Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

A major histocompatibility locus in fish: serological identification and segregation of transplantation antigens in the common carp (Cyprinus carpio L.)

In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic carp showing either slow of fast rejection of skin transplants, the latter were chosen for alloantiserum production by hyperimmunization with peripheral blood leucocytes. The resulting alloantisera were analyzed for hemagglutinating reactivity with gynogenetic siblings and proved to be operationally monoscpecific in absorption experiments. The serologically determined carp erythrocyte specificites were shown to correspond to two codominantly expressed allelic products of a single locus and were designated K1 and K2, respectively. Flow cytometer analysis revealed that the same products are also present on leucocytes from peripheral blood, thymus, spleen, and pronephros.K1-andK2-homozygous second-generation gynogenetic siblings were used to study the histocompatibiligy nature of the K locus products. Skin transplants between K-allogeneic gynogenetic siblings were rejected significantly faster than within K-syngeneic combinations. Taken together, these data suggest that theK locus incorporates MHC class I-like characteristics.     

Tissue-specific expression of a vimentin--desmin hybrid gene in transgenic mice.

We have introduced a hybrid gene, pVVim2, composed of the 5' region of the hamster vimentin gene encoding the head and rod domain of vimentin and the 3' region of the hamster desmin gene encoding the tail domain of desmin, into the germ line of mice by pronuclear injection. RNA and protein analysis of mice transgenic for this construct showed that the pVVim2 gene was expressed at high levels in a developmental and tissue-specific manner. This indicates that the vimentin-derived segment of the fusion gene contains all the regulatory elements required for vimentin-specific expression. Immunohistochemical staining of fibroblast cultures derived from the transgenic mice with antibodies specific for vimentin and desmin demonstrated that the pVVim2 protein is assembled into filaments that co-localize with the endogenous vimentin filaments. The expression of pVVim2 protein in mesenchymal cells does not interfere with the function of vimentin in these cells.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol

Summary 5-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein—Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5mm AMP, 10mm magnesium chloride, 7.2mm lead nitrate, 0.1m Tris—maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8m. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4mm in pericentral zones and 0.8mm in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.     

Chromosome studies in some members of the family Caviidae (Mammalia: Rodentia)

A study of the chromosomes of Cavia aperea and Galea musteloides has been made following the introduction into captivity of these two cavies from Argentina. The evolutionary relationships of the two genera have been considered, and the possible ancestry of C. porcellus from C. aperea was investigated in hybrids of the two species.     

The effect of intrinsic curvature on supercoiling: Predictions of elasticity theory

Elasticity theory of naturally curved rods is employed to study the effects of intrinsic curvature on the properties of the equilibrium conformations of supercoiled DNA. The results stand in sharp contrast to those obtained when the molecule is viewed as being straight in its relaxed form. Starting from very fundamental principles of the theory, we show that the torsion of an open segment with a curved duplex axis can vary when the temperature, and along with it, the intrinsic twist is changed. Conversely, an imposed helicity, such as might be associated with binding to a histone, can change the intrinsic twist. It is also shown that another consequence of the presence of naturally curved sequences is that the twist density will, in general, vary with position along the chain in all equilibrium states. Then portions of the molecule will be more or less susceptible to interaction with other agents sensitive to such a variation. Finally, some closed equilibrium global structures uniquely associated with intrinsic curvature are discussed. © 1993 John Wiley & Sons, Inc.     

Mapping of susceptibility to Marek's disease within the major histocompatibility (B) complex by refined typing of White Leghorn chickens

The major histocompatibility (B) complex of a distinct commercial pure White Leghorn chicken line was characterized using serological, biochemical and restriction fragment length polymorphism (RFLP) typing. Line B chickens displayed a high recombination frequency within the B complex. Three recombinant haplo-types were identified. The influence of these haplotypes was determined in relation to the haplotypes B^l9 and B²¹ on their resistance to Marek's disease (MD) in an experimental infection with the virus. Offspring of sires with a recombinant haplotype in combination with B¹⁹ or B²¹, and dams, which were homozygous B¹⁹/B¹⁹ or B²¹/B²¹ were infected. The B type of the offspring had a significant effect upon survival. Animals with B complex types B²¹/B²¹, B¹³⁴/B²¹ and B²³⁴/B²¹ were relatively resistant to MD (24–32% mortality), whereas B¹⁹/B¹⁹ birds were highly susceptible (68% mortality). Animals with a recombinant halpotype B^19r21 (B-G²¹, B-F¹⁹) were equally susceptible to MD as birds with the complete B¹⁹ haplotype. In contrast to earlier publications, resistance was not inherited as a dominant trait. Apparently, B¹⁹ was associated with a dominant susceptibility. The gene(s) associated with the B complex and involved in resistance to MD were localized within the B-F/B-L region. However, the association with a presumably non-coding subregion of B-G could not be excluded.