Beng-Chuan Ho

N-acetylation of dopamine and tyramine by mosquito pupae (Aedes togoi)

Abstract The N- acetyltransferase (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [ 14 C]-, [ 3 H]dopamine, [ 14 C]tyramine or [ 14 C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn 2+ stimulated and Zn 2+ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, 893 ± 57 and 861 ± 30 pmol [ 14 C]NAcT formed/min per mg protein and 21.9 ± 1.2 and 22.0 ± 1.4 pmol [ 3 H]NADA formed/min per mg protein.

Melanization and encapsulation in Aedes aegypti and Aedes togoi in response to parasitization by a filarioid nematode (Breinlia booliati).

In Aedes aegypti (Singapore strain), s refractory host, there was a marked progressive decline of developing larvae of Breinlia booliati during the incubation period. However, in Aedes togoi, a susceptible host, the filarial larvae developed normally and the number of larvae remained constant throughout the incubation period. Encapsulation and melanization of B. booliati larvae in Ae. aegypti and Ae. togoi were studied. Ae. togoi occasionally mounted a defence reaction to the infection. In Ae. aegypti, various stages of filarial larvae were frequently affected by the defence mechanisms of the host, and a relatively large number of melanized larvae were recovered in dissections throughout the incubation period. The process of melanization is described and the relationship between melanization and haemocytes of mosquitoes is discussed. Haemocytes of the mosquito host appeared to be involved in the encapsulation of filarial larvae, as evidenced by adherence of cells to the cuticle of the larvae and the formation of a translucent, gelatinous envelope which contained intact cells, necrotic cells, spaces and numerous whitish granules. The possible involvement of a humoral reaction in bringing about the degeneration and retardation of the filarial larvae is discussed.

Phenolsulphotransferase activity in the developing mosquito (Aedes togoi)

Phenolsulphotransferase (PST) activity was measured with N-acetyldopamine (NADA) and harmol as substrates in the larvae, pupae and adult mosquito (Aedes togoi). Only the newly emerged pupae showed high PST activity 1–4 hr after pupation. PST activity could also be measured in each individual pupa, with the female exhibiting significantly higher specific activity (30 ± 3.7 pmol NADA [35S]sulphate/min per mg protein) than the males (13.6 ± 2.9 pmol NADA [35S]sulphate/min per mg protein). The optimum pH for the PST reaction was 9.0. The Km values for [35S]PAP were 0.55 and 2.5 μM when measured with NADA and harmol as acceptors, respectively; the corresponding Km values for these two substrates were 2.61 and 16.1 μM. Studies with 2,6-dichloro-4-nitrophenol showed a dose-dependent inhibition of PST. Sulphate conjugation of NADA from ATP and sodium [35S]sulphate was also demonstrated with pupal extracts, with pH optimum between 8.6 and 9.0. The specific activity of this overall sulphate conjugation, measured in the female pupal extract was 5.08 pmol NADA [35S]sulphate/min per mg protein and 1.68 pmol harmol [35S]sulphate/min per mg protein. The importance and possible function of sulphate conjugation of NADA in insects is discussed.

Endotoxin Levels in Rural Thai and Urban Singaporean Homes

Background: Exposure to dust endotoxin and allergens in early childhood may influence the development of allergic diseases. Aims: This study aimed to evaluate dust endotoxin and dust mite allergens in urban Singapore and rural Thai homes of young children and study potential environmental influences. Methods: Mattress dust endotoxin and Der p 1, Der f 1, group 2 (Der f 2 and Der p 2) and Blo t 5 allergen levels were quantified in 101 infant mattress dust samples, 51 from urban Singapore and 50 from rural Thailand. Comprehensive questionnaires on the home environment and cleaning practices were completed. Results: Endotoxin levels in rural Thailand were significantly higher than in urban Singapore (geometric mean 26,334.12 ± 4.60 and 18,377.85 ± 2.52 endotoxin units/g, respectively; p = 0.032). In contrast, higher levels of Der f 1 (p = 0.02), group 2 (p < 0.01) and Blo t 5 (p < 0.01) allergens were found in Singapore homes compared with rural Thai homes. Multiple logistic regression analysis showed that the use of detergents (p = 0.001) and disinfectants (p = 0.024) to clean floors and mattress protectors (p = 0.021) were independently associated with lower endotoxin levels. Conclusion: Endotoxin levels are higher in rural compared with urban homes in South East Asia. The reverse was true for dust mite allergen levels. Certain identifiable home environmental conditions and practices accounted for the differences in endotoxin levels.

Studies on the Malayan forest rat filaria, BreinUa booliati (Filarioidea: Onchocercidae): Course of development in rat host

The development of Breinlia booliati is described in its natural host, Rattus sabanus and in an inbred strain of laboratory albino rat. The growth of the parasite is similar in both the rat hosts. The third moult occurs between six-eight days and the final moult between 24-28 days. Larvae were recovered initially from the skin and carcass. After five weeks, developing stages were seen only in the thoracic and abdominal cavities, the site of development of the adult worms. Worms became sexually mature by 11-12 weeks and there was considerable growth in length of the female worms after this stage.

