Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Expression of GABA Receptor ρ Subunits in Rat Brain

Abstract: The GABA receptor ρ1, ρ2, and ρ3 subunits are expressed in the retina where they form bicuculline-insensitive GABA_C receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of ρ subunits in rat brains. In situ hybridization allowed us to detect ρ-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA_C receptors, ρ2 and ρ1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA_C receptors, ρ2 mRNA is enriched relative to ρ1 mRNA. These results suggest that both ρ1 and ρ2 subunits are necessary to form a functional GABA_C receptor. The use of RT-PCR also showed that, except in the superior colliculus, ρ3 is expressed along with ρ1 and ρ2 subunits. We also raised an antibody against a peptide sequence unique to the ρ1 subunit. The use of this antibody on cerebellum revealed the rat ρ1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA_C receptor subunits to identified neurons paves the way for future electrophysiological studies.     

Money Affects Theory of Mind Differently by Gender

Theory of Mind (ToM) ─ the ability to understand other’s thoughts, intentions, and emotions ─ is important for navigating interpersonal relationships, avoiding conflict, and empathizing. Prior research has identified many factors that affect one’s ToM ability, but little work has examined how different kinds of monetary incentives affect ToM ability. We ask: Does money affect ToM ability? If so, how does the effect depend on the structure of monetary incentives? How do the differences depend on gender? We hypothesize that money will affect ToM ability differently by gender: monetary rewards increase males’ motivation to express ToM ability while simultaneously crowding out females’ motivation. This prediction is confirmed in an experiment that varies the structure of monetary rewards for correct answers in the Reading the Mind in the Eyes Test (RMET). RMET scores decrease for females and increase for males with individual payments, and this effect is stronger with competitively-structured payments. RMET scores do not significantly change when monetary earnings go to a charity. Whether money improves or hinders ToM ability, and, hence, success in social interactions, thus depends on the interaction of gender and monetary incentive structure.     

An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome''s predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.     

The membrane anchor R7BP controls the proteolytic stability of the striatal specific RGS protein, RGS9-2

A member of the RGS (regulators of G protein signaling) family, RGS9-2 is a critical regulator of G protein signaling pathways that control locomotion and reward signaling in the brain. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with its newly discovered partner, R7BP (R7 family binding protein). Interaction with R7BP is important for the subcellular targeting of RGS9-2, which in native neurons is found in plasma membrane and its specializations, postsynaptic densities. Here we report that R7BP plays an additional important role in determining proteolytic stability of RGS9-2. We have found that co-expression with R7BP dramatically elevates the levels of RGS9-2 and its constitutive subunit, Gbeta5. Measurement of the RGS9-2 degradation kinetics in cells indicates that R7BP markedly reduces the rate of RGS9-2.Gbeta5 proteolysis. Lentivirus-mediated RNA interference knockdown of the R7BP expression in native striatal neurons results in the corresponding decrease in RGS9-2 protein levels. Analysis of the molecular determinants that mediate R7BP/RGS9-2 binding to result in proteolytic protection have identified that the binding site for R7BP in RGS proteins is formed by pairing of the DEP (Disheveled, EGL-10, Pleckstrin) domain with the R7H (R7 homology), a domain of previously unknown function that interacts with four putative alpha-helices of the R7BP core. These findings provide a mechanism for the regulation of the RGS9 protein stability in the striatal neurons.     

Aromatase inhibitors--gene discovery

Microarray analysis of tumour RNA is an extremely powerful tool which allows global gene expression to be measured. When used in combination with neoadjuvant treatment protocols in which therapy is given with the primary tumour within the breast, sequential biopsies may be analysed and results correlated with clinical and pathological response. In the present study, a neoadjuvant protocol has been used, administering the third generation inhibitor, letrozole, for 3 months and subjecting RNA extracted from biopsies taken before and after 10–14 days of treatment to microarray analysis. The objectives were to discover: (i) genes that change with estrogen deprivation (the only known biological effect of letrozole is to inhibit aromatase activity and reduce endogenous estrogens in postmenopausal women) and (ii) genes whose basal, on treatment or change in expression differ between tumours which are either responsive or resistant to treatment (so that predictive indices of response/resistance may be developed).

Early changes in gene expression were identified by comparing paired tumour core biopsies taken before and after 14 days treatment in 58 patients using three different approaches based on frequency of changes, magnitude of changes and SAM analysis. All three approaches showed a greater number of genes were down-regulated than up-regulated. Merging of the data produced a total of 143 genes which were subject to gene ontology and cluster analysis. The ontology of the 91 down-regulated genes showed that they were functionally associated with cell cycle progression, particularly mitosis. In contrast, up-regulated genes were associated with organ development and extra-cellular matrix turnover and regulation.

Clinical response was assessable in 52 patients; 37 (71%) tumours were classified as clinical responders (>50% reduction in volume at 3 months). Microarray analysis of pre- and 14-day biopsies identified 291 covariates (84 baselines, 72 14-day and 135 changes) highly predictive of response status. A similarity matrix using the covariates showed responding tumours have a similar genetic profile which was dissimilar to non-responding cancers whereas non-responsive cases were distinctive from each other. Changed genes predicting for response showed no concordance with those changed significantly by treatment in the overall group.     

The genome sequence of the leaf-cutter ant Atta cephalotes reveals insights into its obligate symbiotic lifestyle

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host-microbe symbioses.     

The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.     

Thermometric titration studies of the interaction of block polypeptides with surfactants

The interactions between surfactants and block polypeptides were investigated by titration calorimetry and CD. The polypeptides exhibited signs of an interaction only with surfactants bearing a charge opposite to the charge on the polymer. The stoichiometry of the resulting complex was determined to be approximately equal to the polymer net charge. In general, a decrease in helical content accompanied the interaction between the block polypeptides and the surfactants. Both positive and negative enthalpy changes were noted, depending on the heat of micelle formation. None of the thermal effects noted were preceded by polymer unfolding, as is characteristic of the interaction between surfactants and typical globular proteins.