The N-terminal extension of rusticyanin is not responsible for its acid stability

The N-terminal extension of rusticyanin is a unique structural feature of this protein in the cupredoxin family and has been speculated to be responsible for the extreme acid stability of the protein. We have removed the 35 residues from the N-terminus and show that the resulting -35 mutant is insoluble in aqueous media above pH 5.0 and exists primarily in a hexameric form at lower pHs. Synchrotron radiation circular dichroism (SRCD) and solution X-ray scattering data indicate that much of the beta-sheet structure is retained in acidic solution and indeed there is a small but significant increase in the beta-sheet contribution. We suggest this to be a result of beta-sheet formation between the monomer interfaces. The mutant does not bind copper. These results provide evidence that the unique N-terminus of rusticyanin is not responsible for the acid stability of the hydrophobic beta-barrel core of the protein.     

Green fluorescent protein-labeled recombinant fluobody for detecting the picloram herbicide

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).     

A neuropeptide FF-related gene is expressed selectively in neurons of the terminal nerve in Danio rerio

RFamides constitute a large family of neuromodulatory peptides. We have cloned a zebrafish gene, which is presumably a homologue to the mammalian PQRF subfamily of RFamides, and named it zfPQRF for its species and subfamily allocation. We report that in contrast to its mammalian counterparts zfPQRF is expressed in the olfactory bulb and the nucleus olfactoretinalis in the telencephalon, but absent in more caudal regions, including hypothalamus, brain stem and spinal cord. zfPQRF-expressing neurons originate in the vicinity of the olfactory placode and populate the nuclei of the terminal nerve during later development, as demonstrated by co-expression of zebrafish salmon-type gonadotropin releasing hormone, which was found to exclusively label terminal nerve neurons.     

Identification of a new segment involved in cagA 3' region variation of Helicobacter pylori

The cagA 3' region shows marked variation among Helicobacter pylori strains. Two segments of 102 bp and 57 bp are reportedly responsible for this variation. We analysed the cagA 3' region in 70 H. pylori strains using polymerase chain reaction and sequencing. We found that another segment, namely beta segment, was also involved in the variation of this region. The beta segment was 105 bp long and located between the aforementioned two segments. Six genotypes were identified based on the structure of the cagA 3' region. No relationship was found between these genotypes and the clinical outcomes or vacA genotypes. The numbers of tyrosine phosphorylation sites within the cagA 3' region varied among strains, but this was not related to the cagA genotypes. Our data suggest that the cagA 3' region is significantly variable. It appears that the variation of the cagA 3' region might contribute to the modification of virulence.     

The RHO1-GAPs SAC7, BEM2 and BAG7 control distinct RHO1 functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the small GTPase RHO1 plays an essential role in the control of cell wall synthesis and organization of the actin cytoskeleton. Several regulators for RHO1 are known, including the GTPase-activating proteins (GAPs) SAC7 and BEM2. Here we show that BAG7, identified as the closest homologue of SAC7, also acts as a GAP for RHO1 in vitro and in vivo. Furthermore, we find that BAG7, SAC7, and BEM2 are functionally different in vivo. Overexpression of BAG7 or SAC7,but not BEM2, suppresses the cold sensitivity of a sac7 mutation and the lethality of RHO1 hyperactivation in response to cell wall damage. In contrast, overexpression of BEM2 or SAC7, but not BAG7, downregulates the RHO1-controlled PKC1-MPK1 pathway, and disruption of BEM2 or SAC7, but not BAG7, results in increased MPK1 activation. We conclude that BEM2 and SAC7, but not BAG7, are involved in the control of the RHO1-mediated activation of MPK1, whereas BAG7 and SAC7, but not BEM2, are involved in the regulation of other RHO1 functions. This suggests that different RHO1GAPs control different RHO1 effector pathways, thus ensuring their individual regulation at the appropriate place and time.     

Pulmonary surfactant: phase behavior and function

Pulmonary surfactant functions by first flowing rapidly into the alveolar air/water interface, but then resisting collapse from the surface when the adsorbed interfacial film is compressed during exhalation. Widely accepted models emphasize the importance of phase behavior in both processes. Recent studies show, however, that fluidity is a relatively minor determinant of adsorption and that solid films, which resist collapse, can form by kinetic processes unrelated to equilibrium phase behavior.     

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.     

Pilot plant recovery of catheptic proteases from surimi wash water

Recovery of bioactive compounds, such as proteolytic enzymes, from waste streams is a means to both recuperate value and reduce environmental pollution. Previously optimized lab-scale parameters for the recovery of a stable crude protease fraction from Pacific whiting (Merluccius productus) surimi wash water were tested using pilot plant equipment. Pretreatment of surimi wash water with 60 degrees C heat, acidification to pH 6, and centrifugation doubled ultrafiltration membrane flux and significantly improved protease purity by reducing a majority of the 35-205 kDa proteins. Concentrated crude protease obtained from wash water contained predominantly cathepsin L activity. Enzyme purity was increased about 100-fold, and yield was approximately 80%. Stability (frozen and freeze-dried protease) was maintained for 9 weeks at -80 degrees C. Freeze-dried preparations were also stable for 9 weeks at 4 and -15 degrees C. Successful application of pilot plant conditions allows for sufficient production of protease for further investigations into their applicability.