Chemical modification of hemoglobin improves biocatalytic oxidation of PAHs

Chemical modifications on human hemoglobin were performed with the aim to change both surface and active-site hydrophobicities. The modifications included covalent coupling of poly(ethylene)glycol (5000 MW) on free amino groups and the methyl esterification of free carboxylic groups. The modified hemoglobin was assayed for the oxidation of 11 polycyclic aromatic hydrocarbons (PAHs) and 2 organosulfur aromatic compounds. Acenaphthene, anthracene, azulene, benzo(a)pyrene, fluoranthene, fluorene, phenanthrene, and pyrene were transformed to their respective quinones, while for chrysene and biphenyl no biocatalytic reaction could be detected. Dibenzothiophene and thianthrene were oxidized to form sulfoxides. The doubly modified hemoglobin, PEG-Met-hemoglobin, showed up to 10 times higher activity than the unmodified protein. The kinetic constants show that the PEG-Met-hemoglobin has a significantly higher catalytic efficiency. The equilibrium substrate binding constants for unmodified and PEG-Met-modified hemoglobis and hemoglobin show that this catalytic enhancement could be attributed to the affinity increase for hydrophobic substrates in the modified protein.     

Pollen size evolution: correlation between pollen volume and pistil length in Asteraceae

Based on the assumptions that pollen tube length is predetermined by provisions in the pollen and that it is a function of pistil length, I hypothesise that species with longer pistils will have larger pollen grains than species with shorter pistils, and that pistil length and pollen size will be positively correlated in a linear manner. To test this hypothesis, the relationship between pollen grain volume and pistil length was compared in 43 Asteraceae species from Argentina. A positive linear correlation was found between pollen volume and pistil length. This correlation remained significant even after potential effects of phylogenetic relatedness were removed. The maintenance of this correlation suggests that in Asteraceae the association between pistil length and pollen volume may reflect a functional rather than a phyletic relationship. In addition, the pistil length: pollen volume ratio (PPR) was analysed in relation to the phylogenetic position of the species. High values of PPR would imply a reduction of the male gametophyte in relation to the minimal volume that a pollen grain must have to grow and fertilise an ovule. Thus, the general pattern of pollen volume reduction in relation to pistil length previously found among many angiosperm families will be also present within a family, i.e., PPR values of derived Asteraceae would be higher than those of basal species. Results indicated that reduction of pollen volume in derived Asteraceae was three times greater than the concomitant shortening of pistil length. Consequently, PPR increased with the phylogenetic position of the taxa. This work supports the correlation between pistil and pollen characters previously found for other plant families and confirms the influence of post-pollination processes on pollen size evolution. Received: 4 November 1999 / Revision accepted: 14 February 2000     

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.     

Differential Binding of Escherichia coli O157:H7 to Alfalfa, Human Epithelial Cells, and Plastic Is Mediated by a Variety of Surface Structures

Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was ompA.     

Stochastic Resonance Crossovers in Complex Networks

Here we numerically study the emergence of stochastic resonance as a mild phenomenon and how this transforms into an amazing enhancement of the signal-to-noise ratio at several levels of a disturbing ambient noise. The setting is a cooperative, interacting complex system modelled as an Ising-Hopfield network in which the intensity of mutual interactions or “synapses” varies with time in such a way that it accounts for, e.g., a kind of fatigue reported to occur in the cortex. This induces nonequilibrium phase transitions whose rising comes associated to various mechanisms producing two types of resonance. The model thus clarifies the details of the signal transmission and the causes of correlation among noise and signal. We also describe short-time persistent memory states, and conclude on the limited relevance of the network wiring topology. Our results, in qualitative agreement with the observation of excellent transmission of weak signals in the brain when competing with both intrinsic and external noise, are expected to be of wide validity and may have technological application. We also present here a first contact between the model behavior and psychotechnical data.     

Alterations in adipocytokines and cGMP homeostasis in morbid obesity patients reverse after bariatric surgery

Objective:

Obesity‐associated nonalcoholic fatty liver disease (NAFLD), covering from simple steatosis to nonalcoholic steatohepatitis (NASH), is a common cause of chronic liver disease. Aberrant production of adipocytokines seems to play a main role in most obesity‐associated disorders. Changes in adipocytokines in obesity could be mediated by alterations in cyclic GMP (cGMP) homeostasis. The aims of this work were: (1) to study the role of altered cGMP homeostasis in altered adipocytokines in morbid obesity, (2) to assess whether these alterations are different in simple steatosis or NASH, and (3) to assess whether these changes reverse in obese patients after bariatric surgery.

Design and Methods:

In 47 patients with morbid obesity and 45 control subjects, the levels in blood of adipocytokines, cGMP, nitric oxide (NO) metabolites, and atrial natriuretic peptide (ANP) were studied. Whether weight loss after a bariatric surgery reverses the changes in these parameters was evaluated.

Results:

NO metabolites and leptin increase (and adiponectin decreases) similarly in patients with steatosis or NASH, suggesting that these changes are due to morbid obesity and not to liver disease. Inflammation and cGMP homeostasis are affected both by morbid obesity and by liver disease. The increases in interleukin 6 (IL‐6), interleukin 18 (IL‐18), plasma cGMP, ANP, and the decrease in cGMP in lymphocytes are stronger in patients with NASH than with steatosis. All these changes reverse completely after bariatric surgery and weight loss, except IL‐18.

Conclusion:

Altered cGMP homeostasis seems to contribute more than inflammation to changes in leptin and adiponectin in morbid obesity.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.