First account on the larval biology of a Litomosoides filaria,from a bat

Litomosoides filariae are parasites of unrelated groups of hosts, including bats, marsupials, ancient and modern rodents. The four life cycles to-date elucidated, develop in terrestrial mammals and, at least experimentally, in the mite Ornithonyssus bacoti. A batch of mites was fed on an infected bat, Artibeus jamaicensis captured in Costa Rica, and 18 days later one infective larva was recovered. Its morphology was similar to that of other Litomosoides species, with the characteristic long buccal capsule. These first accounts on the larval biology of Litomosoides from Microchiroptera confirm the unity of the genus which supports the view that it has passed from one group of hosts to another by means of captures.     

Origin and basipetal transport of the IAA responsible for rooting of carnation cuttings

The origin and transport of the IAA responsible for rooting was studied in carnation (Dianthus caryophyllus L.) cuttings obtained from secondary shoots of the mother plants. The presence of mature leaves in the cuttings was essential for rooting. Removal of the apex and/or the youngest leaves did not reduce the rooting percentage as long as mature leaves remained attached. Removal of mature leaves inhibited rooting for a 24-day period during which the basal leaves grew and reached maturity. After this period rooting progressed as in intact cuttings. Auxin (NAA + IBA) applied to the stem base of defoliated cuttings was about 60% as effective as mature leaves in stimulating rooting. Application of NPA to the basal internode resulted in full inhibition of rooting. The view, deduced from these results, that auxin from mature leaves is the main factor controlling the rooting process was reinforced by the fact that mature leaves contained IAA and exported labelled IAA to the stem. The distribution of radioactivity after application of (5-3H)-IAA to mature leaves showed that auxin movement in the stem was basipetal and sensitive to NPA inhibition. The features of this transport were studied by applying 3H-IAA to the apical cut surface of stem sections excised from cuttings. The intensity of the transport was lower in the oldest node than in the basal internode, probably due to the presence of vascular traces of leaves. Irrespective of the localization of the sections and the carnation cultivar used, basipetal IAA transport was severely reduced when the temperature was lowered from 25 to 4 degrees C. The polar nature of the IAA transport in the sections was confirmed by the inhibition produced by NPA. Local application of IAA to different tissues of the sections revealed that polar auxin transport was associated with the vascular cylinder, the transport in the pith and cortex being low and apolar. The present results strongly support the conclusion that IAA originating from the leaves and transported in the stem through the polar auxin transport pathway was decisive in controlling adventitious rooting.     

Heat shock cognate protein 70 is involved in rotavirus cell entry

In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.     

Synthesis and testing of 5-benzyl-2,4-diaminopyrimidines as potential inhibitors of leishmanial and trypanosomal dihydrofolate reductase

Dihydrofolate reductase is a drug target that has not been thoroughly investigated in leishmania and trypanosomes. Work has previously shown that 5-benzyl-2,4-diaminopyrimidines are selective inhibitors of the leishmanial and trypanosome enzymes. Modelling predicted that alkyl/aryl substitution on the 6-position of the pyrimidine ring should increase enzyme activity of 5-benzyl-2,4-diaminopyrimidines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Various compounds were prepared and evaluated against both the recombinant enzymes and the intact organisms. The presence of a substituent had a small or negative effect on activity against the enzyme or intact parasites compared to unsubstituted compounds.     

Fine structure of leydig and sertoli cells in the testis of immature and mature spotted ray Torpedo marmorata

An ultrastructural investigation revealed the presence of true Leydig cells in the testis of sexually mature specimens of Torpedo marmorata. They showed the typical organization of steroid-hormone-producing cells, which, however, changed as spermatocysts approached maturity. In fact, they appeared as active cells among spermatocysts engaged in spermatogenesis, while in regions where spermiation occurred, they progressively regressed resuming the fibroblastic organization typically present in the testis of immature specimens. Such observations strongly suggest that these cells might be engaged in steroidogenesis and actively control spermatogenesis. Sertoli cells, too, appeared to play a role in spermatogenesis control, since, like Leydig cells, they showed the typical aspect of steroidogenic cells. In addition, the presence of gap junctions between Sertoli cells suggests that their activity might be coordinated. After sperm release, most Sertoli cells were modified and, finally, degenerated, but few of them changed into round cells (cytoplasts) or round cell remnants, which continued their steroidogenic activity within the spermatocyst and the genital duct lumen. From the present observations, it can be reasonably concluded that, in T. marmorata, spermatogenesis depends on both Leydig and Sertoli cells, and, as postulated by Callard (1991), in cartilaginous fish, the function of the Leydig cells as producers of steroids might be more recent and subsequent to that of Sertoli cells. In this regard, it is noteworthy that, in immature males, when Leydig cells showed a fibroblastic organization, Sertoli cells already displayed the typical organization of a steroidogenic cell.     

Molecular and Biochemical Diagnosis of Esterase-Mediated Pyrethroid Resistance in a Mexican Strain of Boophilus microplus (Acari: Ixodidae)

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned. This revised version was published online in July 2006 with corrections to the Cover Date.     

Litomosoides yutajensis n. sp., first record of this filarial genus in a mormoopid bat

Twenty-five bats were trapped in Yutaje (Amazonas, Venezuela) and examined for Litomosoides (Filarioidea: Onchocercidae). Of the nine recovered bat species, only Pteronotus parnelli was infected; it is a cave-dwelling species belonging to a family, Mormoopidae, which has not previously been included in the host range of the genus. The new species, L. yutajensis n. sp., has two median cephalic bosses covered with rugosities and differs from the 15 recognized species and subspecies from bats in several characters. Alike L. molossi Esslinger, 1973, L. chandleri Esslinger, 1973 and L. chitwoodi Bain, Guerrero, Rodriguez 2003, the new species has cuticular lateral bosses on the body. Eight of 10 P. parnelli were microfilaraemic, but only three had adult worms, showing that microfilariae survive longer than adults, which could lengthen the period of transmission. No infective larvae were detected in the following macronyssid mites: 58 Ornithonyssus bacoti, Ornithonyssinae, experimentally fed on microfilaraemic bats and dissected 15 days later, and a few Radfordiella sp., Macronyssinae, recovered from P. parnelli.     

Neo-clerodane diterpenoids from Croton schiedeanus

Two new neo-clerodane type furano diterpenoids were isolated from the aerial part of Croton schiedeanus, besides the clerodane diterpenes cis- and trans-dehydrocrotonin, previously isolated from other species of Croton. Structural elucidation was achieved on basis of extensive NMR experiments, including X-ray diffraction analysis and molecular mechanics calculations. The previously known flavonoids ayanin and quercetin-3,7-dimethyl ether were also obtained from the extract of this plant.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.