Isolation and characterization of mutant bacteriophage T7 RNA polymerases.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.     

Successful plant regeneration from callus cultures of Centella asiatica (Linn.) Urban

A successful procedure was established for in vitro plant regeneration from callus derived from stem and leaf explants of Centella asiatica on semisolid modified Murashige and Skoog's [7] medium supplemented with 2.0 mg L³ kinetin and 4.0 mg L³ a-naphthaleneacetic acid. The rate of shoot-bud regeneration was the highest (42.8 and 54.3 shoots/culture in stem and leaf derived callus respectively) after 4 weeks of subculture on 4.0 mg L³ 6-benzyladenine, 2.0 mg L³ Kn, 0.25 mg L³ a-naphthaleneacetic acid and 20 mg L³ adenine sulfate. Differentiated shoots rooted within 11 days in 1/2 strength MS basal salts supplemented with 0.5 mg L³ indole-3-acetic acid and 2% (w/v) sucrose. About 85% of rooted plantlets were acclimatized and transferred to the greenhouse.     

Effect of aminotriazole on antioxidant defense system of tasar silkworm Antheraea mylitta

This study was designed to find out the metabolic consequences of H₂O₂ following catalase inhibition by aminotriazole in the fat body of an Antheraea mylitta pupa. H₂O₂ content in the pupal fat body exhibited a decreasing trend over the experimental period (up to 48 h). However, a substantial decrease in its level was marked after 12, 24 and 48 h of treatment. The level of lipid peroxidation was elevated within 4 h of aminotriazole injection. Nevertheless, its level significantly decreased after 12, 24 and 48 h of treatment. Superoxide dismutase activity was elevated within 4 h, followed by a transient decrease in its activity at 12 h of treatment and again increased over the experimental period. Catalase activity was found to decline in the fat body within 4 h of aminotriazole treatment compared to the control. However, it was surprising to observe that there was a two‐fold increase in catalase activity compared to its previous experimental group after 12 h, followed by a rapid decline in its activity at 24 h of aminotriazole injection and non‐detectable catalase activity at 48 h. Ascorbic acid content was found to be elevated after 12 h of injection and maintained an increasing trend over the rest of the experimental period compared to the respective control. Despite the progressive inhibition of catalase activity beyond 12 h of treatment, H₂O₂ accumulation was not observed as a consequence of catalase inhibition. Hence, catalase depletion by aminotriazole involves compensatory changes in other components of the antioxidant system for the efficient removal of H₂O₂.     

Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth,Motility, and Exopolysaccharide Production

Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher’s equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase–a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics.     

Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.