Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus

A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.     

The active form of the Saccharomyces cerevisiae ribonucleotide reductase small subunit is a heterodimer in vitro and in vivo

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y*) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (beta2) and Y4 (beta'2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (betabeta') is thought to be the active form, several rnr4delta strains are viable. To resolve this paradox, N-terminally epitope-tagged beta and beta' were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-beta2 ((His)beta2) is mixed with beta'2, apo-(His)betabeta' forms quantitatively within 2 min. In contrast, holo-betabeta' fails to exchange with apo-(His)beta2 to form holo-(His)betabeta and beta'2. Isolation of genomically encoded tagged beta or beta' from yeast extracts gave a 1:1 complex of beta and beta', suggesting that betabeta' is the active form. The catalytic activity, protein concentrations, and Y* content of the rnr4delta and wild type (wt) strains were compared to clarify the role of beta' in vivo. The Y* content of rnr4delta is 15-fold less than that of wt, consistent with the observed low activity of rnr4delta extracts (<0.01 nmol min(-1) mg(-1)) versus wt (0.06 +/- 0.01 nmol min(-1) mg(-1)). (FLAG)beta2 isolated from the rnr4delta strain has a specific activity of 2 nmol min(-1) mg(-1), similar to that of reconstituted apo-(His)beta2 (10 nmol min(-1) mg(-1)), but significantly less than holo-(His)betabeta' (approximately 2000 nmol min(-1) mg(-1)). These studies together demonstrate that beta' plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is betabeta'.     

Temporal patterns and variations in phytoplankton community organization and abundance in Narragansett Bay during 1959-1980

Incident irradiance, surface water temperature and phytoplanktonspecies abundances were measured at weekly intervals in NarragansettBay from 1959 through 1980. Stepwise discriminant analyses (SDA)of this 22-year data set indicate that fundamental ecosystemchanges occurred between the 1960s and 1970s, with 1969 beingthe key transitional year in these decadal shifts in phyto planktontaxonomic structure and seasonal abundance. This decadal shiftwas accompanied by the increased summer abundance of small Thalassiosiraspp., which first appeared in 1966 and by 1969 became the sixthmost important phytoplankton component in this bay. Decadaltrends in phyto plankton community organization and abundancewere also accompanied by distinct long-term climatological gradientsof temperature and light. The 1960s were generally colder andbrighter than the 1970s. Prior to 1969, the annual phytoplanktonmaximum occurred most commonly during winter; in the 1970s,the annual maximum generally shifted to a summer event. Three5-year phytoplankton cycles occurred between 1959 and 1974.During each pentade, the phytoplankton community returned toa similar taxonomic organization and abundance cycle after divergingin the intervening years. Pentade cycles did not occur after1974; the phytoplankton community thereafter diverged significantlyfrom each preceding year. Five species [Skeletonema costatum,Detonula confervacea, Asterionellopsis glacialis, Hererosigmaakashiwo (= Olisthodiscus lureus) and Thalassiosira nordenskioeldii]dominated the phytoplankton over the 22-year period. SDA revealeda high degree of similarity and constancy in the annual occurrencepatterns of these taxa. The decadal shifts revealed by SDA weremore directly related to the considerable interannual variabilitythat characterized the abundance and seasonality of the lessabundant species.     

Clinical characteristics of 16 cystic fibrosis patients with the missense mutation R334W,a pancreatic insufficiency mutation with variable age of onset and interfamilial clinical differences

We present the genotype/phenotype correlation analysis for 16 cystic fibrosis (CF) patients who carry mutation R334W. Current age and age of diagnosis was significantly higher in the R334W/any-mutation group (P < 0.05 and P < 0.01), compared with the 508/508 group. A slightly, but not significantly, worse lung function was found in the R334W/any-mutation group, when compared with the 508/508 patients. The proportion of patients with lung colonization with bacterial pathogens was slightly, but not significantly, higher in the R334W/any-mutation group (71.4%), compared with the 508/508 or R334W/508 groups (55.5%). None of the R334W patients had meconium ileus but 60% were pancreatic insufficient (PI), a significantly lower proportion (P 0.001) than 508/508 patients. Two R334W/N1303K compound heterozygous sisters were PI but discrepant for lung function. Two groups of three sibs with genotype R334W/508 showed interfamilial discordant clinical data for lung and pancreatic function. The data provided here for mutation R334W demonstrate that this mutation is responsible for a less severe form of CF than 508. Interfamilial differences for PI and lung function suggest that other factors, viz. genetic, environmental and medical, contribute to the wide spectrum of clinical differences observed in CF patients with the same CF transmembrane conductance regulator genotypes.     

