G-quadruplexes in helminth parasites

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.     

CD8+ T lymphocytes protect SCID mice against Encephalitozoon cuniculi infection

Microsporidia are obligate intracellular parasites that cause opportunistic infections in immunocompromised patients. The role of two main T cell subsets in anti-microsporidial immunity has been studied using an Encephalitozoon cuniculi-severe combined immunodeficient (SCID) mouse model. Whereas SCID mice reconstituted with CD4+ T lymphocyte-depleted naive BALB/c splenocytes resolved the infection, adoptive transfer of CD8+ T cell-depleted splenocytes failed to protect the animals against a lethal E. cuniculi infection. Splenocytes from E. cuniculi-immune mice specifically killed syngeneic infected macrophages in a short-term 51Cr-release assay. These results suggest the crucial role of cytotoxic T lymphocytes in the protection against E. cuniculi infection.     

Reaction mechanism and stereochemistry of gamma-hexachlorocyclohexane dehydrochlorinase LinA

gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide gamma-hexachlorocyclohexane (gamma-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of gamma-HCH by LinA was investigated by GC-MS, NMR, CD, and molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from gamma-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic gamma-PCCH were converted by LinA, each at a different rate. 1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of the biotransformation of biosynthetic gamma-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic gamma-PCCH. delta-PCCH was detected as the only product of dehydrochlorination of delta-HCH. LinA requires the presence of a 1,2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1,2-biaxial HCl pairs present on gamma-HCH and gives rise to a single PCCH enantiomer 1,3(R),4(S),5(S),6(R)-PCCH. Furthermore, LinA enantiomerically differentiates 1,3(S),4(R),5(R),6(S)-PCCH and 1,3(R),4(S),5(S),6(R)-PCCH. The proposed mechanism of enzymatic biotransformation of gamma-HCH to 1,2,4-TCB by LinA consists of two 1,2-anti conformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.     

Potential application of catalase-peroxidase from Comamonas terrigena N3H in the biodegradation of phenolic compounds

Comamonas terrigena N3H is a gram-negative rod-shaped bacterium that was isolated from contaminated soil in Slovakia. This bacterium showed remarkable biodegradation properties. We investigated the expression and functioning of two catalase isozymes in this bacterium. The typical catalase could be induced by cadmium ions, whereas the catalase-peroxidase enzyme was constitutively expressed. Since C. terrigena lacks the key enzyme for complete degradation of phenols (phenolhydroxylase), we analysed the possible removal of phenol by the two catalases of this bacterium. Addition of phenol to the culture medium led to increased expression of the catalase-peroxidase. Applying oxidative stress prior to phenol administration markedly induced the expression of the typical catalase, irrespective of the nature of the added agent. Thus, the rate of phenol degradation is rather reduced under these conditions, while growth of the cells is not impaired. We concluded that phenol peroxidation in C. terrigena can be largely attributed to the action of a catalase-peroxidase. The potential application of this enzyme in the removal of phenol from the environment is discussed.     

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.     

The molecular peculiarities of catalase-peroxidases

SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells. Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity. Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Δ cells but not bet1Δ cells. These data together with structure–function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.     

Treatment of Toxocara canis infections in mice with liposome-incorporated benzimidazole carbamates and immunomodulator glucan

Benzimidazole carbamates (mebendazole, albendazole and fenbendazole) are the most commonly used anthelmintic drugs for the treatment of larval toxocariasis (Toxocara canis) in paratenic hosts. However, the bioavailability of these drugs for tissues is very low due to their extremely low solubility, resulting in the administration of relatively high doses over a long period. To overcome this problem, neutral, negatively or positively charged and stabilized liposome drug carriers were examined in the chronic phase of T. canis infections in mice each orally inoculated with 1000 eggs. Moreover, liposomized albendazole and fenbendazole were co-administered with liposomized immunomodulator glucan. The highest efficacy of both drugs, evaluated 4 weeks after treatment, was recorded after their subcutaneous administration (ten doses of 25 mg kg(-1)) in stabilized liposomes and intramuscular co-administration of liposomized glucan (two doses of 5 mg kg(-1)). Fenbendazole was more effective in muscles (91.5%) whereas albendazole was more effective in the brain (92.2%). Liposomes with incorporated benzimidazole carbamate anthelmintics provide sustained drug-release reservoirs and can considerably enhance drug efficacy. Moreover, despite suppression by T. canis antigens, stimulation of the immune system by the immunomodulator glucan potentiates the effects of these antiparasitic drugs.     

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.     

Larval toxocariasis and its clinical manifestation in childhood in the Slovak Republic

Results are presented of 90 children aged 1-15 years hospitalized with toxocariasis. Blood count analysis and laboratory examination were done by routine clinical laboratory methods. Anti-Toxocara antibodies were detected in the serum of patients using an ELISA method. Demographic analysis of the children's families exposed to the risk of disease allowed estimation of age-specific rates for clinical toxocariasis. The probability of toxocaral infection and the intensity of its clinical manifestations in children are determined by the epidemiology of this zoonosis and by the risk factors in the family. The presence of high titres of specific IgG antibodies in all age categories correlates with the clinical manifestations of toxocariasis. The highest admission rate is in the age categories of 3-5 years (43.3%) and 6-10 years (36.7%). Laboratory findings show that the most conspicuous changes occur in the age category 1-5 years. The high percentage of seropositive dog-keeping and puppy-breeding families and the possibility of infection with repeated doses of larvae stimulate eosinophilia, which prevails in children under the age of five years. We present the percentage of patients whose parameters showed deviations from the reference values for a particular age category. Analyses of laboratory indices and of clinical manifestations will contribute to the accuracy of diagnosis and effectiveness of treatment of this disease.     

Transgenic UCP1 in white adipocytes modulates mitochondrial membrane potential

To test if mitochondrial uncoupling in white adipocytes is responsible for obesity resistance of the aP2-Ucp transgenic mice expressing ectopic uncoupling protein 1 (UCPI) in white fat, mitochondrial membrane potential (delta psi(m)) was estimated by flow cytometry in adipocytes isolated from gonadal fat. Ectopic UCP1 (approximately 0.8 mol UCP1/mol respiratory chain) decreased the delta psi(m) and rendered the potential sensitive to GDP and fatty acids. These ligands of UCP1 had no effect on delta psi(m) in white adipocytes from non-transgenic mice, suggesting that the function of endogenous UCP2 in adipocytes was not affected. The results support the hypothesis that mitochondrial uncoupling in white fat may prevent development of obesity.