The effect of three α-keto acids on 3-mercaptopyruvate sulfurtransferase activity

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three α-keto acids (α-ketobutyrate, α-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the α-keto acid. However, it has also been proposed that α-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three α-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three α-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant K_i was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I₅₀, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three α-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanisms of the α-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate. © 1996 John Wiley & Sons, Inc.     

Ecology of two geographically restricted Astragalus species (Fabaceae), A. bibullatus and A. tennesseensis, of the eastern United States

Astragalus bibullatus is endemic to limestone glades in the Central Basin of Tennessee, and except for one population in Illinois,A. tennesseensis is endemic to limestone glades in the Central Basin and in the Moulton Valley of Alabama. However,A. tennesseensis has been extirpated from four counties in Illinois, from its only known site in Indiana, from one county in Tennessee, and from one county in Alabama.Astragalus bibullatus is closely related to the geographically wide-spread Great Plains taxonA. crassicarpus var.crassicarpus (sectionSarcocarpi), whereasA tennesseensis is the only taxon in sectionTennesseensis. Both species are shallow-rooted, hemicryptophyte perennials without vegetative reproduction, have no effective means of seed dispersal, form long-lived seed banks, have similar life cycle phenologies, are intolerant of heavy shade, and have moderate amounts of genetic diversity. Much. additional information is available on the autecology ofA. tennesseensis. Its primary habitat is the transition zone between open glades and glade woods, where physical environmental factors are intermediate between those of the adjacent zones. Seedling-juvenile survival is low. Plants flower first in their second to fifth year and only a few times before dying, are self-incompatible, respond to drought by shedding leaves and by accumulating large amounts of proline, and compete poorly. Populations exhibit high fluctuations in number of individuals and have high turnover rates. A greenhouse study ofA bibullatus andA crassicarpus var.crassicarpus did not identify any difference in responses of these species to light or soil moisture that could account for the great differences in geographic ranges. Thus, historical factors were also considered to explain the narrow endemism ofA. bibullatus. Finally, we present previously unpublished data on the effect of light level and of competition with the cedar glade dominantSporobolus vaginiflorus in several watering regimes on growth ofA. tennesseensis. The role of these factors in restriction of this species to its transition zone microhabitat is discussed.     

Depressed β-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients’ induced pluripotent stem cell–derived cardiomyocytes

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca²⁺-handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to β-adrenergic stimulation. To test the hypothesis, [Ca²⁺]_i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca²⁺ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca²⁺ signal amplitude and recovery time. In support of the depleted SR Ca²⁺ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the β1-adrenergic receptor (ADRβ1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed β-adrenergic responsiveness.     

CHANGE IN DORMANCY STATUS OF FRASERA CAROLINIENSIS SEEDS DURING OVERWINTERING ON PARENT PLANT

Seeds of the monocarpic perennial Frasera caroliniensis ripen in late summer, and most of them are dispersed in late autumn and winter. However, some viable seeds may remain undispersed for more than a year. Embryos are underdeveloped (ca. 1.1–1.3 mm long) at seed maturity and do not grow while seeds remain on plants in the field. Dormancy in freshlymatured seeds was broken by 12 to 14 weeks of cold stratification at 5 C, during which the embryos elongated. On the other hand, seeds collected in January and March required a period of warm stratification followed by a period of cold stratification to germinate. Seeds collected in September and sown in a nonheated greenhouse germinated to 83% the first spring after maturation, whereas those collected and sown in January and March did not germinate until the second spring. Thus, seeds that remained on plants in the field until winter entered a deepened state of dormancy, and a warm (summer) followed by a cold (winter) stratification period was required to overcome it.     

IL-1 beta modulation of spontaneous autoimmune diabetes and thyroiditis in the BB rat

Long term effects of in vivo treatment with human rIL-1 beta on diabetogenesis and thyroid disease were determined in the Biobreeding rat. Administration of high dose (10 micrograms/kg) IL-1 beta accelerated the onset of insulin-dependent diabetes mellitus compared to saline-injected controls. High dose treatment resulted in goiter development, pronounced LT, reduced serum T4 levels, and overall growth reduction. In contrast, low dose IL-1 beta (0.5 microgram/kg) administration significantly reduced the frequency of insulin-dependent diabetes mellitus (48%) compared to placebo (86%) and high dose IL-1 beta (93%) treatment groups. Rats protected by low dose IL-1 beta had unaffected growth rates and minimal to no pancreatic and thyroid pathology. Our results demonstrate that exogenous administration of IL-1 beta modulates Biobreeding rat idiopathic autoimmune diabetes and thyroid disease in a dose-dependent manner.     

