Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.     

Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis.

We describe the use of gel electrophoresis in studies of equilibrium binding, site distribution, and kinetics of protein-DNA interactions. The method, which we call protein distribution analysis, is simple, sensitive and yields thermodynamically rigorous results. It is particularly well suited to studies of simultaneous binding of several proteins to a single nucleic acid. In studies of the lac repressor-operator interaction, we found that binding to the so-called third operator site (03) is 15-18 fold weaker than operator binding, and that the binding reactions with the first and third operators are uncoupled, implying that there is no communication between the sites. Pseudo-first order dissociation kinetics of the repressor-203 bp operator complex were found to be temperature sensitive, with delta E of 80 kcal mol-1 above 29 degrees C and 26 kcal mol-1 below. The half life of the complex (5 min at 21 degrees C) is shorter than that reported for very high molecular weight operator-containing DNAs, but longer than values reported for much shorter fragments. The binding of lac repressor core to DNA could not be detected by this technique: the maximum binding constant consistent with this finding is 10(5) M-1.     

Efficacy of rapamycin in scleroderma: a case study

Scleroderma is a common autoimmune disorder with no effective therapy. Current concepts of scleroderma include the hypothesis that scleroderma results from excess conversion of endothelial cells to fibroblast like cells, called endothelial mesenchymal transformation. This process is thought to be mediated by cytokines including transforming growth factor beta (TGFb), which causes increased collagen synthesis, resulting in fibrosis, the hallmark of the disease. In vitro studies have hypothesized that rapamycin may be of benefit in scleroderma due to antagonism of collagen synthesis. Given that rapamycin has antiangiogenic activities, inhibits wound healing, and prevents the synthesis of collagen in vivo, we tried rapamycin in a patient with scleroderma. We observed rapid improvement in skin stiffness and mobility. Our results provide the rationale for larger clinical trials of rapamycin in scleroderma and other fibrotic disorders.     

Density-related sterility in Eretmocerus mundus

While studying the reproductive capacity of Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) we found that varying numbers of females were sterile. Investigations showed that sterility occurred also in field populations, but at a very low rate. Laboratory sterility was significantly correlated with crowding of the parental females during oviposition. Whitefly hosts from which fertile of sterile females emerged did not differ in size, neither did the hind tibiae of fertile females differ in dimensions from those of sterile ones. Behavior of sterile females differed from that of the fertile ones in several parameters. They exhibited less leg drumming, used the ovipositor more frequently and for shorter durations, and changed more readily from probing to host stinging, and from a number of activities to walking. Altogether, their behavior appeared more restless and caused them to contact more hosts than fertile females. The possibility that the sterility is caused by crowding alone, or by the activity of microorganisms acting under crowded conditions, and the merits of the phenomenon for biological pest control are discussed.     

A simple method for culturing neonatal Biomphalaria glabrata snails

A simplified method for the culture of neonatal Biomphalaria glabrata snails on a diet of the cyanobacteria Nostoc sp. has been developed. Previous studies reported using Nostoc sp. grown on a mud-based medium to feed neonatal snails; this medium is difficult to prepare and gives inconsistent growth between laboratories due to variations in the soil used in its preparation. Here, we report the use of Bg-11 medium supplemented with sodium nitrate for the culture of Nostoc sp. as a food source for neonatal B. glabrata snails. Bacteria grown in the medium may be maintained in continuous culture and used exclusively as the nutrient source for neonatal snails. Neonatal snails fed with these bacteria exhibit a growth rate of 1-2 mm per week and may be transferred to standard culture conditions after 2-3 wk. Survival of neonatal snails after 2 wk on the Nostoc sp. diet was 60-80%, while survival after switching to standard culture conditions approached 100%. Analysis of the growth rate and survival of neonatal snails maintained on Nostoc sp., as well as the growth rate, survival, and sexual maturation of the resulting juvenile snails, showed that snails maintained using this method are phenotypically comparable to those maintained under other standard laboratory conditions.