Combined spectroscopic and DFT studies on 6-bromo-4-chloro-3-formyl coumarin.

The FTIR and FT-Raman spectra of 6-bromo-4-chloro-3-formyl coumarin (6B4C3FC) have been recorded in the region 4000-400 and 4000-100 cm(-1), respectively. The optimized geometry, frequency and intensity of the vibrational bands were obtained by the density functional theory (DFT) using 6-31G(d,p) basis set. The harmonic vibrational frequencies were scaled and compared with experimental values. The observed and the calculated frequencies were found to be in good agreement. The UV-Visible spectrum was also recorded and compared with the theoretical values. The calculated HOMO and LUMO energies show that charge transfer occurs within molecule. The first order hyperpolarizability (ß0) of 6B4C3FC is 21 times greater than that of urea. Stability of the molecule arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. Information about the charge density distribution of the molecule and its chemical reactivity has been obtained by mapping molecular electrostatic potential surface. In addition, the non-linear optical properties were discussed from the dipole moment values and the excitation wavelength in the UV-Visible region.

Succinate oxidase and fumarate reductase systems of filarial parasite Setaria digitata.

Activities of succinate oxidase, fumarate reductase (FR) and succinate dehydrogenase (SDH) under a set of defined conditions were determined in the mitochondrial isolate from Setaria digitata, the filarial parasite from the cattle Bos indicus. Presence of only two activities namely SDH and succinate--UQ reductase of the succinate oxidase system could be detected in S. digitata. In the absence of cytochromes, the 3rd enzyme of the complex namely cytochrome oxidase is absent and it is proposed that an alternative oxidase is responsible for completing the succinate oxidation expressed as succinate oxidase activity. Though SDH and FR catalyse reverse reactions, they responded differently to modulators such as oxaloacetate, aspartate, alanine, pyruvate and fumarate. The degree of response of the two activities against inhibitors of electron transport was also different. Interestingly fumarate caused only 50% inhibition of succinate oxidation, while the effect against FR was more convincing.

Immune response of electroeluted detergent soluble 29 kDa antigen from Setaria digitata (Von Linstow).

Filariasis is one of the typical parasitic infections which cause immune suppression during the course of infection in both humans and experimental animals. A 29 kDa protein isolated from detergent soluble antigen of S. digitata showed maximum inhibition of cell mediated immune response. The heat inactivated 29 kDa protein was found to be devoid of property of suppression of immune response in the host. Histological study of spleen of BALB/C mice immunized with 29 kDa protein showed changes in regions of spleen such as follicle, trabeculae, capsule, reticuloendothelial cells and eosinophils. The 29 kDa protein, the most reactive of the detergent soluble proteins produced partial suppression of immune response, thereby contributing to the factors responsible for the survival of filarial parasites in hosts.

Presence and formation of heme and occurrence of certain heme proteins in the filarial parasite Setaria digitata.

There is currently renewed interest in the biological significance of heme proteins. The most common heme proteins include hemoglobin, myoglobin, cytochromes, and redox enzymes such as catalase and peroxidase. Setaria digitata is a cattle filarial parasite, which is devoid of typical cytochrome systems. However, studies showed activities of delta Aminolevulinate synthase (ALAS), delta Aminolevulinate dehydratase (ALAD), and heme oxygenase in appreciable amounts, suggesting the presence of necessary equipment for the biosynthesis of heme. This is further confirmed by the end product inhibition of ALAS by heme and the observation of the death of the parasite by succinyl acetone, an inhibitor of the biosynthesis of heme. Though typical cytochrome systems are absent, microsomal cytochrome P 450 and elevated levels of heme containing enzymes such as catalase and peroxidase are present in the parasite. A unique hemoglobin is also detected which shows a difference in biological functions from the host system and that of the much-studied nematode parasite Ascaris sum.

Partial characterization of affinity purified excretory-secretory protease from Setaria digitata.

