Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Synthetic bioregulators of poly-cis carotenoid biosynthesis

Seventeen new bioregulators were synthesized and tested for their ability to induce the biosynthesis of poly-cis carotenes in the flavedo of Marsh white seedless grapefruit. The effects of these new bioregulators are the same as that of the previously reported dibenzylamines, but several of the new compounds are more effective and cause the accumulation of up to 162 μg/g dry wt of poly-cis carotenes in the flavedo as compared to the maximum of 74 μg/g dry wt observed previously. The compounds tested were substituted N-benzyl furfurylamines, N-benzyl, N-methyl furfurylamines and N-alkyl, N-methyl benzylamines. They demonstrate the ability of tertiary as well as secondary amines to stimulate the formation of poly-cis carotenes. The interaction of N-(4-bromobenzyl) furfurylamine, one of the more effective of the new compounds, with lycopene and β-carotene inducers is also reported.     

Effects of amines on the carotenogenesis in Blakeslea trispora

Six amines profoundly affected carotenogenesis in Blakeslea trispora. When cultures were treated with the amines, namely 4-[β-(diethylamino)-ethoxy]-benzaldehyde, 4-[β-(diethylamino)-ethoxy]-acetophenone hydrochloride, 4-β-(diethylamino)-ethoxy]-benzophenone hydrochloride, triethylamine hydrochloride, α-diethylaminopropiophenone hydrochloride and tributylamine hydrochloride, an increase in the lycopene accumulation was observed. The modes of action of these amines appear to be similar to that of 2-(4-chlorophenylthio)triethylamine hydrochloride (CPTA); however, they difrer in relative effectiveness.     

Chemical induction of poly-cis carotenoid biosynthesis

A new class of synthetic bioregulators is reported which cause the accumulation of poly-cis carotenoids in the flavedo of Marsh white seedless grapefruit. The compounds tested were all secondary amines: dibenzylamine, substituted dibenzylamines (4-F; 4-Cl; 4-Br; 2-, 3-and 4-Me; 4-NO₂; 4-CN; 4-Cl, 4′-Me; 4-Me, 4′-NO₂), N-benzyl phenethylamine and N-benzyl 2-naphthalenemethylamine. The most effective, 4-chlorodibenzylamine, caused the accumulation of 74μgg/g dry wt of poly-cis carotenoids. Prolycopene was the predominant pigment but substantial amounts of proneurosporene, poly-cis-γ-carotenes and other cis carotenes were also present. The mode of action of these new bioregulators is probably gene derepression, the same as that of the lycopene inducers. However, the secondary amines probably derepress a recessive gene governing the biosynthesis of poly-cis carotenoids; whereas, the lycopene inducers derepress the dominant gene that gives rise to the normal all-trans carotenoids. The new compounds did not seem to inhibit the cyclase(s), as the lycopene inducers do.     

Characterization of AmphiF-spondin reveals the modular evolution of chordate F-spondin genes

The F-spondin genes are a family of extracellular matrix molecules united by two conserved domains, FS1 and FS2, at the amino terminus plus a variable number of thrombospondin repeats at the carboxy terminus. Currently, characterized members include a single gene in Drosophila and multiple genes in vertebrates. The vertebrate genes are expressed in the midline of the developing embryo, primarily in the floor plate of the neural tube. To investigate the evolution of chordate F-spondin genes, I have used the basal position in chordate phylogeny of the acraniate amphioxus. A single F-spondin-related gene, named AmphiF-spondin, was isolated from amphioxus. Based on molecular phylogenetics, AmphiF-spondin is closely related to a particular subgroup of vertebrate F-spondin genes that encode six thrombospondin repeats. However, unlike these genes, expression of AmphiF-spondin is not confined to the midline but is found through most of the central nervous system. Additionally, AmphiF-spondin has lost three thrombospondin repeats and gained two fibronectin type III repeats, one of which has strong identity to a fibronectin type III repeat from Deleted in Colorectal Cancer (DCC). Taken together, these results suggest a complex evolutionary history for chordate F-spondin genes that includes (1) domain loss, (2) domain gain by tandem duplication and divergence of existing domains, and (3) gain of heterologous domains by exon shuffling.      

Transmission potential,skin inflammatory response,and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background

Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector.

Results

The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs).

Conclusion

Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

     

The three steroidal components of the goldfish preovulatory pheromone signal evoke different behaviors in males

The goldfish sex pheromone system is the best understood among the teleost fishes. Pheromones in this species are unspecialized hormonal products, which are released in ratios that vary with reproductive status. This study examined behavioral responses of male goldfish to three steroidal components of the female preovulatory pheromone: 17,20beta-dihydroxy-4-pregnen-3-one (1720betaP); 17,20beta-dihydroxy-4-pregnen-3-one-20-sulfate (1720betaP-S); and androstenedione (AD). Males were observed during exposure to nanomolar concentrations of each steroid over a 2-h period. We observed chasing, nudging (courtship behaviors) and pushing (an aggressive behavior). Each steroid elicited a different set of behaviors. 1720betaP, which is released by ovulatory females, elicited a low level of chasing and nudging that persisted throughout the experiment. Exposure to 1720betaP-S, which is released primarily by ovulatory females, triggered a large increase in nudging and chasing that lasted for only 5 min. In contrast, AD, which is released by females early in the ovulatory cycle and by mature males, elicited increases in aggressive behavior. 1720betaP and 1720betaP-S both caused increases in GtH-II release while AD did not. These results demonstrate that goldfish can discriminate components found in the female pheromone blend, suggesting that goldfish, and likely other fish species, may employ blends of hormonal products as pheromones.