Voltage-dependent capacitance as a probe for albumin adsorption onto a solid surface

The process of adsorption of bovine serum albumin onto a platinum electrode was monitored through the measurement of a nonlinear electrochemical property. The principle of the new method is that a sinusoidal voltage source is applied to a test solution and the waveform of the output current is analyzed by Fourier transformation. It was found that the intensities of the higher harmonics in the Fourier transformation change depending on the concentration of albumin and with time. From the higher harmonics, voltage dependence of the capacitance was quantitatively evaluated. The change of the state of albumin adsorbed onto the platinum plate was also monitored from the pattern of 'crack' of adsorbed albumin by using scanning electron microscopy. These results were discussed in relation to the mechanism of bimodal adsorption of albumin.     

B-cell epitopes of autoantigenic DNA-binding proteins

Conclusions Autoantibodies to chromatin-associated proteins are frequently present in sera from patients with SLE, and related disorders. Autoantibodies to conformational epitopes may constitute the majority of the immune response to chromatin-associated antigens, suggesting that intact chromatin may be the immunogen in SLE as well as in certain forms of drug-induced lupus (eg. in procainamide-induced lupus). The preferential reactivity of autoantibodies to histones, PCNA, and Ku with antigenic determinants that are exposed on the surface of the native antigens is consistent with this interpretation.Strikingly, autoantibodies to these antigens frequently bind within or near active or functional sites, such as the DNA binding site of Ku [29], the site of PCNA critical for its role in enhancing DNA synthesis by polymerase delta [52], the posttranslational modification sites of the histones [68], and the catalytic site of poly(ADP-ribose) polymerase [69]. The explanation for the frequent observation that autoantibodies inhibit function is not yet known. It is possible that this phenomenon is related to the generation of autoantibodies by molecular mimicry, and that the functional sites of foreign antigens may crossreact with self antigens having similar functional sites [9]. Alternatively, the targeting of functional sites by autoantibodies may reflect merely a similar requirement for active sites and antibody-recognition sites to be exposed on surface. Features that make a site suitable for interacting with other proteins (eg. enzymes) or nucleic acids (eg DNA binding sites) may also make it more easily recognized by antibodies.The amino acids critical for autoantibody binding have not, in any of these cases, been shown to be critical to function. Further mapping and/ or mutagenesis studies will be necessary to determine the significance of the targeting of active or functional sites by autoantibodies.This work was supported by Public Health Service grant AR40391 from the National Institutes of Health     

Salinity tolerance and structure of external and internal gills in tadpoles of the crab-eating frog,Rana cancrivora

Summary Salinity tolerance and histology of gills were studied in Rana cancrivora larvae. The tadpoles at the external gill stages (W stages 21–22) were able to survive in media containing up to 40% seawater, but died in water of higher salinity. Their external gills appear to have no critical role in adaptation to seawater. However, advanced tadpoles with internal gills (T-K stages I–XVIII) were able to tolerate 50% or higher seawater. In the internal gills, there are numerous mitochondriarich cells (MR cells) scattered on the ventral and lateral epithelia of the gill arches and the gill tufts in both freshwater-and seawater-acclimated tadpoles. In freshwater-acclimated tadpoles there are three types of MR cell: (1) microplicated, (2) microvillous, and (3) apically vacuolated. In tadpoles acclimated to dilute seawater, the ratio of type-1 to type-2 cells is lower, although all three types of MR cell are present. In 60%-seawater-acclimated tadpoles, a few MR cells with a lumen and concave cytoplasm at the apical membrane (type 4) are present. The changes in MR cell morphology under ambient conditions of low or high salinity may reflect alterations in the physiological roles of the gills with regard to transport of ions.     

Lack of effect of 2.45-GHz microwave radiation on the development of preimplantation embryos of mice

The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm² and 19 mW/cm² on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 °C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.     