Excretory-secretory protease of S. digitata released along with the microfilariae (mf) during hatching has been purified by affinity chromatography. No other activity could be detected in the affinity purified material. Homogeneity is checked by native PAGE. It has a pH optimum of 5.4 and a molecular weight of 70 kD. The purified material showed positivity against antibodies raised against ES material.

Immunomodulation of excretory secretory materials from the filarial parasiteSetaria digitata.

ES materials released along with the release of mf showed immunosuppression as evidenced by a decrease in T lymphocyte levels in the immunised animals. Lipid fractions of the mf associated ES materials and protein fractions of the detergent soluble materials were found to cause suppression of immune response in the host. Further study revealed that the suppression associated with the lipid fractions is actually the end result and that mf associated ES materials is actually a mixture of immunosuppressive and immunopotentiating components and the release of the components is most likely regulated.

Histological changes in the spleen of BALB/C mice caused by excretory-secretory protein(s) of Setaria digitata (Von Linstow).

The hatching associated materials generally called the excretory-secretory material (ES) released from the filarial parasite S. digitata along with the microfilariae (mf) under in vitro conditions showed immunosuppression. However the protein from lysate of embryo zone, from which ES materials were released along with the mf showed immunopotentiation in BALB/C mice. The histological analysis of spleen of immunized BALB/C mice showed changes confirming the in vitro findings. The main changes observed in the suppressed spleen were decrease in size and number of lymph follicles and increase in number of reticuloendothelial (RE) cells and cosinophil cells, whereas the main change in the potentiated spleen was an increase in the number of lymph follicles as compared to control spleen. These results clearly indicate that there are two types of ES materials in S. digitata, the native material present in the mature embryo causes immunopotentiation, and the materials released during the hatching process into the hostile environment along with mf causes immunosuppression. The difference in the biological effect between the materials released by lysis and hatching of the embryo opens up fresh understanding about the survival strategies of filarial parasites.

Transhydrogenase activities and malate dismutation linked to fumarate reductase system in the filarial parasite Setaria digitata.

Setaria digitata, a cattle filarial parasite, similar to human filarial parasites, possesses significant activities of the 4 transhydrogenases namely NADH-NAD+, NADPH-NAD+, NADH-NADP+, and NADPH-NADP+ in the sonicated mitochondria like particles. The transhydrogenases appear to regulate the metabolic pathways of the parasite in response to the presence of adenyl nucleotides and are non-energy linked. Observations on the transhydrogenase and fumarate reductase activities show the existence of a protein bound NAD in the MLP and a linkage between the fumarate reductase system and malic enzyme through transhydrogenases. The malate dismutation reaction is the result of malic and fumarase enzyme activities. Fumarase and fumarate reductase activities result in succinate formation under anaerobic conditions showing major energy production at the fumarate reductase site. The existence of acetate kinase, phosphotransacetylase, pyruvate carboxylase, propionyl CoA carboxylase and CoA transferase enzymes in the mitochondrial system shows the presence of other energy producing sites in the parasite. The transhydrogenase system, NAD+/NADP+ malic enzyme, fumarase and fumarate reductase are the key enzymes of, production of reducing power for synthetic reactions and regulation of oxidative and reductive stages of the mitochondrial system. Hence, specific drugs targeted against this interconnected complex enzyme system, will be very effective in the control of filarial parasites.

Epitope specific monoclonal antibodies from heterologous antigen for immunodiagnosis of filariasis.

The excretory secretory (ES) antigens produced from Setaria digitata were studied by PAGE and Western blot and compared with human filarial systems. Monoclonal antibodies (K3AE7 and K3BD5) developed against the ES antigen were evaluated by ELISA for the diagnosis of human filariasis. Cent per cent positive reaction was found with the circulating filarial antigen (CFA) of microfilaria carriers, occult and symptomatic patients and clear negative reaction with both the endemic and nonendemic controls. The clones offer promise for the development of highly sensitive diagnostic reagents for the detection of filariasis.

Effect of acetylcholine, L-glutamine and diethyl carbamazine citrate on the release of microfilariae from Setaria digitata.

