Functional properties of carbohydrate-depleted tissue plasminogen activator

In order to evaluate the importance of the carbohydrate moiety of human tissue plasminogen activator (TPA), human melanoma (Bowes) cells were treated with a glycosylation inhibitor, tunicamycin (TM), and cellular fractions were assayed for fibrinolytic activity. Where glycosylation was inhibited by 90% and protein synthesis by 30%, TPA specific activity measured by fibrinolytic assays decreased 6-10-fold in the tissue culture medium and cell cytosol with a concomitant 2-fold increase in the 100000g microsomal pellet. In addition, TPA purified to apparent homogeneity was treated with endo-beta-N-acetylglucosaminidase H (Endo-H), producing a fraction that in contrast to native TPA did not adsorb to concanavalin A-Sepharose (Con A-Sepharose). This fraction represented TPA from which 85-90% of N-linked carbohydrate residues had been removed. Native TPA effectively activated plasminogen in the presence of fibrin (Km = 1 microM, kcat = 0.09 s-1) whereas saturation of the enzyme was not achieved at 100 microM plasminogen in the absence of fibrin. Glycosidase-treated and native TPA activated plasminogen at identical high rates in the presence and at identical negligible rates in the absence of fibrin. These studies indicate that the inhibition of glycosylation of TPA results in the inhibition of secretion of the molecule as has been observed for some other glycoproteins. The enzymatic removal of N-linked carbohydrate from purified TPA does not change its unique fibrin-directed properties.     

Effects of estradiol concentration on levels of nuclear estrogen receptors in MCF-7 breast tumor cells

We have measured the time-course of estrogen receptor levels in nuclei of the estrogen-responsive breast tumor cell line MCF-7 during 90-120 min exposure of the cells to estradiol at physiologic (10(-10)M), pharmacologic (10(-6)M), and an intermediate (10(-8)M) concentration. Cells were preincubated for one week in a serum-free defined medium resembling that of Barnes and Sato, and then incubated in estradiol-containing medium. Nuclei were isolated at various times during the incubation, and filled and unfilled nuclear estrogen receptor levels were assayed. Increasing the concentration of estradiol in the incubation medium from 10(-10)M to 10(-8)M yielded increasing levels of filled nuclear receptor at all times studied, while further increase of the estradiol concentration of 10(-6)M decreased filled receptor levels from 10(-8)M values. Unfilled receptor levels dropped rapidly to zero under 10(-6)M and 10(-8)M estradiol incubation, but remained unchanged under 10(-10)M estradiol incubation. Together these results suggest that high-concentration estradiol may lead to "down-regulation" of filled nuclear receptors, which may be a contributing factor in inhibition of tumor growth. On the other hand, the continued presence of unfilled receptors only under physiological concentrations of estradiol may suggest a role for these receptors in sustaining tumor growth.     

MOLECULAR AND PHYLOGENETIC CHARACTERIZATION OF PHORMIDIUM SPECIES (CYANOPROKARYOTA) USING THE CPCB‐IGS‐CPCA LOCUS1

The accurate determination of species of Cyanoprokaryota/Cyanophyceae has many important applications. These include the assessment of risk with regard to blooms in water reservoirs as well as the identification of species capable of producing valuable bioactive compounds. Commonly, Cyanoprokaryota are classified based on their morphology. However, morphological criteria are not always reliable because they may change, for example, due to environmental factors. Thus, genetic and molecular analyses are a promising additional approach, but their application has so far been limited to relatively few genera. In light of this, we present here the first characterization of species and strains of the genus Phormidium Kütz. based on the cpcB‐IGS‐cpcA locus of the phycocyanin operon. In phylogenetic analyses using deduced amino acid sequences of the cpcB‐cpcA regions, Phormidium was found to be polyphyletic. This analysis appeared to be dominated by the cpcB region, which is characterized by a relatively high percentage of informative substitutions. The percentage of variable positions within the cpcB‐IGS‐cpcA locus overall was 16.5%, thereby indicating a level of divergence remarkably higher than that reported for Nodularia and Arthrospira in previous studies relying on cpcB‐IGS‐cpcA. Further, alignment of informative nucleotide substitutions in the cpcB‐IGS‐cpcA sequences revealed a mosaic distribution, which may be indicative of genetic recombination events. Finally, the length and sequences of the IGS region alone proved useful as markers to differentiate the cyanobacterial genus Phormidium. However, whether the IGS region per se is sufficiently discriminatory to differentiate between Phormidium species or even strains requires further investigation using newly identified Phormidium sequence data.     

Arrival times,laying dates,and reproductive success of Snowy Plovers in two habitats in coastal northern California

ABSTRACT Habitat quality, as indexed by the reproductive success of individuals, can greatly influence population growth, especially for rare species near the limits of their range. Along the Pacific coast, the Snowy Plover (Charadrius alexandrinus nivosus) is a threatened species that, in recent years, has been breeding on both riverine gravel bars and ocean beaches in northern California. From 2001 to 2009, we compared the habitat characteristics, breeding phenology, reproductive success, and abundance of Western Snowy Plovers occupying these two habitats. Similar percentages of yearling and adult plovers returned to gravel bars and beaches, but plovers breeding on gravel bars arrived and initiated first clutches 2–3 weeks later than those breeding on beaches. Despite this delay, however, the mean annual fledging success of plovers on gravel bars (1.4 ± 0.4 [SD]) was double that on beaches (0.7 ± 0.3). Differences in cumulative reproductive success produced a stronger pattern. By their sixth year, males on gravel bars had fledged 14.5 ± 2.1 chicks, more than four times the number of young fledged by males on beaches (3.3 ± 3.1). Over 9 years, local population size decreased by about 75%, coincident with a shift in breeding distribution away from high‐quality gravel bars to ocean beaches. This unexpected population decline and shift to poorer quality beaches may have been related to occasional low survival of plovers that over‐winter exclusively on beaches in our study area. Consistently low productivity of plovers breeding on ocean beaches suggests the need for intensified management to ameliorate the negative impacts of predation and human activity on the recovery of this population.     

Neural Correlates of Post-Conventional Moral Reasoning: A Voxel-Based Morphometry Study

Going back to Kohlberg, moral development research affirms that people progress through different stages of moral reasoning as cognitive abilities mature. Individuals at a lower level of moral reasoning judge moral issues mainly based on self-interest (personal interests schema) or based on adherence to laws and rules (maintaining norms schema), whereas individuals at the post-conventional level judge moral issues based on deeper principles and shared ideals. However, the extent to which moral development is reflected in structural brain architecture remains unknown. To investigate this question, we used voxel-based morphometry and examined the brain structure in a sample of 67 Master of Business Administration (MBA) students. Subjects completed the Defining Issues Test (DIT-2) which measures moral development in terms of cognitive schema preference. Results demonstrate that subjects at the post-conventional level of moral reasoning were characterized by increased gray matter volume in the ventromedial prefrontal cortex and subgenual anterior cingulate cortex, compared with subjects at a lower level of moral reasoning. Our findings support an important role for both cognitive and emotional processes in moral reasoning and provide first evidence for individual differences in brain structure according to the stages of moral reasoning first proposed by Kohlberg decades ago.