Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms

Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three-dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three-dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic-like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N-terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme.     

ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels

During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (DeltaG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (DeltaG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl- from guard cells and a rapid but confined reduction in stomatal aperture.     

Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.     

Conformational analysis of hepatitis B surface antigen fusions in an Agrobacterium-mediated transient expression system

Vaccine antigens have been successfully produced in transgenic plants for oral immunization. Recently, a fusion strategy has been adopted to produce multicomponent vaccines and to target antigens to mucosal sites for enhanced oral immunogenicity. However, antigen fusions may not be folded correctly due to steric hindrance and may thus lose their potency. Here, we describe an Agrobacterium-mediated transient assay that provides enough antigen-expressing material at 2 days post-transfection to evaluate antigen conformation. Using the hepatitis B surface antigen (HBsAg) as a model antigen and the green fluorescent protein (GFP) as a model fusion partner, we showed that transiently expressed HBsAg and an HBsAg fusion with GFP at the N-terminus (GFP:HBsAg), but not the HBsAg fusion with GFP at the C-terminus (HBsAg:GFP), formed the 'a' determinant and virus-like particles (VLPs), similar to yeast-derived vaccine HBsAg. Thus, it is feasible to modify the HBsAg with an N-terminal fusion of up to 239 amino acids without altering its major antigenic properties. Our results also demonstrate that the Agrobacterium-mediated transient expression system can be used to evaluate the conformation of plant-based vaccines or other pharmaceutical proteins in a high-throughput manner.     

Tablet formulation containing meloxicam and β-cyclodextrin: Mechanical characterization and bioavailability evaluation

The purpose of this research was to evaluate β-cyclodextrin (β-CD) as a vehicle, either singly or in blends with lactose (spray-dried or monohydrate), for preparing a meloxicam tablet. Aqueous solubility of meloxicam in presence of β-CD was investigated. The tablets were prepared by direct compression and wet granulation techniques. The powder blends and the granules were evaluated for angle of repose, bulk density, compressibility index, total porosity, and drug content. The tablets were subjected to thickness, diameter, weight variation test, drug content, hardness, friability, disintegration time, and in vitro dissolution studies. The effect of β-CD on the bioavailability of meloxicam was also investigated in human volunteers using a balanced 2-way crossover study. Phase-solubility studies indicated an A_L-type diagram with inclusion complex of 1∶1 molar ratio. The powder blends and granules of all formulations showed satisfactory flow properties, compressibility, and drug content. All tablet formations prepared by direct compression or wet granulation showed acceptable mechanical properties. The dissolution rate of meloxicam was significantly enhanced by inclusion of β-CD in the formulations up to 30%. The mean pharmacokinetic parameters (C_max, K_e, and area under the curve [AUC]_0−∞) were significantly increased in presence of β-CD. These results suggest that β-CD would facilitate the preparation of meloxicam tablets with acceptable mechanical properties using the direct compression technique as there is no important difference between tablets prepared by direct compression and those prepared by wet granulation. Also, β-CD is particularly useful for improving the oral bioavailablity of meloxicam.     

Preparation and physicochemical characterization of 5 niclosamide solvates and 1 hemisolvate

The purpose of the study was to characterize the physicochemical, structural, and spectral properties of the 1∶1 niclosamide and methanol, diethyl ether, dimethyl sulfoxide, N,N' dimethylformamide, and tetrahydrofuran solvates and the 2∶1 niclosamide and tetraethylene glycol hemisolvate prepared by recrystallization from these organic solvents. Structural, spectral, and thermal analysis results confirmed the presence of the solvents and differences in the structural properties of these solvates. In addition, differences in the activation energy of desolvation, batch solution calorimetry, and the aqueous solubility at 25°C, 24 hours, showed the stability of the solvates to be in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > methanol solvate > dimethyl sulfoxide solvate > N,N' dimethylformamide solvate. The intrinsic and powder dissolution rates of the solvates were in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > N,N' dimethylformamide solvate > methanol solvate > dimethyl sulfoxide solvate. Although these nonaqueous solvates had higher solubility and dissolution rates than the monohydrous forms, they were unstable in aqueous media and rapidly transformed to one of the monohydrous forms.     

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (相似文献