Release of microfilariae from adult filarial parasite S. digitata was reversibly arrested in presence of 2 mM L-glutamine. Diethylcarbamazine also showed a similar effect, but at a lower concentration (0.26 mM). Both compounds inhibited activities of protease and leucine amino peptidase enzymes. The only difference being, different concentrations of the two materials were required for comparable effects. Levels of histamine and acetylcholine in parasite were lowered during incubation under in vitro conditions. Direct effect of L-glutamine on the release of mf was also confirmed.

Immune response in mice against hatching associated materials from the filarial parasite Setaria digitata (Von Linstow).

The hatching associated materials (excretory-secretory materials (ES)) from filarial parasite S. digitata showed immunosuppression in BALB/C mice on immunization. The material released along with microfilariae (mf) in Tyrode medium showed maximum immunosuppression while that released in presence of sublethal concentration of diethyl carbamazine (DEC, 0.25 mM) showed initial potentiation followed by suppression. While, protein from lysate of embryo zone from which ES materials were released along with the release of mf originate, showed generalized immunopotentiation in BALB/C mice. The latter suggest a change in the nature of materials of embryonic origin before and after the release of mf.

Biosynthesis of isoprenoid compounds in cattle filarial parasite Setaria digitata.

The biological significance of isoprenoid compounds such as ubiquinones, prenols and sterols have been well established. The presence and biological function of the two quinones Q6 and Q8 in the cattle filarial parasite Setaria digitata have already been reported. Inhibition of the function of quinone was already shown to be an effective means of controlling the filarial parasite. Detailed investigations of the non-saponifiable lipids from S. digitata using column, thin layer, reverse phase and high performance liquid chromatography showed the presence and formation of isoprenoid compounds such as prenols and sterols, in addition to the two quinones. Blocking of the biosynthesis of these useful compounds may prove to be an additional means of control of filarial parasites.

Lactate oxidation coupled to energy production in mitochondria like particles from Setaria digitata, a filarial parasite.

In the filarial parasite, Setaria digitata, the mitochondria like particles (MLP) show NAD reduction with sodium lactate. The MLP also reduces dye and ferricyanide with lactate. The ferricyanide reduction by lactate is found to be sensitive to the cytochrome o inhibitor orthohydroxy diphenyl (OHD) and complex I inhibitor rotenone, modulated by ADP (+) and ATP (-) and inhibited by pyruvate and oxaloacetate. MLP shows lactate oxidation sensitive to OHD, rotenone and sodium malonate. Thus, the lactate utilizing complex system, consisting of an NADH generating MLP bound lactate dehydrogenase and a lactate flavocytochrome reductase tightly linked to complex I and cytochrome o, produces ATP in functional association with fumarate reductase complex and other enzyme systems. Hence, this study provides new dimensions to the study of metabolism in filarial parasites.

Ontogenetic transformation of surface epitope expression, an adaptive immunoevasive strategy of filarial parasites.

Filarial nematodes are highly successful in invading, persisting and propagating in human body and eliciting severe ailments. The exact mechanism by which, filarial nematodes evade the host immunity is still ill-defined. The present investigation on the surface antigens of S. digitata revealed the occurrence of shared antigens in the egg, embryo, mf and adult stages. All these stages showed exposed carbohydrate moieties on their surface. In situ localization studies proved that the egg and embryo have exposed surface epitopes whereas the microfilariae and adults did not have any such epitopes. Based on these observations, a model has been proposed on "the surface epitope hiding", as an immunoevasive strategy of the filarial parasite which explains why the naturally shed surface antigens evoke antifilarial immune response in the host even though the system could not recognize the microfilariae or adult parasite due to lack of exposed surface epitopes, permitting the parasite to escape successfully from immune rejection. As treatment with detergents leads to exposure of surface epitopes of parasites, a safe intervention of parasite surface would be an effective strategy for detection and ultimate control of filariasis.

Quinone analogues: a drug of choice for the control of filariasis.

