Charles J. Epstein

A convenient method of preparing polyacrylamide gels for liquid scintillation spectrometry.

Abstract A convenient method is described for the preparation for liquid scintillation spectrometry of slices of polyacrylamide gel containing isotopically labeled protein. Slices in counting vials are incubated with hydrogen peroxide (0.1 ml) at 50°. Complete solubilization of the gels occurs in several hours, and the aqueous material is combined with toluene-based scintillation fluid by the addition of NCS (1.0 ml). Efficiencies of counting are extremely high, and recoveries of radioisotope usually exceed 90%.

Brain injury after perinatal hypoxia-ischemia is exacerbated in copper/zinc superoxide dismutase transgenic mice

The role of superoxide radical formation in the pathogenesis of perinatal hypoxic-ischemic injury was examined using transgenic (Tg) mice expressing three times normal amounts of copper/zinc-superoxide dismutase (CuZn/SOD). Fourteen litters of postnatal d 7 strain 218/3 mice were subjected to right common carotid artery ligation followed by 90 min of hypoxia in an 8% oxygen/humidified chamber maintained at 37°C. Both Tg mice (n = 32) and their nontransgenic (nTg) littermates (n = 30) survived the injury equally. Evaluation of infarcted brain areas measured by video image analysis of three coronal brain sections through the anterior hippocampus from each animal revealed that the Tg animals suffered brain infarction more frequently than did nTg mice. Blinded histologic scoring of cerebral cortex and striatum 5 d after injury revealed that Tg mice were more likely to have higher histologic severity scores than their nTg littermates (p = 0.0463, Mann-Whitney U test). These findings suggest that brain injury in perinatal hypoxia-ischemia may be mediated in part by free radical formation from excessive hydrogen peroxide or nitric oxide production.

Genetic Dissection of Region Associated with Behavioral Abnormalities in Mouse Models for Down Syndrome

Two animal models of Down syndrome (human trisomy 21) with segmental trisomy for all (Ts65Dn) or part (Ts1Cje) of human chromosome 21-homologous region of mouse chromosome 16 have cognitive and behavioral abnormalities. To compare these trisomies directly and to assess the phenotypic contribution of the region of difference between them, Ts65Dn, Ts1Cje, and a new segmental trisomic (Ms1Ts65) for the region of difference (App to Sod1) have been generated as littermates and tested in parallel. Although the performance of Ts1Cje mice in the Morris water maze is similar to that of Ts65Dn mice, the reverse probe tests indicate that Ts65Dn is more severely affected. By contrast, the deficits of Ms1Ts65 mice are significantly less severe than those of Ts65Dn. Therefore, whereas triplication of Sod1 to Mx1 plays the major role in causing the abnormalities of Ts65Dn in the Morris water maze, imbalance of App to Sod1 also contributes to the poor performance. Ts65Dn mice are hyperactive and Ts1Cje mice are hypoactive; the activity of Ms1Ts65 mice is not significantly above normal. These findings indicate that genes in the Ms1Ts65 trisomic region must interact with others in the Ts1Cje region to produce hyperactivity in Ts65Dn mice.

X-chromosome inactivation during differentiation of female teratocarcinoma stem cells in vitro.

Evidence is presented that both X chromosomes are genetically active in clonal cultures of undifferentiated female mouse teratocarcinoma stem cells derived from a spontaneous ovarian tumour. As the cells differentiate in vitro one of the X chromosomes becomes inactivated.

Spectral studies on the denaturation of myoglobin

Absorption and fluorescence spectroscopy and optical rotation measurements have been used to follow the denaturation of sperm whale skeletal muscle and horse heart myoglobins by urea and guanidine hydrochloride. The spectral properties of the metmyoglobin forms of each of these proteins change concomitantly during denaturation, and the transitions are compatible with a one-step, cooperative denaturation process. The apomyoglobins from sperm whale and horse undergo transitions at lower concentrations of denaturants than do their respective metmyoglobins, and the data are again compatible with a one-step process. For both metmyoglobins and apomyoglobins, the protein from the sperm whale is significantly more stable than that from the horse. These results are discussed with respect to the chemical bases of the spectral properties, the location and relationship of the chromophores in the atomic model of the protein, and the forces which lead to structural stability.

Preimplantation lethality of monosomy for mouse chromosome 19.

IT remains a mystery why autosomal trisomy is found more frequently than autosomal monosomy in man. Several surveys of spontaneous abortions have revealed these chromosomal abnormalities in proportions of 26% (ref. 1) and 0.1% (refs 1, 2), respectively, while a prospective study of 47,000 newborn babies revealed no full monosomy and only five cases in which parts of chromosomes were missing3. Similarly, in mouse embryos from translocation stocks bred to produce aneuploid progeny, high frequencies of trisomy but no monosomy are found after 12–13 days4,5. This is hard to understand because most autosomal aneuploidy is assumed to result from meiotic nondisjunction, so that for every gamete carrying two copies of a particular chromosome there should be a complementary gamete carrying none. However, the deficiency of monosomic embryos could be because nullisomic gametes are less able than disomic gametes to participate in fertilisation, or because the resulting monosomic embryos are less viable early in gestation. We report here the investigation of these possibilities in a mouse system which is known to produce a high frequency of nondisjunction. Equal numbers of monosomic and trisomie progeny were found early in gestation, but while some monosomies were compatible with survival to implantation, monosomy for the smallest mouse autosome, chromosome 19, was lethal at the early to mid-blastocyst stage. This lethality did not seem to be the result of the expression of recessive lethal genes.

