Glycosylation of catalase inhibitor necessary for activity

A protein isolated from maize scutella which inhibits catalase in vitro has been shown to contain 12% carbohydrate in the form of galactose. This corresponds to four galactose molecules per inhibitor subunit. Removal of the carbohydrate with β-galactosidase or blockage with a galactose-specific lectin abolished activity of the inhibitor.     

Optimizing purebred selection for crossbred performance using QTL with different degrees of dominance

A method was developed to optimize simultaneous selection for a quantitative trait with a known QTL within a male and a female line to maximize crossbred performance from a two-way cross. Strategies to maximize cumulative discounted response in crossbred performance over ten generations were derived by optimizing weights in an index of a QTL and phenotype. Strategies were compared to selection on purebred phenotype. Extra responses were limited for QTL with additive and partial dominance effects, but substantial for QTL with over-dominance, for which optimal QTL selection resulted in differential selection in male and female lines to increase the frequency of heterozygotes and polygenic responses. For over-dominant QTL, maximization of crossbred performance one generation at a time resulted in similar responses as optimization across all generations and simultaneous optimal selection in a male and female line resulted in greater response than optimal selection within a single line without crossbreeding. Results show that strategic use of information on over-dominant QTL can enhance crossbred performance without crossbred testing.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Nonenzymatic isolation and culture of adult islets from atrophic pancreata of copper-deficient rats: A morphologic analysis

Summary The purpose of this study was to develop a nonenzymatic method of isolating adult islets using atrophied pancreata from copper-deficient rats and to analyze their morphologic characteristics and behavior in culture. This unusual model of isolation was studied because islets remain intact in the course of dietary copper deficiency while the acinar glandular component of the pancreas undergoes selective atrophy and lipomatosis. Small fragments containing islets were readily microdissected from atrophied glands and placed in culture. Within 24 h the fragments congealed into small irregular- to spherical-shaped masses within which the darker profile of islets could be distinguished. Within a period of 3 to 5 d, islet tissue began to bud from the lipocytic mass until by Day 7 spherical aggregates of intact islet tissue separated from the residual fragments. Subsequent to further in vitro treatment, these islets could be maintained as free viable spherical masses if periodically agitated, as attached stationary islets which developed monolayer growth if left undisturbed and as aggregated masses of islet tissue forming megaislets if combined in small groups. Grouped islets treated with actinomycin D and cycloheximide did not exhibit aggregation when incubated with these inhibitors. This suggests that megaislet formation was an active process requiring protein-RNA synthesis rather than passive clumping or aggregation that can accompany metabolically altered or dying islets undergoing cellular shedding and adhesion. Immunohistochemical localization demonstrated that insulin, glucagon, somatostatin, and pancreatic polypeptide-immunoreactive cell types were present within the islets derived from this technique. The cellular topography of these islets was not unlike that described by others for islets cultured from enzymatic isolation. This culture model may serve as a resource for mature, viable islets isolated without mechanical or enzymatic disaggregation which can have attenuating effects on islet function. This work was supported by a research grant from the Diabetes Research and Education Foundation.     

Copper(II) (3,5-diisopropylsalicylate)2 oxidizes thiols to symmetrical disulfides and oxidatively converts mixtures of 5-thio-2-nitrobenzoic acid and nonsymmetrical 5-thio-2-nitrobenzoic acid disulfides to symmetrical disulfides

L-cysteine, D-penicillamine, and L-glutathione were oxidized to symmetrical disulfides in the presence of Cu(II)(3,5-DIPS)2 and air-oxygen at physiologic pH, 7.3. Air-oxygen caused the oxidation of thiol reduced copper, Cu(I), to Cu(II), as evidenced by expected spectrophotometric changes in these reaction mixtures. L-cysteine, D-penicillamine, and L-glutathione formed mixed disulfides and TNB with the addition of DTNB to solutions of these thiols. The observed order of reactivity for these thiols with DTNB was: L-cysteine greater than D-penicillamine greater than L-glutathione. Surprisingly, Cu(II)(3,5-DIPS)2 converted these mixed disulfides to their symmetrical disulfides and DTNB, and although the initial conversion rate was rapid, complete conversion required more than two hours. These observations suggest caution with regard to the spectrophotometric determination of thiols immediately after the addition of Ellman's reagent. These results also clarify an earlier report concerning the oxidation of thiols by Cu(II)(o-phenanthroline)2 and offer caution with regard to the determination of thiols using DTNB in the presence of copper complexes. Spectrophotometric data are provided in support of the suggestion that analysis of plasma or cellular samples for thiols be done in the absence of copper(II) complexes to avoid false negative results.     

Effects of CuDIPS and tween 80 on SJL/J tumorigenesis

Plasma copper and zinc levels were measured in SJL/J mice, an inbred strain characterized by a high, spontaneous incidence of reticulum cell sarcoma (RCS). The changes with age in mean concentrations of these metals were consistent with a physiological response that is required for remission of neoplasia. Treatment of SJL/J mice with a copper complex, Cu(II)(3,4-diisopropylsalicylate)₂ (Cu 3,5-DIPS), dissolved in a 10% Tween 80-saline solution revealed a decrease in survival and decline in the incidence of RCS at 52 wk of age. The toxic effects of Cu 3,5-DIPS therapy appeared to be related to the intraperitoneal route of administration and to extracellular deposition of collagen. The inhibitory effect on tumor development was not related to Cu 3,5-DIPS. Rather, Tween 80 was found to be the factor of importance.     

Production of aflatoxin on rice

A method has been developed for the production of aflatoxin by growing Aspergillus flavus strain NRRL 2999 on the solid substrate rice. Optimal yields, more than 1 mg of aflatoxin B₁ per g of starting material, were obtained in 5 days at 28 C. A crude product containing aflatoxins was isolated by chloroform extraction and precipitation with hexane from concentrated solutions. The crude product consisted of 50% aflatoxin in the following ratio: B₁-B₂-G₁-G₂, 100:0.15:0.22:0.02. Aflatoxin B₁ was separated from almost all the impurities and from the other aflatoxins by chromatography on silica gel with 1% ethyl alcohol in chloroform. Analytically pure aflatoxin B₁ was recrystallized from chloroform-hexane mixtures.     

The Human Serum Paraoxonase/Arylesterase Gene (PON1) Is One Member of a Multigene Family

A physiological role for paraoxonase (PON1) is still uncertain, but it catalyzes the hydrolysis of toxic organophosphates. Evidence that the human genome contains twoPON1-like genes, designatedPON2andPON3,is presented here. HumanPON1andPON2each have nine exons, and the exon/intron junctions occur at equivalent positions.PON1andPON2genes are both on chromosome 7 in human and on chromosome 6 in the mouse. Turkey and chicken, like most birds, lack paraoxonase activity and are very susceptible to organophosphates. However, they have aPON-like gene with 70% identity with humanPON1, PON2,andPON3.Another unexpected finding is that the deduced amino acid sequences of PON2 in human, mouse, dog, turkey, and chicken and of human PON3 are all missing the amino acid residue 105, which is lysine in human PON1. The expanded number ofPONgenes will have important implications for future experiments designed to discover the individual functions, catalytic properties, and physiological roles of the paraoxonases.