Human filariasis is caused by Wuchereria bancrofti, Brugia malayi and B. timori. Of the several recommended model filarial parasites by WHO, Setaria digitata a bovine one has characteristics such as cyanide insensitivity, lack of detectable cytochromes, presence of two quinones Q8 and Q6. Of the two quinones Q8 seems to have a predominant role in energy production. In vitro inhibitory studies using quinone analogues, coenzyme Q0 and menadione have shown that these compounds paralyse the worms in very low concentrations compared to diethyl carbamazine, the drug of choice for filariasis. The mitochondrial energy production associated with electron transfer is intercepted by quinone analogues. Hence for the treatment of filariasis, this study paves a chemotherapeutic target for the design of drugs which can control the parasites by interacting at the subcellular level by energy depletion.

Effect of diethylcarbamazine on acetylcholine and gamma amino butyric acid in Setaria digitata.

In vitro studies on the effect of neurotransmitter amino acids and amines on the motility of S. digitata showed that acetylcholine (Ach) had a stimulatory and gama amino butyric acid (GABA) an inhibitory effect on the parasite. When the worms were incubated in different concentrations of diethylcarbamazine there was a significant dose related increase in the level of Ach, and the level of GABA remained unchanged. Inhibition of acetylcholine esterase activity by diethylcarbamazine caused the accumulation of Ach in the synapses resulting in receptor desensitization and after a momentary stimulation causes paralysis of the parasite.

Quinone dependent NADH dehydrogenation in mitochondria-like particles from Setaria digitata, a filarial parasite.

In the cattle filarial parasite, Setaria digitata, the mitochondria-like particles have been shown to possess site I associated oxidative phosphorylation and rotenone sensitive and insensitive pathways for the dehydrogenation of NADH. Quinone depleted mitochondria-like particles show a loss of activity of these NADH dehydrogenases and also a complete loss of fumarate reductase activity. Reconstitution with quinone restores both NADH linked oxygen uptake and fumarate reductase activity. Thus activities of complex I and fumarate reductase are linked to quinone. Hence an inhibitor at the level of quinone can simultaneously block both aerobic and anaerobic pathways which drive ATP production and may prove useful in the effective control of filariasis.

Antigenic analysis of purified surface antigens of filarial nematode Setaria digitata.

The surface antigens of S. digitata were isolated by treatment with Triton X-100. In non SDS-PAGE the surface antigen preparation resolved into more than 6 protein bands. Electroelution of gel slices corresponding to the protein bands with relative mobilities 0.09, 0.32, 0.41, 0.53, 0.61 and 0.76 gave 6 purified surface antigen fractions (SAF). Analysis of SAFs by SDS-PAGE showed that the proteins with molecular weights 17, 29 and 36 KD were the three major polypeptides and different combination of these gave rise to the 6 native surface proteins. The 29 KD protein existed as a monomer and as cross-linked with the 17 and 36 KD proteins. All surface antigen fractions showed antigenicity, where as 29 KD protein remained as a high avidity surface antigen.

Fumarate reductase system of filarial parasite Setaria digitata.

In the cattle filarial parasite Setaria digitata the mitochondria like particles have been shown to possess NADH dependent fumarate reduction coupled with site I electron transport associated phosphorylation. This reduction is catalysed by the fumarate reductase system. The Km for fumarate is 1.47 mM and that for NADH is 0.33 mM. This activity is sensitive to rotenone, antimycin A and o-Hydroxy diphenyl. One ATP is produced for each pair of electrons transferred to fumarate. The fumarate reductase system consisting of NADH-coenzyme Q reductase, cytochrome b like component(s) and succinate dehydrogenase/fumarate reductase is thus very important and hence specific inhibitors of the system may prove useful in the effective control of filariasis.

Changes in the level of biogenic amines in experimentally infected and DEC treated proxy host mice.

The levels of biogenic amines, viz., epinephrine, histamine and dopamine were significantly altered in the blood and brain of the proxy host mice which were infected with the mf of Setaria digitata, a filarial parasite. When DEC, the drug of choice was administered to the infected mice, serotonine level was enhanced while the histamine level was decreased. The results were similar in normal mice which were given DEC.