Thymus-dependent antiidiotype and anti-antiidiotype responses to a dinitrophenyl-specific monoclonal antibody

BALB/c mice were inoculated i.p. with graded doses of a DNP-specific, IgM mAb (designated 57.1). Injection with unmodified 57.1 in the absence of adjuvants resulted in the generation of an anti-Id response (Ab2) and an anti-anti-Id response (Ab3). The generation of serum anti-Id antibodies was found to be thymus dependent. Nude mice immunized with 57.1 were unable to produce a serum Ab2 response above nonimmunized controls whereas euthymic mice receiving identical doses of 57.1 produced strong Ab2 responses. To examine the specificity of serum anti-Id, sera from mice receiving 57.1 were screened against a panel of mAb representing at least five distinct VH gene families. Serum titers were significantly higher against 57.1 than against any of the other antibodies in the panel. Three of the antibodies in this panel bind FD5-1, a monoclonal anti-Id (Ab2) that also binds 57.1. However, sera from mice receiving 57.1 bound 57.1 only. Thus, the serum Ab2 response appears to be highly specific for idiotopes on 57.1. The predominant isotype of these anti-Id antibodies was IgG1. The number of isotypes detected increased in a dose dependent manner with all IgG subclasses having anti-Id specificity in sera from animals receiving the higher doses of 57.1. Further analysis of the serum demonstrated that approximately 8% of the Ab2 response was paratope-specific (inhibitable by the monovalent hapten DNP-lysine). The same sera were analyzed for the presence of Ab3 by binding to the monoclonal anti-Id antibody FD5-1. Lower serum titers of Ab3 were generated in comparison to serum titers of Ab2. Analysis of the binding specificity of the Ab3 response revealed that DNP-BSA was able to partially inhibit the binding of serum IgM and IgG Ab3 to FD5-1. A subset of the Ab3 response. Ab1' that is specific for DNP was observed in a direct binding assay where detectable amounts of DNP binding IgM, IgG1, and IgG3 isotypes were present. We have thus described a complete circuit (Ab1----Ab2----Ab3) of antibodies within the Id network by immunizing animals with an unmodified mAb in the absence of Ag or adjuvants.     

Rigorous analysis of light diffraction by a striated muscle fibre.

A rigorous theory describing the diffraction of light by a muscle fibre has been formulated. The basis of this analysis is the rigorous coupled-wave approach of T. K. Gaylord & M. G. Moharam (Proc. IEEE 73, 894 (1985)); however, we obtain here a closed-form analytical result that is both mathematically simple and physically easy to understand. We have compared our results on striated muscle fibres with the analytical results obtained by A. F. Huxley (Proc. R. Soc. Lond. B 241, 65 (1990)) using the normal mode approach, and with those obtained by R. A. Thornhill, N. Thomas & N. Berovic (Eur. Biophys. J. 20, 87 (1991)) using a multiwave first-order coupled-wave approximation. For an equivalent set of assigned fibre parameters, our results are consistent with these mentioned. Extension of this analysis to a fibre with different structures showed that the differences in diffraction efficiencies of different orders for a frog skeletal fibre and for an insect flight fibre are clear; the sensitivity to distinct structural organization of the fibre is very good.     

Does dormancy protect seeds against attack by the pathogenic fungus <Emphasis Type="Italic">Fusarium tricinctum</Emphasis> in a semiarid grassland of Northwest China?

Aims

Soil fungal pathogens can result in the failure of seedling establishment, but the effects of fungicide applications on seed/seedling survival have differed among studies. We assumed that the variation may relate to seed dormancy/germination characteristics and hypothesized that nondormant germinating seeds are more likely to be killed by fungal pathogens than dormant seeds.

Methods

Dormant and nondormant seeds of Stipa bungeana and Lespedeza davurica were inoculated with a pathogenic fungus Fusarium tricinctum under laboratory and field conditions. The outcomes of seed/seedling fate and other parameters were evaluated.

Results

In the laboratory, nondormant seeds inoculated with F. tricinctum developed white tufts of mycelium on the radicles of germinating seeds causing them to quickly die, but dormant seeds remained intact. In contrast, in the field inoculation with F. tricinctum did not cause higher mortality of nondormant than dormant seeds but resulted in higher percentages of seedling death before they emerged from soil than the controls.

Conclusions

Our results suggest that dormancy protects seeds from being attacked by some pathogens by preventing germination, but the protection is lost once germination has commenced. Further study involving various plant species with more seeds is needed to assess the generality of this pathogen-seed interaction hypothesis.

     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.