Rao UR et al. (1987) Immunol Cell Biol "Complement activation by eggs and microfilariae of filarial parasites."

Rao UR, Subrahmanyam D, Chandrashekar R

[Immunol Cell Biol, 1987]

The complement of fresh normal rat serum was activated by filarial eggs and microfilariae (mf). C3 was deposited on the surface of Litomosoides carinii, Brugia pahangi, Brugia malayi and Dipetalonema viteae as seen by immunofluorescence. Intra-uterine and in vitro-derived mf did not bind C3. In contrast, C3 bound to the blood-derived mf of B. pahangi and B. malayi as well as exsheathed mf of L. carinii and B. malayi. Significant consumption of complement was observed with eggs of all filarial species, as well as sheathed mf of B. pahangi, B. malayi and exsheathed mf of L. carinii and B. malayi. These experiments indicated that complement was activated by filarial parasites via the alternative pathway. The bound complement promoted neutrophil-mediated adherence and cytotoxicity.

Rao UR et al. (1996) J Submicrosc Cytol Pathol "Effect of Brugia malayi infections on endothelial cells: a morphological study."

Zometa CS, Vickery AC, Rao UR, Sutton ET

[J Submicrosc Cytol Pathol, 1996]

Athymic mice (C3H/HeN) parasitized by Brugia malayi develop gross lymphatic dilations at the chronic stage of infection. The hyperplastic endothelial cells and low fluid pressure of the lymphatics, characteristic of these infections, suggest that abnormal changes in these cells may play an important role in the dilation. We studied the lymphatic and vascular endothelium of parasitized mice for morphological changes by scanning and/or transmission electron microscopy. The lymphatic endothelium of dilated lymphatics was perturbed, scalloped, bulbous and highly indented. Numerous mononuclear and giant cells were closely apposed to the endothelial wall. Endothelial cells of both the lymphatics and the adjacent venules revealed no focal cytoplasmic lesions. Growth factor-dependent cell proliferation was significantly suppressed in vitro in endothelial cell cultures containing adult female worms, male worms or microfilariae. The actin cytoskeletal network appeared intact in these cells, and no gross changes in distribution were evident. Although the lymphatic walls were highly tortuous, our examination revealed no significant alterations in their morphology. Perivascular infiltration of activated mast cells, lymphocytes and monocytes/macrophages indicated polarization of inflammatory cells into the lymphatic tissue. It is possible that these inflammatory cells might induce temporal functional changes in the lymphatics of infected athymic mice.

Rao UR et al. (1991) Antimicrob Agents Chemother "Brugia malayi and Acanthocheilonema viteae: antifilarial activity of transglutaminase inhibitors in vitro."

Rao UR, Mehta K, Vickery AC, Subrahmanyam D

[Antimicrob Agents Chemother, 1991]

The possible involvement of transglutaminase-catalyzed reactions in survival of adult worms, microfilariae (mf), and infective larvae of the filarial parasite Brugia malayi was studied in vitro by using the specific pseudosubstrate monodansylcadaverine (MDC) and the active-site inhibitors cystamine or iodoacetamide. These inhibitors significantly inhibited parasite mobility in a dose-dependent manner. This inhibition was associated with irreversible biochemical lesions followed by filarial death. A structurally related, inactive analog of MDC, dimethyldansylcadaverine, did not affect the mobility or survival of the parasites. Adult worms failed to release mf when they were incubated in the presence of MDC or cystamine, and this inhibitory effect on mf release was concentration dependent. Similar embryostatic and macrofilaricidal effects of MDC were observed in Acanthocheilonema viteae adult worms. These studies suggest that transglutaminase-catalyzed reactions may play an important role in the growth, development, and survival of filarial parasites.

Rao UR et al. (2000) Parasitol Res "Identification and localization of glutathione S-transferase as a potential target enzyme in Brugia species."

