The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.     

Purification of porphobilinogen deaminase from Euglena gracilis and studies of its kinetics.

1. Porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] from Euglena gracilis was purified more than 200-fold. 2. The enzyme has a molecular weight of 41 000 +/- 2000, does not contain a chromophoric prosthetic group, and appears not to require metal ions for activity. 3. The stoicheiometry of the overall reaction at pH 7.4 was shown to be: 4 Porphobilinogen leads to uroporphyrinogen-I + 4 NH4+. This stoicheiometry for porphobilinogen and uroporphyrinogen was also observed over a wide range of pH values. 4. Initial-velocity studies showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. 5. Vmax. varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 6.1 and 8.9 are important for catalysis. A plot of Vmax./Km against pH showed a single ionization (pK 8.2) to influence binding of substrate.     

MEASUREMENTS OF PHYTOPLANKTON-PROTEIN CONTENT WITH THE HEATED BIURET-FOL1N ASSAY1,2

The heated biuret-Folin method for determining protein consistently measures 90% of the total nitrogen of filtered algae samples as protein-N without the need of mechanical disruption as long as the heating period in biuret is 100 min at 100 C. Data indicate this protein assay measures total protein on all species tried and for naturally occurring mixtures of species plus detritus. Dilute algal suspensions with as little as 0.05 μg-atom particulate protein N.liter ^-1 can he concentrated by fltration on glass fiber filters to 1.0 μg-atom particulate protein-N per filter, the optimal amount of sample for a 5 ml volume of biuret. The filtered algae samples can be stored for several weeks frozen before assaying, if necessary.     

Glycogen synthesis by amylosucrase from Neisseria perflava.

Amylosucrase (sucrose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase; EC 2.4.1.4) which mediates the transfer of the glucosyl moiety of sucrose to a growing alpha-1,4-glucan chain is a constitutive enzyme of Neisseria perflava. The products of enzymic action are insoluble glycogenlike polysaccharides and fructose, the latter being a competitive inhibitor of the enzyme (Ki=20 mM). The enzyme is extremely stable and appears to bind very tightly to its polymerized product. Properties of product-bound enzyme reflect those of the native complex.     

Treatmenf of optic neuritis by retrobulbar injection of triamcinolone.

In a single-blind controlled clinical trial patients with optic neuritis caused by demyelination were given a single retrobulbar injection of triamcinolone. Though the treated group showed a trend towards more rapid recovery of vision than the controls, there was no significant difference in visual acuity, colour vision, or visual fields during the first six months after treatment. We conclude that routine use of corticosteroids is not justified in unilateral optic neuritis when vision in the other eye is good. Shortening the period of visual disability in bilateral disease or unilateral disease when vision in the other eye is poor, however, may be justifiable.     

Excitotoxic Brain Injury Suppresses Striatal High-Affinity Glutamate Uptake in Perinatal Rats

In immature rodent brain, the glutamate receptor agonist N-methyl-D-aspartate (NMDA) is a potent neurotoxin. In postnatal day (PND)-7 rats, intrastriatal injection of 25 nmol of NMDA results in extensive ipsilateral forebrain injury. In this study, we examined alterations in high-affinity [3H]glutamate uptake (HAGU) in NMDA-lesioned striatum. HAGU was assayed in synaptosomes, prepared from lesioned striatum, the corresponding contralateral striatum, or unlesioned controls. Twenty-four hours after NMDA injection (25 nmol), HAGU declined 44 +/- 8% in lesioned tissue, compared with the contralateral striatum (mean +/- SEM, n = 6 assays, p less than 0.006, paired t test). Doses of 5-25 nmol of NMDA resulted in increasing suppression of HAGU (5 nmol, n = 3; 12.5 nmol, n = 3; and 25 nmol, n = 5 assays; p less than 0.01, regression analysis). The temporal evolution of HAGU suppression was biphasic. There was an early transient suppression of HAGU (-28 +/- 4% at 1 h; p less than 0.03, analysis of variance, comparing changes at 0.5, 1, 2, and 3 h after lesioning); 1 or 5 days postinjury there was sustained loss of HAGU (at 5 days, -56 +/- 11%, n = 3, p less than 0.03, paired t test, lesioned versus contralateral striata).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cost of predation avoidance in young-of-year lake trout (Salvelinus namaycush): growth differential in sub-optimal habitats