Linking inventories and impact assessment models for addressing biodiversity impacts: mapping rules and challenges

The International Journal of Life Cycle Assessment - An adequate matching between the nomenclature of elementary flows in life cycle inventory (LCI) databases and life cycle impact assessment...     

Influence of Ozone-Related Increases in Ultraviolet Radiation on Antarctic Marine Organisms

Every spring for the past two decades, depletion of stratosphericozone has caused increases in ultraviolet B radiation (UVB,280–320 nm) reaching Antarctic terrestrial and aquatichabitats. Research efforts to evaluate the impact of this phenomenonhave focused on phytoplankton under the assumption that ecosystemeffects will most likely originate through reductions in primaryproductivity; however, phytoplankton do not represent the onlysignificant component in ecosystem response to elevated UVB.Antarctic bacterioplankton are adversely affected by UVB exposure;and invertebrates and fish, particularly early developmentalstages that reside in the plankton, are sensitive to UVB. Thereis little information available on UV responses of larger Antarcticmarine animals (e.g., birds, seals and whales). Understandingthe balance between direct biological damage and species-specificpotentials for UV tolerance (protection and recovery) relativeto trophic dynamics and biogeochemical cycling is a crucialfactor in evaluating the overall impact of ozone depletion.After more than a decade of research, much information has beengathered about UV-photobiology in Antarctica; however, a definitivequantitative assessment of the effect of ozone depletion onthe Antarctic ecosystem still eludes us. It is only obviousthat ozone depletion has not had a catastrophic effect in theAntarctic region. The long-term consequences of possible subtleshifts in species composition and trophic interactions are stilluncertain.     

Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic

Cervical cancer is the second most prevalent cancer in women worldwide. It is linked to infection with human papillomavirus (HPV). As the virus cannot be propagated in culture, vaccines based on virus-like particles have been developed and recently marketed. However, their high costs constitute an important drawback for widespread use in developing countries, where the incidence of cervical cancer is highest. In a search for alternative production systems, the major structural protein of the HPV-16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (approximately 3 mg L1/g fresh weight; equivalent to 24% of total soluble protein). This is the highest expression level of HPV L1 protein reported in plants. A single mature plant synthesized approximately 240 mg of L1. The chloroplast-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles, visible by transmission electron microscopy. Furthermore, leaf protein extracts from L1 transgenic plants were highly immunogenic in mice after intraperitoneal injection, and neutralizing antibodies were detected. Taken together, these results predict a promising future for the development of a plant-based vaccine against HPV.     

The role of mating history and male size in determining mating behaviours and sexual conflict in a water strider

We conducted three experiments to test the effects of mating history of both sexes and of male body size on mating behaviours in the water strider, Gerris buenoi. Our manipulations influenced the interests of both sexes and, thus, the degree of conflict over mating behaviours. Mating history was a dichotomous variable (deprived/mated), depending on holding conditions in the laboratory. Experiment 1 considered and found independent effects of male and female mating history on latency to copulation and copulation duration. In experiment 2, we manipulated only female mating history, using unsuccessful struggle rates as evidence for female reluctance and conflict over mating. Finally, we investigated the relation between male body size and mating history on copulation duration. We predicted that intersexual conflict over mating would be lowest when females were deprived, because female interests under these conditions should more closely match those of males. Deprived females began mating in half the time of mated females and were twice as likely to mate because of reduced reluctance. Furthermore, copulation duration for deprived males was about one and a half times longer than that for mated males. Although previous studies examining nonrandom mating patterns by size predicted longer copulations for small males, we found that small males prolonged copulation when deprived more than large males. We conclude that females primarily influence copulation frequency, but males primarily influence copulation duration. Our results favour the hypothesis that reduced mating opportunity for small males accounts for their extended copulation duration. Finally, our findings provide evidence for strong effects of male body size on selection mechanisms in water striders, and support the hypothesis of conflicting pre- and postcopulatory selection mechanisms in this group. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.