Studies on the malayan forest rat filaria, Breinlia booliati (Filarioidea: Onchocercidae): Transmission to laboratory rats

Abstract Breinlia booliati , a new filarial parasite of Rattus sabanus , has been successfully transmitted to 3 strains of laboratory rats by various routes of inoculation. Out of a total of 222 rats, 54 showed microfilaraemia with a mean prepatent period of 12·5 weeks. Out of 168 amicrofilaraemic rats necropsied at 20 weeks after inoculation, 67 harboured adult worms localized in the thoracic and abdominal cavities. Unisexual infections occurred frequently ( 58 67 rats). In bisexual amicrofilaraemic infections, adult male and female worms were recovered either in the same location or separately in the thoracic and abdominal cavities. Female rats were more susceptible to infection than male rats. The Charles River inbred rats and outbred albino rats were more susceptible than the HS (Hooded) inbred rats. This rat-filaria combination may provide a useful experimental model for studying various aspects of filariasis.

Studies on filariasis. IV. The rate of escape of the third-stage larvae of Brugia pahangi from the mouthparts of Aedes togoi during the blood meal.

The rate of escape of the third-stage larvae of Brugia pahangi from Aedes togoi which were allowed to probe on a cat and a mouse at time intervals of 5, 10, 20, 30 and 60 seconds was studied. The rate of escape of third-stage larvae at all time intervals was greater during probing on the cat than on the mouse, and was in a logarithmic linear relation to the length of probing time by the mosquito host. The greatest loss (91.35%) of third-stage larvae was in mosquitoes which fed on a cat until fully gorged. There was a remarkable rate of escape (57.41%) within 5 seconds. This striking rate of escape of third-stage larvae may have important implications on filariasis transmission. Most larvae migrated towards the proboscis and head of mosquitoes during these feeding periods. Nearly equal numbers of third-stage larvae escaped from mosquitoes which fed to repletion for more than 60 seconds on a mouse and from those which probed fro more than 60 seconds on the same mouse but did not engorge. This confirmed our view that filling up of the stomach with blood does not constitute the single factor in causing the release of third-stage larvae from the mosquito host.

Drug Inhibition of Anaphylactic Histamine Release from Peritoneal Cells of Rats Infected with Toxocara canis

An experimental model for studying anaphylactic histamine release from rat peritoneal cells in vitro is described. Rats were sensitized by feeding the infective eggs of ttoxocara canis, the antigen being prepared from the infective eggs. The characteristics of histamine release are comparable with those found for other anaphylactic systems. Addition of 0.2% inactivated normal rat serum to the incubation media reduced the spontaneous histamine release from 11.5 +/- 1.9% to 3.1 +/- 1.6% (mean +/- SD) of total histamine, whereas the corresponding figure for anaphylactic histamine release was 41.3 +/- 19.7%. Both disodium cromoglycate (DSCG) and levamisole produced a dose-related inhibition of histamine release, with DSCG being the more potent drug.

Some ultrastructural observations on the microfilaria of Breinlia sergenti—The excretory complex, rectal cells and anal vesicle

Abstract The present investigations describe the fine structure of the excretory complex, rectal cells and the anal vesicle of the microfilaria of Breinlia sergenti. The structure of the excretory cell and rectal cells is found to be similar to nerve cells. Axonal (digitiform) processes in the excretory and anal vesicles are described and a possible sensory function is ascribed to these structures.

Infections of forest rat filaria, Breinlia booliati, in neonate and juvenile laboratory white rats.

The kinetics of Breinlia booliati infection in 3 inbred rat strains (Lewis, Wistar and Sprague Dawley) were investigated. One group of rats was infected as neonates (less than 24 hours of age) with third-stage larvae of B. booliati and the other group was infected as juveniles (4 weeks of age). The results showed that infection in the neonates were significantly different from the infection in the juveniles. The 60 rats infected as neonates, when necropsied between 8 to 10 months postinfection, yielded adult worms. The 2 neonatal infection groups of Lewis and Wistar strains showed highest susceptibility to the infections. The mean prepatent period was 85 days. Ninety to 95% of the infected rats were patent with microfilaraemia and a large percentage (33 to 47%) of them had high microfilaraemia counts exceeding 3000 mff/20 mm3 of blood and larger sizes (mean 157.11 mm for female adult worms and 61.88 mm for male adult worms. The adult worms were distributed equally in both the pleural (57%) and peritoneal cavity (43%). In most aspects, the neonatal infection group of the Sprague-Dawley strain was intermediate in susceptibility between the 2 neonatal infection groups of the Lewis and Wistar strains and the 3 juvenile infection groups. In contrast to neonatal infection groups, the 3 juvenile infection groups exhibited low infection rates (37%, 58% and 47% for the Lewis, Wistar and Sprague Dawley strains respectively), longer prepatent periods (mean 101 days), lower recovery rates (2 to 4%), lower adult worm loads (mean 0.4 to 0.8 female worms, and 0.2 to 0.8 male worms per rat), and smaller sizes (mean 141.24 mm for female adult worms and 53.75 mm for male adult worms). Forty-four to 57% of these infected rats harboured either single male or single female adult worms in the body cavity. Most (92%) of the adult worms recovered from the juvenile infection groups resided in the pleural cavity and the remaining 8% were recovered from the peritoneal cavity. Microfilaraemia could be detected in only 3/20 Lewis rats, 5/20 Wistar rats and 5/20 Sprague Dawley rats. The mean peak microfilaraemia of the 3 pooled juvenile infection groups was 632 mff/20 mm3 of blood, ranging from 7 mff/20 mm3 to 1856 mmf/20 mm3. Our results indicate that the susceptibility to B. booliati infection in white rats is both genetic and age-associated. The responses of the 2 distinct infection groups to B. booliati infections are discussed.