Selection of habitat to avoid predation may affect the diet of young-of-year (YOY) lake trout (Salvelinus namaycush). YOY lake trout may use inshore habitat to avoid predation; this habitat may be sub-optimal for growth. To test this, YOY lake trout were penned in nearshore and offshore pelagic areas of two arctic lakes. Toolik Lake had a lake trout population, the other lake, S6, did not. YOY lake trout in Toolik Lake lost weight, but those offshore lost less weight. The YOY lake trout in Lake S6 gained weight and those offshore gained more weight. The primary diet item of the YOY lake trout in both lakes during this experiment was the zooplankter Diaptomis probilofensis; it was also one of the most abundant species. However, its density inshore in Lake S6 was similar to inshore and offshore densities in Toolik Lake. The increased availability of alternative zooplankton prey in Lake S6 may account for the growth differential of YOY lake trout in Lake S6 relative to Toolik Lake. Bioenergetic modeling of YOY lake trout suggests that growth similar to that in the offshore of Lake S6 would be necessary for successful recruitment. If the reduced zooplankton availability in Toolik Lake leads to the reduced growth of YOY in the inshore and offshore pelagic areas, then these fish will be more susceptable to winter predation/starvation. For YOY lake trout to survive in Toolik Lake they most likely shift to feeding on benthic prey before the end of their first summer. Dept. of Chemical Engineering     

Thrombopoietin from human embryonic kidney cells causes increased thrombocytopoiesis in sublethally irradiated mice.

Previous work showed that mice treated with platelet-specific antiserum prior to whole-body irradiation did not suffer the degree or duration of thrombocytopenia as did irradiated control mice. We now report that a partially purified preparation of a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) mimics the biological effects of platelet-specific antiserum treatment in hematopoietically suppressed mice. Male C3H mice were exposed to 3.0 or 4.5 Gy of 137Cs gamma radiation and injected with a total dose of 4 units (U) of TSF. Human serum albumin (HSA) and rabbit anti-mouse platelet serum-injected mice, along with unirradiated mice, served as controls. Packed cell volumes (PCV), RBC counts, WBC counts, platelet counts, and percentage 35S incorporation into platelets were measured in mice at various days (7-14) following treatment. The results showed that irradiated mice treated with TSF had increased 35S uptake into platelets and higher platelet counts than HSA-treated controls. Also, PCV, RBC counts, and WBC counts of irradiated mice treated with TSF were significantly higher than values for HSA-treated mice. Additional experiments using 40,000 U/mouse of Interleukin-6 (IL-6), 227 U/mouse of granulocyte macrophage-colony stimulating factor (GM-CSF), or a combination of GM-CSF and IL-6 did not show increased platelet counts or 35S incorporation into platelets on Days 10 and 14 when compared to other mice treated with control substances. These results suggest that the radioprotective effects of platelet antibodies reported previously may be due to the release and action of thrombopoietin. These studies also demonstrate that thrombopoietin therapy will modulate the severe thrombocytopenia that occurs in radiation-induced bone marrow suppression.     

Two Types of Genetic Interaction Implicate the Whirligig Gene of Drosophila Melanogaster in Microtubule Organization in the Flagellar Axoneme

The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an alpha-tubulin locus, alpha 1t, mutations in a beta-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known alpha- or beta-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrlnc4 and B2tn are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2tn, for a deficiency of alpha 1t, or for the haync2 allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrlnc4 or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.     

Bromocriptine in management of large pituitary tumours.

Bromocriptine has an accepted place in the management of small pituitary tumours that secrete either prolactin or growth hormone. The treatment of large tumours with extrasellar extensions is more difficult, however: though surgery is the standard treatment, it is often unsuccessful in returning excessive hormone secretion to normal and may cause hypopituitarism. A prospective trial was undertaken to assess the frequency with which changes in pituitary function and size of large tumours occurs. Nineteen patients were studied before and during treatment with bromocriptine (7.5 to 60 ml/day) for three to 22 months, using contrast radiology and a detailed assessment of pituitary function. Eighteen patients had hyperprolactinaemia and two of these also had raised concentrations of growth hormones; one patient had an apparently non-functioning tumour. In 12 patients (63%) tumour size decreased with bromocriptine and no tumour enlarged. Nine patients had visual-field defects, which improved in seven, becoming normal in five. Pituitary function improved in nine patients (47%) becoming entirely normal in three. Bromocriptine should be the treatment of choice in patients with large pituitary tumours with extrasellar extensions, provided close supervision is maintained.