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.     

Control of concanavalin A receptor mobility by cytoplasmic actin in human tumour cells.

Tissue culture monolayers of seven human intracranial tumours comprising 2 astrocytomas, 3 meningiomas, 1 secondary squamous cell carcinoma and 1 secondary adenocarcinoma were examined by a double immunofluorescent staining technique to demonstrate Concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell. Tumour cells, treated with fluoresceinisothiocyanate-labelled Con A (FITC-Con A) showed staining in cell margins or in a random distribution over the cell surface. Incubating the cells with FITC-Con A at 37 degrees for increasing periods of time resulted first in staining of clusters and later of perinuclear globules. Cells, pretreated with 4% paraformaldehyde at 4 degrees for 10 min or with cytochalasin B at 37 degrees for 30 min showed staining restricted to cell margins. In the cytochalasin B-treated cells, the peripheral staining was in the form of coarse clusters. Double fluorochrome studies showed that the anti-actin antibody (AAA) staining occurred in sites closely related to those stained by FITC-Con A both in untreated as well as in cytochalasin B-treated cells. The findings suggest that Con A receptors, as an example of a stable cell membrane determinant in human tumour cells, are associated with actin and that their mobility on the cell surface is dependent on an intact cytoplasmic actin system.     

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.     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

Changes in nucleic acids, protein-nitrogen and amino-nitrogen of excised winter wheat embryos during vernalization

Embryos excised from winter wheat grains were vernalized for10–50 days with or without sugar (sucrose). Determinationswere made of fresh weight, protein-nitrogen, amino-nitrogen,RNA and DNA. There was no change in the contents of RNA of wheatembryos during the vernalization. The incorporation of ³²P intonucleic acid in wheat embryos during vernalization in the presenceof sugar was much higher than that of embryos vernalized withoutsugar. From these results we assumed that RNA turnover occurredduring the vernalization. There was no significant differencein the nucleotide composition of RNA extracted between the vernalizedand unvernalized embryos. The RNA of wheat embryos was separatedinto two fractions. Proportions of these two RNA fractions variedin the course of cold treatment, and similar changes were foundin developing wheat leaves. (Received July 25, 1974; )     

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.     

Fetal monitoring during maternal cardiac surgery with cardiopulmonary bypass.

Fetal cardiac activity was monitored with an external ultrasound transducer in two patients with clinical class III heart disease due to severe mitral stenosis complicated by pulmonary hypertension, undergoing open heart surgery with cardiopulmonary bypass in the 2nd trimester of pregnancy. Fetal distress was detected in one patient, who had mitral valvuloplasty, and was corrected by increasing the rate of blood flow, and the other patient had a mitral valve replacement but no fetal distress was noted. The postoperative course of both mothers and fetuses was uneventful.     

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.