Engineering a novel, stable dimeric streptavidin with lower isoelectric point

We have engineered a soluble, stable two-chain dimeric streptavidin (TCD) in Escherchia coli. Examination of the three-dimensional structure of streptavidin aided by empirical binding free-energy calculations helped us to select mutations at subunit interfaces that dissociate the native tetramer and stabilize the desired dimer. We chose positions W120, L124, V125 and H127 and mutated them to 120D/124D/125D/127D (TCD-1); 120D/124N/125S/127D (TCD-2); and 120D/124D/125S/127D (TCD-3). The H127D mutation creates electrostatic repulsion that disrupts the dimer-dimer interface, but leaves it very hydrophobic. Therefore, W120, L124 and V125 were mutated to hydrophilic residues to increase dimer solubility. Among the three candidates, TCD-2 gave the best result: a stable, active dimer with K(d) for biotin of approximately 1x10(-7)M after purification by gel-filtration chromatography. The experimental results confirm the possibility of rational engineering of low-pI dimeric streptavidins. Reduced-size streptavidin mutants with a net negative charge may be more suitable than antibodies or wild-type streptavidin for the targeting step in radioimmunotherapy because they should clear faster from the bloodstream and the kidney.     

Coupled reductive and oxidative sulfur cycling in the phototrophic plate of a meromictic lake

Mahoney Lake represents an extreme meromictic model system and is a valuable site for examining the organisms and processes that sustain photic zone euxinia (PZE). A single population of purple sulfur bacteria (PSB) living in a dense phototrophic plate in the chemocline is responsible for most of the primary production in Mahoney Lake. Here, we present metagenomic data from this phototrophic plate – including the genome of the major PSB, as obtained from both a highly enriched culture and from the metagenomic data – as well as evidence for multiple other taxa that contribute to the oxidative sulfur cycle and to sulfate reduction. The planktonic PSB is a member of the Chromatiaceae, here renamed Thiohalocapsa sp. strain ML1. It produces the carotenoid okenone, yet its closest relatives are benthic PSB isolates, a finding that may complicate the use of okenone (okenane) as a biomarker for ancient PZE. Favorable thermodynamics for non‐phototrophic sulfide oxidation and sulfate reduction reactions also occur in the plate, and a suite of organisms capable of oxidizing and reducing sulfur is apparent in the metagenome. Fluctuating supplies of both reduced carbon and reduced sulfur to the chemocline may partly account for the diversity of both autotrophic and heterotrophic species. Collectively, the data demonstrate the physiological potential for maintaining complex sulfur and carbon cycles in an anoxic water column, driven by the input of exogenous organic matter. This is consistent with suggestions that high levels of oxygenic primary production maintain episodes of PZE in Earth's history and that such communities should support a diversity of sulfur cycle reactions.     

Estimating downwind concentrations of viable airborne microorganisms in dynamic atmospheric conditions.

A Gaussian plume model has been modified to include an airborne microbial survival term that is a best-fit function of laboratory experimental data of weather variables. The model has been included in an algorithm using microbial source strength and local hourly mean weather data to drive the model through a summer- and winter-day cycle. For illustrative purposes, a composite airborne "virus" (developed using actual characteristics from two viruses) was used to show how wind speed could have a major modulating effect on near-source viable concentrations. For example, at high wind speeds such as those occurring during the day, or with short travel times, near-source locations experience high viable concentrations because the microorganisms have not had time to become inactivated. As the travel time increases, because of slow wind speed or longer distances, die-off modulation by sunshine, relative humidity, temperature, etc., potentially becomes increasingly predominant.     

Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP

The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of development is incomplete. We present here a detailed expression profile of this promoter investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development, and in all the vegetative and floral cell and tissue types. GFP expression was not detected during the early stages of embryogenesis. The first perceptible GFP expression was observed in a small area at the junction of hypocotyl and cotyledons in embryos at around 13 days after anthesis. The GFP fluorescence progressively became stronger and expanded throughout the cotyledon and hypocotyl as embryo development advanced. After germination, varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen exhibited a low level of greenish autofluorescence, it was possible to discern GFP-dependent fluorescence in some of the pollen from all the T₀ plants examined. Developing cotton fibers also exhibited GFP fluorescence suggesting that the 35S promoter was active in these specialized epidermal cells. Thus, we show that the expression of the 35S promoter was developmentally regulated during embryogenesis and that beyond a certain stage during embryogenesis, the promoter was expressed in most cell and tissue types in cotton albeit at different levels.     