Salinas G, Mehta K, Klei TR, Rao UR

[Parasitol Res, 2000]

Brugia filarial nematodes are pathogenic lymphatic-dwelling parasites that, like other helminths, may modify the host's defense mechanisms by a major detoxification process involving glutathione-binding proteins such as glutathione S-transferases (GSTs). In the present study, soluble extracts of third-stage larvae, adult male and female worms, microfilariae of either B. pahangi or B. malayi or the adult worm excretory-secretory products of B. malayi were used to determine GST activity. These extracts and affinity-purified fractions of B. pahangi adult worms had a specific enzymatic activity when 1-chloro-2,4-dinitrobenzene was used as a substrate. The observance of this enzyme in all life cycle stages of Brugia spp. demonstrates its ubiquitous nature. Lavage of intraperitoneally infected jirds, but not that of uninfected jirds, also showed increased enzymatic activity, suggesting that GST is secreted in vivo. Soluble proteins of both Brugia spp. were strongly recognized by antibodies in sera from rabbits immunized with affinity-purified native GST of Onchocerca volvulus. Immunohistochemical studies localized these proteins in adult worms, demonstrating cross-reactivity between the GST of these two filarial nematodes. The effect of this enzyme on the motility and viability of adult worms, microfilariae, and larvae was tested in vitro using a battery of known GST inhibitors. Of all those tested, ethacrynic acid, N-ethylmalemide, 4-nitropyridine-oxide, or 1-chloro-2,4-dinitrobenzene at micromolar concentrations reduced the viability and motility of microfilariae, third-stage larvae, and adult worms. These results suggest that Brugia GSTs are major metabolic enzymes and may play an important role in the parasite's survival.

Rao UR et al. (1990) Int J Parasitol "A direct fluorescence technique for the rapid detection of sheathed microfilariae in blood smears."

Rao UR, Vickery AC, Nayar JK, Lowrie RC, Kwa BH

[Int J Parasitol, 1990]

Thick and thin blood smears containing microfilariae of Wuchereria bancrofti, Loa loa, Brugia malayi, Brugia pahangi, Brugia patei or Acanthocheilonema viteae were prepared from either cryopreserved blood samples or from freshly collected blood, fixed in methanol and treated with a fluoresceinated lectin wheat germ agglutinin. Sheathed microfilariae of W. bancrofti, L. loa, B. malayi, B. pahangi and B. patei in the blood smears could be easily detected and counted using a fluorescence assay. The unsheathed microfilaria of Acanthocheilonema viteae did not fluoresce. The possibility of adapting this technique, which does not require the use of parasite specific antibody for the sensitive, parasitological detection of filarial infections, is discussed.

Rao UR et al. (1984) Acta Trop "The effect of p-aminobenzoic acid and folic acid on the development of infective larvae of Brugia malayi in Aedes aegypti."

Chandrashekar R, Subrahmanyam D, Parab PB, Rao UR

[Acta Trop, 1984]

Adult Aedes aegypti mosquitoes, infected with the subperiodic Brugia malayi, were found to enhance the development of the filarial parasites to the infective stage when they were exposed to a cotton pad soaked in 10% sucrose solution containing p-aminobenzoic acid (PABA) in 0.001, 0.005, 0.01, 0.05 and 0.1% concentrations. Similarly, larval development increased when the mosquitoes were fed with folic acid at 0.001, 0.01 and 0.1% concentrations. This stimulation was more when PABA or folic acid was given prior to the infected blood meal through the developmental period of the larvae. The data thus suggest that PABA and folic acid are nutrients for the development of B. malayi-microfilariae to the infective stage in A. aegypti.

Rao UR et al. (1992) Parasitol Res "Effect of carrageenan on the resistance of congenitally athymic nude and normal BALB/c mice to infective larvae of Brugia malayi."

