P=0.001) and significant negative correlation with patients’ age (P<0.001). Expression of MAGE-A 3/4 and NY-ESO-1 in primary tumors and lymph node metastases showed no significant correlation with prognostic parameters such as tumor grade and TNM stage (P>0.05). We have shown different levels of MAGE-A 3/4 and NY-ESO-1 expression in almost all specimens of primary tumor and lymph node metastases, suggesting that ESCC may be possible target of immunotherapy and anti-tumor vaccination. High levels of expression in lymph node metastases indicate possible clinical benefit of postoperative vaccine with MAGE-A3 and NY-ESO-1 in advanced stage of disease.     

The role of fat dormouse (Glis glis L.) as reservoir host for spirochete Borrelia burgdorferi sensu lato in the region of Gorski Kotar, Croatia

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.     

Dendrosoter protuberans (Hymenoptera: Braconidae), An Important Elm Bark Beetle Parasitoid

The most favourable period for Dendrosoter protuberans development occurred when wasps were introduced 16 days after the beginning of Scolytus multistriatus development (the greatest number of emerged wasps per a single parasitoid female with a very positive sexual index and high percentage of parasitised elm bark beetle larvae). No significant differences were observed in parasitoid efficiency between the 11^th and the 21^st day. The most unfavourable period for elm bark beetle parasitisation was six days after the beginning of S. multistriatus development (a low reproduction of D. protuberans, a negative sexual index and a low number of destroyed S. multistriatus larvae). The reproduction of both the elm bark beetle and D. protuberans was greater in the first, than in the second generation. The developmental period of D. protuberans in the first generation was approximately two days longer than in the second generation. Furthermore, the development of females was 1-2 days longer than the development of males. D. protuberans developed most successfully on S. multistriatus (the most abundant parasitoid reproduction with the longest developmental period and a very positive sexual index that resulted in a high percentage of parasitised larvae) and then on S. ensifer, S. pygmaeus and Pteleobius kraatzi. On the other hand, S. scolytus larvae were the poorest host of all elm species - the reproduction of D. protuberans in this species was the scarcest with the shortest period of development and a negative sexual index, hence larval parasitisation was the lowest.     

Sublethal effects of NeemAzal- T/S botanical insecticide on Dutch and Serbian populations of Encarsia formosa (Hymenoptera: Aphelinidae)

Sublethal effects of an azadirachtin-based botanical insecticide (commercial product NeemAzal-T/S) on behaviour, life history traits and population growth of one commercial (Dutch strain, D) and two Serbian populations (Bujanovac, B; Negotin, N) of Encarsia formosa Gahan were evaluated in laboratory bioassays. In a two-choice test, parasitoids from all tested populations preferred to parasitise the untreated whitefly nymphs over those treated with 50, 25, 12.5, 6.25 and 3.12?mg/l of azadirachtin. All concentrations caused significant deterrent effects after 24?h, and the following deterrence indices were calculated: 9.7–60.9% (B), 5.5–57.5% (N) and 12.9–63.5% (D). The longevity of adults exposed for 48?h to residues of the botanical insecticide (applied at LC₅₀) was shorter (1.5–1.7 days) than that of control wasps. Both daily and total parasitism were significantly reduced (by 41.7, 48.3 and 60.1% for N, D and B, respectively) compared to control, as well as adult emergence in the F₁ generation. The instantaneous rate of increase (r_i) of surviving adults was also significantly reduced (by 13.2, 19. 3 and 20.5%, for populations N, B, and D, respectively). Adults from all tested populations, emerged from pupae treated with the botanical insecticide applied at LC₅₀, showed reduced parasitism, adult emergence, and r_i levels, but the reduction was significant only for adult emergence of B and r_i of B and N. Juvenile development of the parasitoid in treated pupae was significantly extended, compared to control. The implications of these results on integrated control of the greenhouse whitefly are discussed.     

Combining precision spin-probe partitioning with time-resolved fluorescence quenching to study micelles. Application to micelles of pure lysomyristoylphosphatidylcholine (LMPC) and LMPC mixed with sodium dodecyl sulfate

Micelles of lysomyristoylphosphatidylcholine (LMPC) and mixed micelles of LMPC with anionic detergent sodium dodecyl sulfate (SDS) have been characterized by spin-probe-partitioning electron paramagnetic resonance (SPPEPR) and time-resolved fluorescence quenching (TRFQ) experiments. SPPEPR is a novel new method to study structure and dynamics in lipid assemblies successfully applied here for the first time to micelles. Several improvements to the computer program used to analyze SPPEPR spectra have been incorporated that increase the precision in the extracted parameters considerably from which micelle properties such as effective water concentration and microviscosity may be estimated. In addition, with this increased precision, it is shown that it is feasible to study the rate of transfer of a small spin probe between micelles and the surrounding aqueous phase by SPPEPR. The rate of transfer of the spin probe di-tert-butyl nitroxide (DTBN) and the activation energy of the transfer process in LMPC and LMPC-SDS micelles have been determined with high precision. The rate of transfer increases with temperature and SDS molar fraction in mixed micelles, while it remains constant with LMPC concentration in pure LMPC micelles. The activation energy of DTBN transfer in pure lysophospholipid micelles does not change with LMPC concentration while it decreases with the increasing molar fraction of SDS in mixed LMPC-SDS micelles. Both this decrease in activation energy and the increase in the rate of transfer are rationalized in terms of an increasing micelle surface area per molecule (decreasing compactness) as SDS molecules are added. This decreasing compactness as a function of SDS content is confirmed by TRFQ measurements showing an aggregation number that decreases from 122 molecules for pure LMPC micelles to 80 molecules for pure SDS micelles. The same increase in surface area per molecule is predicted to increase the effective water concentration in the polar shell of the micelles. This increase in hydration with SDS molar fraction is confirmed by measuring the effective water concentration in the polar shell of the micelles from the hyperfine spacing of DTBN. This work demonstrates the potential to design mixed lysophospholipid surfactant micelles with variable physicochemical properties. Well-defined micellar substrates, in terms of their physicochemical properties, may improve the studies of protein structure and enzyme kinetics.     

An EPR study of the interfacial properties of phosphatidylcholine vesicles with different lipid chain lengths

The hydrophobic spin probe 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl octadecanoate (TEMPO-stearate) is used to study the interfacial properties of a variety of phosphatidylcholine vesicles. Since the spin probe exhibits a fast motional electron paramagnetic resonance (EPR) spectrum above the phase transition, the EPR spectrum of the spin probe is analyzed by nonlinear least-squares spectral fitting. EPR spectral line fitting provides high precision spectral parameters, which can be used to construct a detailed picture of the dynamics of the probe and its environment. The hyperfine coupling spacing is used to estimate the effective water concentration in the polar shell of vesicles, while the rotational correlation times give the information on the motion of the spin probe. The effective water concentration of the polar shell of dimyristoyl-phosphatidylglycerol (DMPG) vesicles is greater on average by about 4.0M than the effective water concentration of the polar shell of dimyristoyl-phosphatidylcholine (DMPC) vesicles. The effective water concentration decreases by about 0.5M for an increase of two carbons in the chain, and increases noticeably with hydrocarbon chain unsaturation, which is in good agreement with literature values. The nitroxide moiety rotates preferentially along the N-O bond, that is, parallel to its hydrocarbon chain.