Diffusion Tensor Imaging of Brain Abnormalities Induced by Prenatal Exposure to Radiation in Rodents

We assessed brain abnormalities in rats exposed prenatally to radiation (X-rays) using magnetic resonance imaging (MRI) and histological experiments. Pregnant rats were divided into 4 groups: the control group (n = 3) and 3 groups that were exposed to different radiation doses (0.5, 1.0, or 1.5 Gy; n = 3 each). Brain abnormalities were assessed in 32 neonatal male rats (8 per group). Ex vivo T₂-weighted imaging and diffusion tensor imaging (DTI) were performed using 11.7-T MRI. The expression of markers of myelin production (Kluver–Barrera staining, KB), nonpyramidal cells (calbindin-D28k staining, CaBP), and pyramidal cells (staining of the nonphosphorylated heavy-chain neurofilament SMI-32) were histologically evaluated. Decreased brain volume, increased ventricle volume, and thinner cortices were observed by MRI in irradiated rats. However, no abnormalities in the cortical 6-layered structure were observed via KB staining in radiation-exposed rats. The DTI color-coded map revealed a dose-dependent reduction in the anisotropic signal (vertical direction), which did not represent reduced numbers of pyramidal cells; rather, it indicated a signal reduction relative to the vertical direction because of low nerve cell density in the entire cortex. We conclude that DTI and histological experiments are useful tools for assessing cortical and hippocampal abnormalities after prenatal exposure to radiation in rats.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.     

Isolation of morphologically and functionally intact gastric mucosal microvessels rapid communication.

Gastric mucosal microvessels were isolated after arterial perfusion of the rat stomach with magnetized iron oxide suspension. After homogenization of scrapped gastric mucosa, microvessels were initially separated with a high power magnet and further separated and purified by using a nylon sieve. Aliquots of purified microvessels were assessed for viability, histologic appearance, ultrastructure and generation of prostacyclin. Microvessels were plated on Matrigel and cultured in DMEM with high glucose and 10% FBS for 1, 3 or 5 days. After 1, 3 and 5 days of culturing, endothelial viability was assessed with Fast green exclusion, and the basal and stimulated (with calcium ionophore) generation of prostacyclin was determined by assaying aliquots of the incubating medium for 6-keto PGF(1alpha). At 1 and 3 hrs after isolation, microvessels demonstrated intact morphologic structures as reflected by transmission EM and 92+/-4% of viable endothelial cells. The microvessels plated on Matrigel maintained good viability for at least 5 days and generated prostacyclin at the baseline and following ionophore stimulation. These data demonstrate that isolated microvessels cultured under optimal conditions are fully viable and functional.     

Comparison of an antiviral activity of recombinant consensus interferon with recombinant interferon-alpha-2b

To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-alpha-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-alpha-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.     

Occurrence of singlet oxygen oxygenation of oleic acid and linoleic acid in the skin of live mice

To assess the contribution of singlet molecular oxygen [O₂ (¹Δ_g)] to lipid peroxidation in vivo, this study combined gas chromatography-mass spectrometry with thin layer chromatography to analyse peroxidized lipids in the skin of hairless mice. Hydroxyoctadecenoate isomers and unconjugated hydroxyoctadecadienoate isomers derived from peroxidized oleic acid and linoleic acid, respectively, which are specific to O₂ (¹Δ_g)-dependent oxygenation, were detected in the skin of live mice under ordinary feeding conditions. Short-term ultraviolet A (UVA)-irradiation of the skin in vivo elevated levels of the unconjugated hydroxyoctadecadienoate isomers significantly, whereas the irradiation of skin homogenate in vitro increased levels of all isomers derived from both O₂ (¹Δ_g) and free radical-dependent oxygenation to a much greater extent. This is the first report to demonstrate the occurrence of O₂ (¹Δ_g)-specific oxygenation of unsaturated fatty acids in living animals.     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Thermodynamic analysis of the effect of concentrated salts on protein interaction with hydrophobic and polysaccharide columns

An attempt was made to explain the effect of concentrated salts on protein interaction with hydrophobic columns. From the previously observed results of preferential interactions for salting-out salts with proteins, it was shown that the free energy of the protein is increased by addition of the salts and this unfavorable free energy is smaller for the proteins bound to the columns because of their smaller surface area exposed to solvent; i.e., the bound form of the proteins is thermodynamically more stable. This explains the protein binding to the hydrophobic columns at high salt concentrations and the elution by decreasing the salt concentration. The unfavorable interaction free energy was greater for Na2SO4 or (NH4)2SO4 than for NaCl, which explains the stronger effect of the former salts on the protein binding to the columns. The observed favorable interaction between KSCN or guanidine hydrochloride and the proteins explains the decreasing effect of these salts on the protein binding to the hydrophobic columns.     

Development of a highly sensitive chemiluminescent assay for hydrogen peroxide under neutral conditions using acridinium ester and its application to an enzyme immunoassay

We developed a highly sensitive chemiluminescent (CL) assay for hydrogen peroxide using 10‐methyl‐9‐(phenoxycarbonyl) acridinium fluorosulfonate (PMAC) that produced chemiluminescence under neutral conditions and applied it to an enzyme immunoassay (EIA). One picomole of hydrogen peroxide could be detected using the optimized PMAC‐CL method and 6.2 × 10^‐20 mol β‐d ‐galactosidase (β‐gal) could be detected by combining an indoxyl derivative substrate and the proposed PMAC‐CL method. This highly sensitive CL β‐gal assay was applied to an EIA for thyroid‐stimulating hormone (TSH) using β‐gal as a label enzyme; 0.02–100.0 μU/mL TSH in human serum could be assayed directly and with high reproducibility. Copyright © 2013 John Wiley & Sons, Ltd.