AN APPROACH TO THE PROBLEM OF CONFORMATIONAL ISOZYMES

A. Evolutionarily unrelated: “convergent” evolution B. Evolutionarily related 1. Genetically unrelated: “divergent” evolution of duplicated genes 2. Genetically related a. Covalent differences 1) Introduced during translation 2) Introduced after translation a ) Deamination b) Attachment of carbohydrate c ) Phosphorylation, sulfation d) aand c-NH2 acetyls, formyls, Schiff‘s bases e ) Oxidation of sulfhydryl groups f ) Oxidation and reduction of prosthetic group g) Cleavage of peptide chain b. Mixed multimers c . Noncovalent differences 1) Aggregation 2) Binding of small molecules 3) “Stable” conformational variants Recently, many investigators have invoked the concept of stable variations in three-dimensional structure, or conformation, to explain the existence of certain sets of proteins, especially enzymes. These conformational variants are distinct from all other types of isozymes in that their origin does not involve differences in the covalent structures of the molecules or detectable alterations of their molecular weights. I t is assumed that the only differences among conformational isozymes, or “conformers” as they have recently been termed,‘ are the precise ways in which the polypeptide chains are folded. I t is now generally believed that the conformation of a protein in solution 2 9

Lipid peroxidation and superoxide dismutase-1 and glutathione peroxidase activities in trisomy 16 fetal mice and human trisomy 21 fibroblasts

ABSTRACT: An increase in lipid peroxidation has been reported in fetal human trisomy 21 brains. To determine whether this change can be regarded as a consequence of the increase in soluble Cu, Zn-superoxide dismutase (SOD-1) activity caused by the trisomy, we have made use of the trisomy 16 mouse, a model for human trisomy 21. Lipid peroxidation, as malonaldehyde, and the activities of SOD-1 and glutathione peroxidase were studied in diploid and trisomy 16 mouse fetuses and fetal brains and, for comparison, in diploid and trisomy 21 human fibroblasts. SOD-1 activity in diploid mouse brain increased during fetal and postnatal development, but glutathione peroxidase activity was unchanged. Mean SOD-1 activity was almost exactly 50% increased in trisomy 16 fetuses and fetal brains and in human trisomy 21 fibroblasts, confirming the gene dosage effect in both species. The SOD-1 activity in the trisomic fetuses was correlated with that in their matched diploid littermates, suggesting that factors other than the gene dosage also determine activity. Mean glutathione peroxidase activity was not increased in trisomy 16 fetuses or brains and only slightly increased in human trisomy 21 fibroblasts. Mean lipid peroxidation was decreased in fetal trisomy 16 brains but was increased in human trisomy 21 fibroblasts. These results do not lend support to the notion that increased SOD-1 activity is developmentally deleterious and necessarily increases lipid peroxidation and, secondarily, the activity of glutathione peroxidase. The difference between the human and mouse data concerning lipid peroxidation in trisomic brains may be related to structural differences in the lipids which provide the substrate for lipid peroxidation.

Mitochondrial Malate Dehydrogenase: Reversible Denaturation Studies

The malate dehydrogenase isoenzymes from the mitochondria of chicken hearts have been partially resolved into separate pools. Reversible denaturation, in concentrated guanidine hydrochloride, does not change the isoenzyme distribution in each pool. This result suggests that the electrophoretic differences among the isoenzymes are not solely conformational in origin.

Role of CuZn superoxide dismutase in regulating lymphocyte apoptosis during sepsis.

Objective The lymphocyte is a principal mediator of the inflammatory response, and lymphocyte depletion via apoptosis may be an important mechanism of modulating inflammation. Increased oxygen consumption occurs during sepsis and results in the generation of reactive oxygen species. Although reactive oxygen species initiate apoptosis in many biological systems, their role in controlling lymphocyte apoptosis during sepsis is unclear. The objective of this study was to better characterize the role of oxidative stress in precipitating lymphocyte apoptosis during sepsis and to specifically define the role of the CuZn superoxide dismutase (SOD) enzyme complex, a major antioxidant defense, in modulating this process. Design Prospective, randomized, controlled study. Setting Research laboratory at an academic medical center. Subjects Mice that were either genetically normal or that were deficient in or overexpressed the enzyme CuZn SOD. Interventions Mice from each genetic group were randomized to no manipulation (control), sham surgery, or cecal ligation and puncture. Mice were killed 18–24 hrs after study entry, and the thymi and spleen were removed for analysis of apoptosis. Measurements and Main Results Lymphocyte apoptosis was assessed by three independent methods: light microscopy, fluorescent terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and DNA gel electrophoresis. Comparisons were performed using standard parametric statistical tests. Lymphocyte apoptosis was present in mice after CLP but not in control mice or in mice after sham surgery (p < .05). Mice completely lacking CuZn SOD developed significantly more lymphocyte apoptosis than did either partially CuZn SOD-deficient or genetically normal mice (p < .05). This apoptosis was more pronounced in the thymus than the spleen and, within the thymus, more prominent in the cortex than medulla (p < .05 for all). In contrast, mice that overexpressed CuZn SOD did not differ in the amount of apoptosis after CLP compared with genetically normal mice (p = NS for all). Conclusions Oxidative stress occurs in sepsis and appears to be one stimulus for the development of lymphocyte apoptosis, a process that is partly regulated by CuZn SOD. However, we were unable to demonstrate that overexpression of this enzyme suppressed lymphocyte apoptosis, suggesting that either other antioxidant defenses or other pathways independent of oxidative stress may mediate lymphocyte elimination in this syndrome.