Use of monoclonal anti-light subunit antibodies to study the structure and function of theEntamoeba histolytica Gal/GalNAc adherence lectin

Adherence ofEntamoeba histolytiea trophozoites to host cells is medicated by a galactose (Gal) andN-acetylgalactosamine (GalNAc)-specific surface lectin. The lectin is a heterodimeric protein composed of heavy (170kDa) and light (35-31 kDa) subunits linked by disulfide bonds. Polyclonal and monoclonal antibodies (mAb) raised against a light subunit-glutathione-S-transferase fusion protein were used to probe its structure and function. Four light subunit-specific mAb were produced which recognized distinct epitopes on five different light subunit isoforms. Immunoblots with these mAb demonstrated co-migration of light and heavy subunits when nonreduced trophozoite proteins were analysed by SDS-PAGE, indicating that the subunits do not exist free of the heterodimer in significant quantities. While anti-heavy subunit antibodies had previously been shown to alter adherence, anti-light subunit antibodies did not, suggesting that the heavy subunit contains the carbohydrate recognition domain.     

12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1.

P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is an energy-dependent efflux pump associated with treatment failure in some hematopoietic malignancies. Its expression is regulated during normal hematopoietic differentiation, although its function in normal hematopoietic cells is unknown. To identify cellular factors that regulate the expression of MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1 promoter in K562 cells. Transient-transfection assays demonstrated that an MDR1 promoter construct containing nucleotides -69 to +20 conferred a TPA response equal to that of a construct containing nucleotides -434 to +105. TPA induced EGR1 binding to the -69/+20 promoter sequences over a time course which correlated with increased MDR1 promoter activity and increased steady-state MDR1 RNA levels. The -69/+20 promoter region contains an overlapping SP1/EGR site. The TPA-responsive element was localized to the overlapping SP1/EGR site by using a synthetic reporter construct. A mutation in this site that inhibited EGR protein binding blocked the -69/+20 MDR1 promoter response to TPA. The expression of a dominant negative EGR protein also blocked the TPA response of the -69/+20 promoter construct. Finally, the expression of EGR1 was sufficient to activate a construct containing tandem MDR1 promoter SP1/EGR sites. These data suggest a role for EGR1 in modulating MDR1 promoter activity in hematopoietic cells.     

Effects of density and cellular aging on collagen synthesis and growth kinetics in keloid and normal skin fibroblasts

Summary Cell proliferation and collagen synthesis were analyzed in high, medium, and low density keloid and normal skin fibroblasts and also at various times during the course of in vitro aging to expand previous findings of increased collagen synthesis in keloid compared to normal skin fibroblasts. It was found that both keloid and normal fibroblasts (<20 population doublings) responded similarly to high, medium, and low initial plating densities; however, sparsely plated keloid fibroblasts exhibited a loss of replicative capacity earlier in their in vitro lifespan than did sparsely plated normal skin fibroblasts. When analyzed at population doubling levels 2 to 38, collagen synthesis was elevated in keloid compared to normal skin fibroblasts but decreased at the same rate in both cell types throughout this in vitro interval. Supported by NIH Grant GM-20298.     

A New Ichnospecies of Arthrophycus from the Late Carboniferous (Pennsylvanian) of Michigan,USA

A unique ichnofossil assemblage from Pennsylvanian-age sandstones near Eaton Rapids, Michigan, USA, comprises straight-to-curved traces preserved in convex hyporelief, with the transverse ridges and median grove associated with the ichnogenus Arthrophycus. The Michigan traces show some branching or pseudo-branching (also known from other Arthrophycus specimens) and are among the smallest structures (millimeters in diameter) attributed to this ichnogenus. The orientation of the Michigan Arthrophycus burrows is distinct from other ichnospecies of this taxon in the pronounced co-planar alignment of the burrows, as opposed to the multiplanar, interweaving, “bundled” nature typical of the ichnogenus. On this basis we assign the Michigan specimens to a new ichnospecies of Arthrophycus. The “paralleling behavior” of the new taxon may reflect a strategy of the tracemaker to avoid previous burrows and reflect differences in resource availability or current energy. This new taxon supports previous records of the occurrence of this ichnogenus in Upper Carboniferous strata.     

Capsaicin does not attenuate bombesin-induced suppression of operant responding for food reward.

