LacZ transgenic mice and immunoelectron microscopy: an ultrastructural method for dual localization of beta-galactosidase and horseradish peroxidase.

Transgenic animals bearing the reporter gene, LacZ, encoding the histochemical enzyme, beta-galactosidase, are increasingly becoming available. Similarly, antibody conjugates consisting of specific IgGs coupled to horseradish peroxidase (HRP) are widely used for Western blotting, ELISA, and immunohistochemistry. Here we provide a detailed fixation and histochemical protocol for the simultaneous electron microscopic visualization and discrimination of beta-galactosidase and peroxidase reaction products within mouse kidney. After incubation of transgenic LacZ tissues with IgG-HRP conjugates, samples were lightly fixed with 2% paraformaldehyde and 0.4% glutaraldehyde and processed for peroxidase histochemistry. Tissues underwent beta-galactosidase histochemistry, were refixed with glutaraldehyde, osmicated, and embedded. In Flk1/LacZ mice, we immunolocalized anti-laminin beta1 chain IgG-HRP specifically to developing glomerular basement membranes, whereas Flk1/LacZ was expressed only by glomerular endothelial cells. In Epas1/LacZ mice, we immunolocalized anti-platelet endothelial cell adhesion molecule-1 specifically to glomerular endothelial plasma membranes, whereas Epas1/LacZ was expressed by both glomerular endothelial and mesangial cells. This dual ultrastructural localization technique should be broadly applicable for immunoelectron microscopic studies in LacZ transgenic animals, particularly those where LacZ expression and antibody-HRP binding are both relatively abundant.     

Cloning and Expression of the Vitreoscilla Hemoglobin Gene in Enterobacter Aerogenes: Effect on Cell Growth and Oxygen Uptake

The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxi-dative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5–2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2–5-fold higher activity for these enzymes than cells at the exponential phase.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.     

L-Arginine inhibits xanthine oxidase-dependent endothelial dysfunction in hypercholesterolemia

Xanthine oxidase (XO)-derived superoxide contributes to endothelial dysfunction in humans and animal models of hypercholesterolemia (HC). Since L-arginine supplementation prevents defects in NO signaling, we tested the hypothesis that L-arginine blunts the inhibitory effect of XO on vascular function. Acetylcholine-mediated relaxation was significantly impaired in ring segments of HC rabbits, a response that was associated with an increase in plasma XO activity. L-Arginine treatment of HC rabbits reduced plasma XO and improved endothelial function. L-Arginine also modestly prolonged the lag time for oxidation in isolated lipoprotein samples. These results reveal that the principal action of L-arginine is to protect against the XO-dependent inactivation of NO in arteries of HC rabbits.     

The sorbitol phosphotransferase system is responsible for transport of 2-C-methyl-D-erythritol into Salmonella enterica serovar typhimurium

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.     

Type II isopentenyl diphosphate isomerase from Synechocystis sp. strain PCC 6803

Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His(6)-tagged protein was produced in Escherichia coli and purified by Ni(2+) chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg(2+) for activity; K(m)(IPP) was 52 microM, and k(cat)(IPP) was 0.23 s(-1).     

Simultaneous blockade of both the epidermal growth factor receptor and the insulin-like growth factor receptor signaling pathways in cancer cells with a fully human recombinant bispecific antibody

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 degrees C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.     

Regulation of angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5, Rab7, and Rab11 GTPases

Previous studies have demonstrated that the interaction of the angiotensin II type 1A receptor (AT(1A)R) carboxyl-terminal tail with Rab5a may modulate Rab5a activity, leading to the homotypic fusion of endocytic vesicles. Therefore, we have investigated whether AT(1A)R/Rab5a interactions mediate the retention of AT(1A)R.beta-arrestin complexes in early endosomes and whether the overexpression of Rab7 and Rab11 GTPases influences AT(1A)R lysosomal degradation and plasma membrane recycling. We found that internalized AT(1A)R was retained in Rab5a-positive early endosomes and was neither targeted to lysosomes nor recycled back to the cell surface, whereas a mutant defective in Rab5a binding, AT(1A)R-(1-349), was targeted to lysosomes for degradation. However, the loss of Rab5a binding to the AT(1A)R carboxyl-terminal tail did not promote AT(1A)R recycling. Rather, it was the stable binding of beta-arrestin to the AT(1A)R that prevented, at least in part, AT(1A)R recycling. The overexpression of wild-type Rab7 and Rab7-Q67L resulted in both increased AT(1A)R degradation and AT(1A)R targeting to lysosomes. The Rab7 expression-dependent transition of "putative" AT(1A)R.beta-arrestin complexes to late endosomes was blocked by the expression of dominant-negative Rab5a-S34N. Rab11 overexpression established AT(1A)R recycling and promoted the redistribution of AT(1A)R.beta-arrestin complexes from early to recycling endosomes. Taken together, our data suggest that Rab5, Rab7, and Rab11 work in concert with one another to regulate the intracellular trafficking patterns of the AT(1A)R.     

Structure-function studies of the Vitreoscilla hemoglobin D-region

The D-region connecting helices C and E of Vitreoscilla hemoglobin (VHb) appears disordered in the crystal structure. Six site-directed mutants in this region were made to investigate its possible functions. The mutant VHb's were analyzed using UV-visible and FTIR spectroscopy, using primarily the CO liganded forms, and their heme/protein ratios were determined. The results implicate Asp44, Arg47, and Glu49 as especially important in heme-globin interactions and ligand binding, and enabled construction of a model in which the D-region forms a loop that protrudes upward over the heme. Interactions between VHb (wild type and the D-region mutants) with the flavin domain of 2,4-DNT dioxygenase from Burkholderia were tested using bacterial two-hybrid screening. There was a correlation between the extent of the D-loop perturbation predicted for each mutant and the amount of the reduction in VHb-flavin domain interaction, suggesting that this region may be more generally involved in binding of VHb to flavoproteins.