Tryptophan 5-monooxygenase from mouse mastocytoma P815. A simple purification and general properties

Tryptophan 5-monooxygenase was purified 880-fold with a 48% yield from mouse mastocytoma cells (P815) by only a one-step purification procedure of pteridine affinity chromatography. The specific activity of the final preparation was 5280 nmol min-1 mg-1. It gave a single protein band on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecylsulfate. The molecular weight of the enzyme was determined to be 270,000 by gel filtration and 280,000 by gradient polyacrylamide gel electrophoresis. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the enzyme to be composed of identical subunits with a molecular weight of 53,000. Tetrameric structure of the enzyme was suggested by cross-linking studies using dimethyl suberimidate as a bifunctional reagent. The isoelectric point of the enzyme was estimated to be 6.0. Amino acid analysis showed a residue composition similar to that reported for rat liver phenylalanine 4-monooxygenase. The enzyme activity was stimulated approximately fivefold by preincubation with dithiothreitol and Fe2+. The purified enzyme had an activity of phenylalanine hydroxylation and also a weak activity of tyrosine hydroxylation. The kinetic properties of the enzyme are also presented.     

Efficient selection of transgenic mouse embryos using EGFP as a marker gene

We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/β‐actin/EGFP fusion gene were cultured in vitro until they developed into the morulae‐ or blastocyst‐stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals. Mol. Reprod. Dev. 54:43–48, 1999. © 1999 Wiley‐Liss, Inc.     

Carcinogenicity test of 50 Hz sinusoidal magnetic fields in rats

Male and female F344 rats, 48 per exposure group, were sham exposed (Group A) or exposed to 0.5 (Group B) and 5 mT (Group C) magnetic fields for two years. Animals were exposed from 5–109 weeks of age in SPF conditions according to the OECD test guideline No. 451. Average exposure was 22.6 hr/day. No significant differences in body weight and food consumption were observed between the sham and exposed groups. At the end of the exposure period, survival rates of the male rats were 73, 83, and 79%, and those of the females, 77, 79, and 75% for Groups A, B, and C, respectively, with no significant differences between groups. Differential counts of leukocytes were measured at the 52nd, 78th, and 104th weeks of exposure and no significant differences were observed between the exposure groups. All survivors were euthanized on schedule, and all the organs and tissues suspected of tumoral lesions were examined histopathologically. Incidences of mononuclear cell leukemia in the male and the female rats were 5, 4, 4 and 8, 6, 7 for Groups A, B and C, respectively; incidences of malignant lymphoma in the female rats were 0, 1 and 1. Neither significant increases nor acceleration of incidence of leukemia were observed. Incidences of brain and intracranial tumors did not increase in the exposed groups. Incidences of both benign and malignant neoplasms showed no significant difference between the exposed and sham exposed groups with one exception: fibroma of the subcutis in the male rats, which was considered not to be a statistically significant when evaluated with respect to the historical control data in our laboratory. Bioelectromagnetics 18:531–540, 1997. © Wiley-Liss, Inc.     

High avidity cytokine autoantibodies in health and disease: Pathogenesis and mechanisms

Numerous reports have documented the presence of autoantibodies working against naturally occurring cytokines in humans in health and disease. In most instances, their physiological and pathophysiological significance remains unknown. However, recent advances in the methodologies for detecting cytokine autoantibodies and their application in research focused on specific disorders have shown that some cytokine autoantibodies play an important role in the pathogenesis of disease. Additionally, levels of cytokine autoantibodies may also correlate with disease severity and progression in certain infectious and autoimmune diseases but not in others. This suggests that cytokine-specific pathogenic differences exist. While multiple lines of evidence support the notion that high avidity cytokine autoantibodies are present and likely to be ubiquitous in healthy individuals, their potential physiological role, if any, is less clear. It is believed that they may function by scavenging pro-inflammatory cytokines and thereby inhibiting deleterious ‘endocrine’ effects, or by serving as carrier proteins, providing a ‘reservoir’ of inactive cytokines and thus modulating cytokine bioactivity. A central hypothesis is that sustained or repeated high-level exposure to cytokines triggers defects in T-cell tolerance, resulting in the expansion of existing cytokine autoantibody-producing B cells.