Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from Dihydroneopterin triphosphate

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed enzyme A (MW 82,000) and enzyme B (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl₂, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K _m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (PCM75-19513 A02). G. G. K. was supported as a predoctoral trainee by National Institutes of Health Training Grant GM00515.     

Lysinonorleucine cross-link formation in alpha amino heptenoic acid-substituted peptide derivatives

Studies on the cross-linking of a tripeptide (t-butyloxycarbonyl-L -alanyl-D ,L -2-amino-6-heptenoyl-L -alanine methyl ester) have shown that it is possible to form specific cross-links in good yields through Schiff base formation of the ε amino group of lysine. The heptenoic acid residue has been ozonized to an aldehyde and condensed with the ε amino of lysine in the compounds alpha-t-butyloxycarbonyl-L -alanyl-L -lysine methyl ester and alpha-t-butyloxycarbonyl-L -lysine methyl ester to form the cross-link, lysinonorleucine. This compound has been stabilized by reduction with sodium borohydride and quantitated on the amino acid analyzer. This technique converts from 60 to 98% of the available aldehyde to lysinonorleucine.     

Characteristics of the induction of aldehyde dehydrogenase in rat liver

The activity of rat liver supernatant aldehyde dehydrogenase is increased by phenobarbital treatment in one selected strain (RR) but not in another strain (rr) of animals derived from randomally bred populations (Deitrich, Collins, and Erwin (1972) J. Biol. Chem., 247, 7232). Before 14 days of age, increased enzyme activity after phenobarbital treatment is minimal but between 30 and 60 days of age there is a maximal increase in activity after phenobarbital treatment. Using animals of this age, it was shown that both cycloheximide and actinomycin D block this response to phenobarbital. Phenobarbital treatment decreases heat stability of crude preparations of the enzyme from RR rats, but increases heat stability of the enzyme from rr animals.     

Perturbation analysis of competition and overlap in habitat utilization between Dipodomys ordii and Dipodomys merriami

Summary The populations of two coexisting species of Dipodomys (Heteromyidae, Rodentia) were manipulated on 10, large, unenclosed, trapping grids. These manipulations revealed that, although many kangaroo rats are established residents in an area, a large number are transient individuals who quickly occupy vacated habitats. On plots from which residents had been removed, transients settled at rates of up to 5% of carrying capacity per day. These immigrants were invariably of the same species that was removed, indicating a strong element of intraspecific competition with little or no evidence of competition between the species.Trapping records suggest that these species avoid competition through habitat selection. Dipodomys ordii prefer a grassier habitat, and D. merriami a habitat dominated by creosote bush. Apparent overlap in their utilization of habitats, based on sites of capture, predicts competition coefficients to be higher than those permitted by the theory of limiting similarity and much higher than those actually shown by the perturbation experiments.This study demonstrates the dangers of estimating alpha without experimentation. This is especially true in cases where habitat selection may be important, since organisms may travel in habitats without collecting resources therein. Our results are discussed in light of a theory which examines the optimal (rather than tolerable) amount of overlap in habitat utilization between two potential competitors in a mixed habitat. This theory predicts that the pressure of natural selection should eliminate the interspecific competition entirely.However, the conclusion that the interspecific competitive alpha is zero does not lead to the conclusion that interspecific competition is unimportant in the system. Instead, if our interpretation is correct, such competition has molded the system, and were there not a continual threat of interspecific competition, the habitat specializations would soon disappear.     

On the statistical significance of primary structural features found in DNA-protein interaction sites

Probabilities of occurrence for a number of the symmetries and other sequence regularities found in DNA-protein interaction site sequences have been calculated for segments of random DNA sequence. Results show that many of the symmetrical and repetitive features seen in these interaction sites are likely to have occurred by chance. Other features are so unlikely to have occurred by chance that they are probably involved in the DNA-protein interaction processes.     

ZAKβ antagonizes and ameliorates the cardiac hypertrophic and apoptotic effects induced by ZAKα

ZAK (sterile alpha motif and leucine zipper containing kinase AZK), a serine/threonine kinase with multiple biochemical functions, has been associated with various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy. In our previous reports, we found that the activation of ZAKα signaling was critical for cardiac hypertrophy. In this study, we show that the expression of ZAKα activated apoptosis through both a FAS‐dependent pathway and a mitochondria‐dependent pathway by subsequently inducing caspase‐3. ZAKβ, an isoform of ZAKα, is dramatically expressed during cardiac hypertrophy and apoptosis. The interaction between ZAKα and ZAKβ was demonstrated here using immunoprecipitation. The results show that ZAKβ has the ability to diminish the expression level of ZAKα. These findings reveal an inherent regulatory role of ZAKβ to antagonize ZAKα and to subsequently downregulate the cardiac hypertrophy and apoptosis induced by ZAKα.     

Indication of dynamic neurovascular coupling from inconsistency between EEG and fMRI indices across sleep–wake states

Sleep and Biological Rhythms - Neurovascular coupling (NVC), the transient regional hyperemia following the evoked neuronal responses, is the basis of blood oxygenation level-dependent techniques...     

Genetic and Anatomical Basis of the Barrier Separating Wakefulness and Anesthetic-Induced Unresponsiveness

A robust, bistable switch regulates the fluctuations between wakefulness and natural sleep as well as those between wakefulness and anesthetic-induced unresponsiveness. We previously provided experimental evidence for the existence of a behavioral barrier to transitions between these states of arousal, which we call neural inertia. Here we show that neural inertia is controlled by processes that contribute to sleep homeostasis and requires four genes involved in electrical excitability: Sh, sss, na and unc79. Although loss of function mutations in these genes can increase or decrease sensitivity to anesthesia induction, surprisingly, they all collapse neural inertia. These effects are genetically selective: neural inertia is not perturbed by loss-of-function mutations in all genes required for the sleep/wake cycle. These effects are also anatomically selective: sss acts in different neurons to influence arousal-promoting and arousal-suppressing processes underlying neural inertia. Supporting the idea that anesthesia and sleep share some, but not all, genetic and anatomical arousal-regulating pathways, we demonstrate that increasing homeostatic sleep drive widens the neural inertial barrier. We propose that processes selectively contributing to sleep homeostasis and neural inertia may be impaired in pathophysiological conditions such as coma and persistent vegetative states.     

Ecological Consequences of Shifting the Timing of Burning Tallgrass Prairie

In the Kansas Flint Hills, grassland burning is conducted during a relatively narrow window because management recommendations for the past 40 years have been to burn only in late spring. Widespread prescribed burning within this restricted time frame frequently creates smoke management issues downwind. A potential remedy for the concentrated smoke production in late spring is to expand burning to times earlier in the year. Yet, previous research suggested that burning in winter or early spring reduces plant productivity and cattle weight gain while increasing the proportion of undesirable plant species. In order to better understand the ecological consequences of burning at different times of the year, plant production and species abundance were measured for 20 years on ungrazed watersheds burned annually in autumn, winter, or spring. We found that there were no significant differences in total grass production among the burns on either upland or lowland topographic positions, although spring burned watersheds had higher grass culm production and lower forb biomass than autumn and winter burned watersheds. Burning in autumn or winter broadened the window of grass productivity response to precipitation, which reduces susceptibility to mid-season drought. Burning in autumn or winter also increased the phenological range of species by promoting cool-season graminoids without a concomitant decrease in warm-season grasses, potentially widening the seasonal window of high-quality forage. Incorporating autumn and winter burns into the overall portfolio of tallgrass prairie management should increase the flexibility in managing grasslands, promote biodiversity, and minimize air quality issues caused by en masse late-spring burning with little negative consequences for cattle production.