An analysis of the action of light on betalain synthesis in the seedling of Amaranthus caudatus,var. viridis

Accumulation of betalain (amaranthin) in the seedling of Amaranthus caudatus, var. viridis, is inducible by light. Since the apparent lag-phase of amaranthin accumulation after the onset of light is of the order of 3 h, light induction experiments could be performed up to 3 h after the onset of light without interference with actual synthesis. The intricate induction phenomena can be explained as follows: The inductive light operates through phytochrome and through a blue/UV photoreceptor (cryptochrome). A phytochrome-dependent High Irradiance Reaction is of minor importance. However, there is a strong, specific interaction between the light effects mediated through phytochrome and cryptochrome in the sense that the extent of the reversible response — (response obtained with a particular light treatment terminated with a saturating red light pulse) minus (response obtained with the same light treatment when terminated with a saturating 756 nm light pulse) —increases with increasing P_fr level and total fluence rate during the induction period. It is concluded that light induced amaranthin synthesis is, in fact, a convenient biochemical model system of photomorphogenesis in the case when phytochrome and cryptochrome operate simultaneously in mediating photomorphogenesis. Abbreviations: see Table 1 and 2 Materials and Medthods     

Different handling of parathyrin by basal-lateral and brush-border membranes of the bovine kidney cortex.

The two parts of the bovine kidney cortex plasma membrane, the basal-lateral and the brush-border membrane, were simultaneously prepared from the same organ. Both types of membrane bound parathyrin, but only from the basal-lateral fraction was the hormone displaceable by its bioactive N-terminal fragment. In parallel, parathyrin-stimulated adenylate cyclase was predominantly found in basal-lateral membranes. The hormone was fragmented by both membrane types. Basal-lateral membranes generated fragments with a rather uniform size distribution (somewhat smaller than the intact peptide) and apparently preferred the hormone itself as a substrate. In contrast, the fragments produced by brush-border membranes were numberous small peptides.     

Eukaryotic translation initiation factor 4F architectural alterations accompany translation initiation factor redistribution in poxvirus-infected cells

Despite their self-sufficient ability to generate capped mRNAs from cytosolic DNA genomes, poxviruses must commandeer the critical eukaryotic translation initiation factor 4F (eIF4F) to recruit ribosomes. While eIF4F integrates signals to control translation, precisely how poxviruses manipulate the multisubunit eIF4F, composed of the cap-binding eIF4E and the RNA helicase eIF4A assembled onto an eIF4G platform, remains obscure. Here, we establish that the poxvirus infection of normal, primary human cells destroys the translational repressor eIF4E binding protein (4E-BP) and promotes eIF4E assembly into an active eIF4F complex bound to the cellular polyadenylate-binding protein (PABP). Stimulation of the eIF4G-associated kinase Mnk1 promotes eIF4E phosphorylation and enhances viral replication and protein synthesis. Remarkably, these eIF4F architectural alterations are accompanied by the concentration of eIF4E and eIF4G within cytosolic viral replication compartments surrounded by PABP. This demonstrates that poxvirus infection redistributes, assembles, and modifies core and associated components of eIF4F and concentrates them within discrete subcellular compartments. Furthermore, it suggests that the subcellular distribution of eIF4F components may potentiate the complex assembly.     

Protective effect of bixin on carbon tetrachloride-induced hepatotoxicity in rats

Background

The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl₄) in rats.

Results

The animals were divided into four groups with six rats in each group. CCl₄ (0.125 mL kg^-1 body wt.) was injected intraperitoneally, and bixin (5.0 mg kg^-1 body wt.) was given by gavage 7 days before the CCl₄ injection. Bixin prevented the liver damage caused by CCl₄, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl₄ by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment.

Conclusion

Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl₄ is related to the antioxidant activity of the compound.