Structural analysis of the operator binding domain of Tn10-encoded Tet repressor: a time-resolved fluorescence and anisotropy study.

An engineered Tn10-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative alpha-helix-turn-alpha-helix motif of the operator binding domain, was studied by time-resolved fluorescence and anisotropy. Fluorescence intensity decay data suggested the existence of two classes of Trp-43, defined by different lifetimes. Analysis of anisotropy data were consistent with a model in which each class was defined by a different lifetime, rotational correlation time, and fluorescence emission maximum. The long-lifetime class had a red-shifted spectrum, similar to that of tryptophan zwitterion in water, and a short rotational correlation time. In contrast, the spectrum of the short-lifetime class was blue-shifted 10 nm compared to that of the long-lifetime class. Its correlation time was similar to that of the protein, which showed that Trp in this class was entirely constrained. Trp in this latter class could not be quenched by iodide, whereas most of the long-lifetime class was easily accessible. Presence of disruptive agents, such as 1 M GuCl or 3 M KCl, did not alter markedly the lifetimes but increased the weight of the short-lifetime component. In the same time, the rotational correlation time of this component was dramatically reduced. Taken together, our data suggest that the long-lifetime class could correspond to the tryptophan residues exposed to solvent whereas the short-lifetime class would correspond to the tryptophan residues embedded inside the hydrophobic core holding the helix-turn-helix motif. Destabilization of hydrophobic interactions would lead to an increase in the weight of the latter class for entropic reasons. Analysis of the fluorescence parameters of Trp-43 could provide structural information on the operator binding domain of Tet repressor.     

High dose antioxidant supplementation to MS patients. Effects on glutathione peroxidase, clinical safety, and absorption of selenium.

High-dose antioxidant supplementation has recently been recommended for multiple sclerosis (MS) patients. This study tests the clinical safety, the glutathione peroxidase (GSH-px) activity, and the absorption of selenium during such supplementation. Eighteen MS patients were given 6 tablets especially made for this study, equivalent to 6 mg sodium selenite, 2 g vitamin C, and 480 mg vitamin E a day for five wk. GSH-px, which was lower than in non-MS controls before the start of treatment, increased fivefold during 5 wk of treatment. Side effects were scarce. Ten MS patients were subjected to a 24-h selenium absorption study after ingestion of 2 active tablets, equivalent to 2 mg sodium selenite. Selenium, which was low initially, increased 24% during the first 3 h and then stabilized. It is concluded that the tested antioxidant treatment seems to be safe and that MS patients have low GSH-px, which may be increased by the tested antioxidant treatment.     

Bacteria associated with a marine planktonic copepod in culture. I. Bacterial genera in seawater, body surface, intestines and fecal pellets and succession during fecal pellet degradation

In laboratory experiments, the bacterial flora of the zooplanktonmicrobial environments seawater, fecal pellets and associatedwith the external and internal surfaces of the copepod Acartiatonsa(Dana) were examined. The bacteria associated with fecal pelletswere dominated by Bacillus spp., Cytophaga/Flavobacterium spp.,Vibrio spp. and Pseudomonas spp. The same genera were foundin the seawater (0.22 7mu;m filtered) in which the pellets wereincubated. The bacteria showed a characteristic growth succession,and the abundance increased several orders of magnitude in theseawater during incubation of the pellets, indicating growthand proliferation based on the disintegrating/degrading fecalpellets. A carbon budget calculation revealed that organic matterfrom degrading fecal pellets could cover the carbon demand forthe growing bacterioplankton. The composition of the bacterialcommunity in the seawater and the fecal pellets also indicateda colonization of the pellets from bacterioplankton. The compositionof the bacteria associated with the copepods showed that bacterialgenera characterized as surface associated were preferentiallyassociated with fecal pellets, animal surfaces and intestines.This suggests a specific intestinal flora in the cultivatedcopepods composed of 10³ culturable bacteria per intestine (colony-formingunits, c.f.u.) or 10⁵ bacteria per intestine (acridine orangedirect counts, AODC), possibly colonizing the intestine passivelyduring filtration of algae. The activity of the bacterial communitieswas examined by the numencal ratio c.f.u.:AODC, where 1–19%of the bacteria were found active, with no significant differencebetween microbial environments.     

MORBILLIVIRUS INFECTION IN BOTTLENOSE DOLPHINS: EVIDENCE FOR RECURRENT EPIZOOTICS IN THE WESTERN ATLANTIC AND GULF OF MEXICO

Morbillivirus infection is widespread among odontocetes of the western Atlantic and Gulf of Mexico. Serologic evidence of infection in bottlenose dolphins, Tursiops truncatus , was first detected during an epizootic along the mid-Atlantic coast in 1987. Here, we report recurrent epizootics in the coastal dolphin population since at least the early 1980s based on serological surveys and regional stranding frequencies. The first observed epizootic of this series occurred in the Indian and Banana Rivers in 1982 and was followed by others on the mid-Atlantic coast in 1987–1988 and in the Gulf of Mexico between 1992 and 1994. This temporal pattern of infection is likely facilitated by the population size and its fragmentation into relatively discrete coastal communities. Introduction of morbillivirus into a community with a sufficient number of naive hosts may precipitate an epizootic, depending on the potential for transmission within the group. Propagation of an epizootic along the coast is probably determined by frequency of contact between adjacent communities and seasonal migrations.
Morbillivirus antibodies were also detected in serum from offshore bottlenose dolphins. The sero-prevalence in the latter may be higher than in coastal dolphins because of their close association with enzootically infected pilot whales ( Globicephala spp.). Occasional contact between offshore and coastal dolphins may provide an epizootiologic link between pilot whales and coastal dolphin communities.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Magnetic-field flux density and spectral characteristics of motor-driven personal appliances

Flux density and spectral measurements were carried out on magnetic fields generated by several types of motor-driven personal appliances used near the body. Among the units tested were several for which the average flux densities, as determined at the surfaces of the appliance, exceeded 0.4 mT. Time-rates-of-change (dB/dt) for several units exceeded 1000 T/s, and several units exhibited high-frequency components in the low-MHz range. Use of such appliances, although normally of short duration, can represent exposure to magnetic fields of relatively high flux density, which may also have high-frequency components. Compared to other household and commercial sources of magnetic fields, those generated by certain motor-driven personal appliances may represent a significant contribution to time-weighted average exposure and may represent an important source of local induced currents in the body. Furthermore, high-frequency transients that represent only a minor contribution to time-weighted average exposure may generate significant instantaneous induced currents. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Concordance between Parental Origin of Chromosome 13q Loss and 6p Duplication in Sporadic Retinoblastoma

Two hypotheses are capable of explaining nonrandom loss of one parent's alleles at tumor suppressor loci in sporadic cases of several pediatric cancers, including retinoblastoma—namely, preferential germ-line mutation or chromosome imprinting. We have examined 74 cases of sporadic retinoblastoma for tumors in which at least two genetic events—loss of heterozygosity for chromosome 13q markers and formation of an isochromosome 6p—have occurred. Sixteen cases were found to contain both events. In 13 of 16 such tumors, the chromosomes 13q that were lost and chromosomes 6p that were duplicated are derived from the same parent. These data may be explained within the framework of the genome imprinting model but are not predicted by preferential germ-line mutation.