Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration.

Bradyrhizobium japonicum cytochrome c(550), encoded by cycA, has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c(550) is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum, as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c(550) is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c(550) mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb(3)-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

RNA removal from Candida utilis by chemical and thermal treatments

The removal of the content of nucleic acids of fodder yeast (Candida utilis) by treatment with HCl or heat shock was investigated Acid concentrations between 5 and 15% (on dried matter basis) were used. A maximal removal of the content of nucleic acids of 88% was realized wheńn was worked with 15% HCl, 90 °C during 30 minutes. But under this conditions were observed high demanges of protein and also of some essential amino acids Good results for diminishing the content of nucleic acids were reached with the highest concentration of acid and a treatment during 20 minutes The experiments with heat shock were carried out at 68 °C, different times for heating and different contents of yeast In this way better results than for treatment with acid according to diminishing the content of nucleic acids and yield of protein and essential amino acids were reached A removal of over 80% of the content of nucleic acids was achieved in all cases.     

Nucleic acid reduction from yeast activation of intracellular rnase

The study of a heat-shock process for RNA reduction was carried out for different yeast strains. Different results were obtained from each of them. Candida utilis NRRL Y-660 shows its best performance after a 8-s. heat-shock in the presence of 3% NaCl. For commercial baker's yeast Saccharomyces cerevisiae and Kluyveromyces fragilis L-1930, similar results were obtained with only 1% of NaCl. The latter needed longer heat-shock periods. e.g. 15s. to give such an RNA reduction. Biomass recovery ranged from 60 to 75%, being higher for C. utilis and K. fragilis while excessive losses were observed in S. cerevisiae cells. No significant protein deterioration was obtained in the best performance samples. The aminoacid profile appears to be improved in comparison to the starting material in these strains after RNA reduction.     

Effects of corticotropin-releasing factor, corticotropin and cortisol on gastrointestinal motility in dogs

Gastrointestinal motor activity following intracerebroventricular (ICV) and intravenous (IV) administration of corticotropin releasing factor (CRF), corticotropin (ACTH) and cortisol was investigated in fasted dogs with strain-gauge transducers chronically implanted on the antrum and proximal jejunum. ICV but not IV administration of CRF (20 to 100 ng/kg) suppressed the gastric cyclic migrating motor complex (MMC) for 3 to 6 hours without affecting the jejunum. Similar disruptive effects on the gastric MMC were observed after ICV administration of ACTH (0.5 U/kg) or cortisol (0.1 micrograms/kg) but not after IV administration of 10 times higher doses. These results suggest that in dog CRF may be involved in the central control of the interdigestive gastric motility, these effects were not probably due to the release of ACTH and cortisol the other hormones of the pituitary adrenocortical system change the gastric motility when centrally administered through a possible feed-back mechanism affecting brain CRF level.     

Toxic effects of Canavalia ensiformis L. (Leguminosae) on laboratory colonies of Atta sexdens L. (Hym., Formicidae)

Laboratory colonies of the leaf-cutting ants Atta sexdens that were fed daily with leaves of Canavalia ensiformis showed a high ant mortality, and a significant decrease in fungal garden volume, with complete depletion of nests after 11 weeks of treatment.     

OpenAWSEM with Open3SPN2: A fast,flexible, and accessible framework for large-scale coarse-grained biomolecular simulations

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.     

Novel model for “calcium paradox” in sympathetic transmission of smooth muscles: Role of cyclic AMP pathway

It is well established that reduction of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-type VDCC), or increase of cytosolic cAMP concentration ([cAMP]c), inhibit contractile activity of smooth muscles in response to transmitters released from sympathetic nerves. Surprisingly, in this work we observed that simultaneous administration of L-type VDCC blocker (verapamil) and [cAMP]c enhancers (rolipram, IBMX and forskolin) potentiated purinergic contractions evoked by electrical field stimulation of rat vas deferens, instead of inhibiting them. These results, including its role in sympathetic transmission, can be considered as a “calcium paradox”. On the other hand, this potentiation was prevented by reduction of [cAMP]c by inhibition of adenylyl cyclase (SQ 22536) or depletion of Ca²⁺ storage of sarco-endoplasmic reticulum by blockade of Ca²⁺ reuptake (thapsigargin). In addition, cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evaluated by fluorescence microscopy in rat adrenal medullary slices was significantly reduced by verapamil or rolipram. In contrast, simultaneous incubation of adrenal slices with these compounds significantly increased [Ca²⁺]_c. This effect was prevented by thapsigargin. Thus, a reduction of [Ca²⁺]_c due to blockade of Ca²⁺ influx through L-type VDCC could stimulate adenylyl cyclase activity increasing [cAMP]c thereby stimulating Ca²⁺ release from endoplasmic reticulum, resulting in augmented transmitter release in sympathetic nerves and contraction.