Systemic treatment with capsaicin, a neurotoxin which damages unmyelinated peptide-containing sensory neurons, has been shown to attenuate bombesin (BBS)-induced suppression of food intake. To determine whether capsaicin-sensitive fibers mediate the effect of BBS on appetitive motivation, we examined BBS-induced suppression of operant responding in rats pretreated neonatally with capsaicin (50 mg/kg; SC) or control vehicle. At 8-10 weeks of age, rats were trained to bar press for food. After achieving a stable level of performance, the animals were injected with BBS (10 micrograms/kg), normal saline, or prefed with 20 Noyes 45-mg pellets. Animals were then tested in an operant chamber on an FR 5 schedule of reinforcement for one hour. The results indicated that BBS suppressed bar pressing, regardless of whether animals were pretreated with capsaicin or control vehicle. These findings are inconsistent with the hypothesis that BBS induces satiety via capsaicin-sensitive neurons. The results suggest the possibility that more than one mechanism may mediate the effects of BBS: a neural mechanism involved in consummatory responses and a humoral mechanism involved in the operant response.     

Fire alters patterns of genetic diversity among 3 lizard species in Florida Scrub habitat

The Florida Sand Skink (Plestiodon reynoldsi), the Florida Scrub Lizard (Sceloporus woodi), and the Six-lined Racerunner (Aspidoscelis sexlineata) occur in the threatened and fire-maintained Florida scrub habitat. Fire may have different consequences to local genetic diversity of these species because they each have different microhabitat preference. We collected tissue samples of each species from 3 sites with different time-since-fire: Florida Sand Skink n = 73, Florida Scrub Lizard n = 70, and Six-lined Racerunner n = 66. We compared the effect of fire on genetic diversity at microsatellite loci for each species. We screened 8 loci for the Florida Sand Skink, 6 loci for the Florida Scrub Lizard, and 6 loci for the Six-lined Racerunner. We also tested 2 potential driving mechanisms for the observed change in genetic diversity, a metapopulation source/sink model and a local demographic model. Genetic diversity varied with fire history, and significant genetic differentiation occurred among sites. The Florida Scrub Lizard had highest genetic variation at more recently burned sites, whereas the Florida Sand Skink and the Six-lined Racerunner had highest genetic variation at less recently burned sites. Habitat preferences of the Florida Sand Skink and the Florida Scrub Lizard may explain their discordant results, and the Six-lined Racerunner may have a more complicated genetic response to fire or is acted on at a different geographic scale than we have investigated. Our results indicate that these species may respond to fire in a more complicated manner than predicted by our metapopulation model or local demographic model. Our results show that the population-level responses in genetic diversity to fire are species-specific mandating conservation management of habitat diversity through a mosaic of burn frequencies.     

Collectrin/tmem27 is expressed at high levels in all segments of the developing Xenopus pronephric nephron and in the Wolffian duct

Collectrin/tmem27 encodes a transmembrane protein that plays a critical role in amino-acid transport. Originally described as being expressed only in collecting ducts, it has subsequently also been shown to also be expressed in the S1 segment of the proximal tubule of mammalian metanephric nephrons. In this report we describe the expression of collectrin in the simple embryonic kidney of amphibians, the pronephros. Each pronephros contains a single large nephron with a proximo-distal segmentation very similar to that of mammalian metanephric nephrons. Analysis of collectrin expression in pronephroi at a variety of embryonic stages indicates that this gene is expressed at very high levels throughout the pronephric system, including proximal and distal segments and the Wolffian duct. Expression in the pronephros commences at Xenopus embryonic stage 28 which corresponds to when epithelialization begins within the pronephric mesenchyme. Like the Na+K+ATPase/atp1a1, another highly expressed pronephric marker, collectrin is also expressed in the cloaca but not in the cloacal derived posterior segment of the Wolffian duct, the rectal diverticulum. Unlike the Na+K+ATPase, which is expressed at lower levels in proximal portions of the pronephric nephron, expression of collectrin is even throughout all of the pronephric epithelia. This expression domain extends far beyond that shown to express amino-acid transporters and indicates collectrin may function in facilitating additional transport processes. Its high level of expression and broad distribution make it an excellent marker with which to examine pronephric kidney development.     

Evaluation of a minimal-therapist-contact thermal biofeedback treatment program for essential hypertension

We compared a clinic-based regimen of 16 individual sessions (2 per week) of thermal biofeedback with a largely home-based regimen of 5 sessions (spread over 8 weeks) for the treatment of essential hypertension in patients who required at least two drugs to maintain control of blood pressure (BP). On the basis of the clinical end point of being successfully withdrawn from the second stage medication while BP remained under control, the clinic-based regimen (5 of 9) was superior (chi less than (1) = 4.0, p less than .05) to the home-based regimen (1 of 9). Internal analyses point to more frequently obtaining a hand temperature of at least 95 degrees F by the office-based patients as possibly the reason for the difference.     

Long-term evolution and short-term adaptation of microbiota strains and sub-strains in mice

1. Download : Download high-res image (138KB)
2. Download : Download full-size image