Vickery AC, Rao UR, Kwa BH, Nayar JK, Subrahmanyam D

[Parasitol Res, 1992]

Resistance of BALB/c mice to infective third-stage larvae (L3) of the human filarial parasite Brugia malayi is thymus-dependent, although the actual effector mechanisms that mediate larval killing are unknown. The present study examined the effect of carrageenan (CGN) on the mechanisms of resistance to B. malayi infection in heterozygous (nu/+) and nude (nu/nu) mice. Mice were treated with CGN at a single dose of 20 or 200 mg/kg and were inoculated intraperitoneally 1 day later with 100 L3. The results showed a dose-dependent increase in the numbers of L4 and L5 that were recovered from nu/+ and nu/nu mice. CGN treatment also enhanced the recovery of mature adult worms from nu/nu mice and appeared to abolish partially the dichotomy of resistance between the usually more susceptible male and the more resistant female nu/nu mouse. Microfilariae were found in the peripheral blood and the peritoneal cavity of CGN-treated male and female nu/nu mice and in the peritoneal cavity of male but not female nu/+ mice. Fewer larval granulomas were recovered from the peritoneal cavity of treated mice. CGN-treated, parasitized nu/+ and nu/nu mice showed high titers of IgM and IgG antibodies. An experimental compound, CGP 20376, showed 100% larvicidal activity following the administration of a single dose of 20 mg/kg to CGN-treated mice. From this study, we conclude that macrophages alone or in conjunction with other cells are actively involved in the resistance of mice to B. malayi L3.

Rao UR et al. (1990) Exp Parasitol "Brugia malayi and Brugia pahangi: transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti."

Nayar JK, Kwa BH, Rao UR, Vickery AC

[Exp Parasitol, 1990]

Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 micrograms/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L2, and L3 larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L3 larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L3's recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microfilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microfilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microfilariae to develop to the L3 stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microfilariae also failed to develop to L3 in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 ng/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microfilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath.(ABSTRACT TRUNCATED AT 250 WORDS)

Liu Y et al. (2003) RNA "An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing."

Blumenthal T, Huang T, MacMorris M, Kuersten S, Liu Y, Larsen A

[RNA, 2003]

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3' ends of mature mRNAs. This is accompanied by trans-splicing with SL2apprx100 nucleotides downstream of the 3' end formation sites to create the 5' ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located apprx70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3' end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3' end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5' or 3' direction alters the location of the 5' end of the Ur-RNA. We propose that a 5' to 3' exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability

Kuersten S et al. (1997) International C. elegans Meeting "AN INTERCISTRONIC SEQUENCE INVOLVED IN PROCESSING OF C. ELEGANS OPERON PRE-mRNAS"

MacMorris MA, Blumenthal TE, Spieth J, Kuersten S, Lea K

[International C. elegans Meeting, 1997]

Polycistronic pre-mRNAs in C. elegans are processed into monocistronic mRNAs by 3' end formation and SL2-specific trans-splicing. We are studying the mechanism of this type of RNA processing in vivo, using a heat-shock driven transgenic gpd-2/gpd-3 operon. We have identified a U-rich sequence (Ur) located within the intercistronic region, 75 bp upstream of the gpd-3 trans-splice site, but nevertheless required for trans-splicing of gpd-3. A Ur mutation (DELTAUr) weakly inhibits 3' end formation, resulting in some polycistronic RNA accumulation, but more dramatically prevents the accumulation of trans-spliced gpd-3 mRNA. In the absence of the Ur sequence and 3' end formation of gpd-2 , trans-spliced gpd-3 mRNA accumulates, but is only spliced to SL1. Insertion of a 10 base oligo(U) tract in place of the normal Ur sequence (DELTAUr/pU) partially rescues SL2 trans-splicing and completely restores 3' end formation. Under extreme heat-shock conditions that inhibit trans-splicing, we detect a primer extension product that extends from gpd-3 into the intercistronic region and ends just upstream of the Ur sequence. Accumulation of this product requires an intact Ur region and is strongly enhanced by 3' end formation. In the DELTAUr/pU mutant, multiple primer extension products that map within and just upstream of the inserted poly(U) accumulate. These products do not appear to represent a Y-branch intermediate (with the Ur sequence playing a role as a polypyrimidine tract) since it is stable to debranching treatment with S100 extract. Instead, we hypothesize that the Ur sequence may act to prevent transcription termination following 3' end formation of the upstream mRNA by protecting the uncapped downstream product from degradation