Social Cues and Following Behavior in the Forest Tent Caterpillar

Colonies of the social caterpillar Malacosoma disstria Hubner (Lepidoptera: Lasiocampidae) travel in groups following silk trails marked with pher-omone. This study examined first, the cues involved in following behavior and second, the responses to these cues at different larval stadia. Both second and fourth instar larvae discriminated between fresh and older trails, and travelled faster in the presence of trails. In addition to trail following, young caterpillars exhibited leader following, which might be particularly important in exploring unmarked territory. Indeed, second instar caterpillars were more likely to travel together when trails were absent. Fourth instar larvae exhibited greater independent locomotion in the absence of trails than did younger larvae. These findings help explain patterns of social behavior observed in forest tent caterpillar colonies in the field.     

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.     

Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system

The process of DNA donation for natural transformation of bacteria is poorly understood and has been assumed to involve bacterial cell death. Recently in Neisseria gonorrhoeae we found that mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. To better characterize the GGI and the process of DNA donation, the 57 kb genetic island was cloned, sequenced and subjected to insertional mutagenesis. DNA sequencing revealed that the GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. The GGI was found to be incorporated near the chromosomal replication terminus at the dif site, a sequence targeted by the site-specific recombinase XerCD. Using a plasmid carrying a small region of the GGI and the associated dif site, we demonstrated that this model island could be integrated at the dif site in strains not carrying the GGI and was spontaneously excised from that site. Also, we were able to delete the entire 57 kb region by transformation with DNA from a strain lacking the GGI. Thus the GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. We made mutations in six putative type IV secretion system genes and assayed these strains for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Our data indicate that N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response.     

Using 2H2O to study the influence of feeding on protein synthesis: effect of isotope equilibration in vivo vs. in cell culture

We previously reported that 2H2O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau BR. Am J Physiol Endocrinol Metab 286: E665-E672, 2004). The underlying premise of our method is that following the administration of 2H2O, 2H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether 2H2O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of 2H2O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between 2H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that approximately 50% of the plasma albumin that is synthesized over the course of 24 h is made within approximately 5 h after eating (in rats trained to eat a complete 24-h ration of food in a single meal). Contrary to what we observed in vivo, feeding (the replenishment of cell culture medium) does influence the use of 2H2O for in vitro studies. In particular, since there can be slow equilibration of 2H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro.     

Differences in the neurotoxicity profile induced by ATP and ATPgammaS in cultured cerebellar granule neurons

Extracellular ATP and P2 receptors may play a crucial role in the neurodegeneration of the CNS. Here, we investigated in neuronal cerebellar granule cultures the biological effect of the quite stable P2 receptor agonist ATPgammaS and compare it to the cytotoxic action of ATP. Time-course experiments showed that 500 microM ATPgammaS causes 50-100% cell death in 15-24 h. As proved by pharmacological means, ATPgammaS toxicity apparently involves neither indirect activation of NMDA receptors, nor ectonucleotidase activities, nor nucleoside transport and intracellular purine metabolism. Moreover, ATPgammaS induces detrimental effects without modifying the expression of several P2X and P2Y receptor proteins. Cell death instead occurs after extracellular release of the cytosolic enzyme lactic dehydrogenase and inhibition of the overall activity of the intracellular dehydrogenases. Moreover, ATPgammaS causes transient outflow of cytochrome c from mitochondria (maximal 2.5-fold stimulation in 4 h), it raises the intracellular reactive oxygen species (about four-fold in 1 h) and cAMP levels (about 40% in 15 min-4 h). Among several P2 receptor antagonists, only pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium promotes 80-100% neuroprotection.     

Facilitating genome navigation: survey sequencing and dense radiation-hybrid gene mapping

Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences lack long-range continuity and provide only a fragmented view of a genome. Here we show the usefulness of combining survey sequencing with dense radiation-hybrid (RH) maps for extracting maximum comparative genome information from model organisms. Based on results from the canine system, we propose that from now on all low-pass sequencing projects should be accompanied by a dense, gene-based RH map-construction effort to extract maximum information from the genome with a marginal extra cost.     

Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor

When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.     

Synthesis of biologically active steroid derivatives by the utility of Lawesson's reagent

Steroid derivatives V, VI, VII and VIII reacted with Lawesson's reagent (LR) to produce spiro-oxazaphosphole-4',17-androstene derivative XI, diazaphospholoandrostane XIV and the thionated derivatives XVI and XVII, respectively. The structures of the new compounds were confirmed by analytical and spectroscopic evidence. A mechanism accounting for the formation of the new compounds was given. The in vitro antimicrobial activity of the new compounds were tested.