Turnover of Brain Monoamine Oxidase Measured In Vivo by Positron Emission Tomography Using l-[11C]Deprenyl

The distribution of carbon-11-labeled L-deprenyl, an irreversible inhibitor of monoamine oxidase type B (MAO-B), was determined in the baboon brain by positron emission tomography. The irreversible blood-to-brain transfer constant (influx constant, K_i) was measured using a complete metabolite-corrected arterial plasma concentration curve. This influx constant was used as a measure of functional enzyme activity for sequential determinations of MAO-B recovery following a single high dose of unlabeled l -deprenyl. The half-life for turnover of MAO-B was thus determined to be 30 days. Using appropriate irreversible inhibitors, this procedure should be generally useful for determining enzyme turnover rates in any organ in vivo and can be applied to some human studies as well.     

Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site

The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.     

Reversible effects of moderately elevated temperature on the distribution of excitation energy between the two photosystems of photosynthesis in intact avocado leaves

Initial (F_o), maximum (F_m) and steady-state (F_s) levels of modulated chlorophyll fluorescence were measured in intact avocado leaves (Persea americana Mill.) during state 1-state 2 transitions using a combination of modulated and non-modulated lights with synchronized detection. Under normal temperature conditions (20°C), transition from state 2 to state 1 was associated with a substantial increase (about 20%) in F_m and F_o whereas the F_m/F_o ratio remained constant, reflecting increased absorption cross-section of PS II. On the contrary, at moderately elevated temperature (35°C), these fluorescence changes were very limited, indicating marked inhibition of the state regulation. The fraction of light distributed to PS II () was calculated from the F_o, F_m and F_s levels for both types of leaves. In control leaves, varied from 48% (in state 2) to values as high as 58% (in state 1). In contrast, mild heat treatment resulted in values close to 50% in both states, indicating the inability of heated leaves to reach extreme state 1. The results suggested that avocado leaves under moderately elevated temperature conditions are blocked in a state close to state 2. This effect was shown to occur in a non-injurious temperature range (as shown by the preservation of the (photoacoustically monitored) oxygen evolution activity) and to be rapidly reversed upon lowering of the temperature. Thermally induced development of state 2 (independent on the light spectral quality) could possibly be a protective mechanism to avoid photodamage of the heat-labile PS II by high light intensities which usually accompany heat stress in the field.     

Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Gene regulation during dedifferentiation in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire the capacity to rapidly recapitulate morphogenesis. Therefore, when cells at the loose aggregate stage are disaggregated and challenged to reaggregate, they do so in a tenth of the original time. If loose aggregate cells are disaggregated and resuspended in buffered dextrose solution (erasure medium), they retain the capacity of rapid recapitulation for 80 min, then completely lose this capacity in a single, synchronous step referred to as the "erasure event." The erasure event sets in motion a program of dedifferentiation during which cells lose developmentally acquired characteristics at different times. The erasure event is inhibited by the addition of 10(-4) M cAMP to erasure medium. The synthesis of 33 growth-associated polypeptides, the synthesis of 53 development-associated polypeptides, and the level of 2 development-associated RNAs have been monitored during the erasure program and in cultures inhibited from erasing by the addition of 10(-4) M cAMP. Growth-associated polypeptides begin to be resynthesized and development-associated polypeptides exhibit dramatic decreases in rate of synthesis at different times throughout the first 240 min in erasure medium. Inhibiting the erasure event with cAMP has no major effect in the resynthesis of the majority of growth-associated polypeptides. Only one growth-associated polypeptide, V28, is completely inhibited by cAMP, suggesting that it may play a role in the erasure process. In contrast, inhibiting the erasure event with cAMP has a marked effect on the synthesis of development-associated polypeptides, causing a dramatic reduction in the rate at which synthesis decreases for 6 polypeptides, and completely inhibits the decrease in the synthetic rate of 8 polypeptides. The two development-associated RNAs, 16G1 and 10C3, exhibit two distinctly different patterns of loss during erasure, but in both cases cAMP added at time zero of the erasure process dramatically retards or inhibits loss. In addition, when cAMP is added just prior to the erasure event, it inhibits the erasure event and stimulates a rapid increase in the level of 16G1 RNA back to the developmental level. The level of 16G1 RNA then remains at this level for at least 400 min. When cAMP is added after the erasure event, it causes a low, transient increase in the level of 16G1 RNA. These results are considered both in relation to the program of erasure, and in relation to the role of cAMP in the expression of developmental genes during the forward program of development.     

Metal ion size selectivity in ligands with groups containing the neutral oxygen donor atom. A crystallographic and thermodynamic study

The coordinating properties of open-chain ligands containing alcoholic or ethereal oxygen donors are examined. Addition of oxygen donors usually leads to complex stabilisation for large metal ions (Pb²⁺, Cd²⁺) and to less favourable effects on complex stability for small metal ions (Cu²⁺, Ni²⁺). The formation constants of these metal ions with the set of ligands RN(CH₂CHOH·CH₃)₂ where R is ---H, ---CH₂CHOH·CH₃, ---CH₂CH₂OCH₃, ---CH₂CH₂OCH₂CH₂OH, and ---CH₂---CHOCH₂CH₂CH₂ are reported. The largest stabilisation for each case where R is an O-donor group relative to R = H occurs for Pb²⁺, the largest metal ion, while Cu²⁺, the smallest metal ion, shows the smallest stabilisation. The crystal structure of [Ni(HOCH₂CH₂NHCH₂CH₂NH₂)₂] (NO₃)₂ is reported. The space group is P , with cell constants a = 13.098(3), b = 8.737(4), and c = 7.746(3) Å, β = 112.66(3), β = 90.65(3), and γ = 85.03(2), and Z = 2. Disorder of the nitrate anions hindered refinement, with the result that a final conventional R factor of 0.0903 was achieved. The Ni---N bond lengths average 2.06(1) (secondary nitrogen) and 2.10(2) (primary nitrogen). The Ni---O bond lengths are rather long, averaging 2.15(1) Å, which is used to support the idea that the steric effects are responsible for destabilising the complexes of small metal ions such as Ni(11) when neutral oxygen donors are present.     

Vertical nutrient transport and its effects on epilimnetic phosphorus in four calcareous lakes

The influence of vertical nutrient transport on epilimnetic phosphorus was studied comparatively in four calcareous Wisconsin lakes during 1972. In the two lakes with steep metalimnetic nutrient gradients (Mendota, Delavan), upward fluxes by entrainment and eddy diffusion exceeded all other influxes combined; here epilimnetic total-P concentrations increased during period of high windpower and thermocline migration, and decreased during comparatively stagnant intervals. In the two lakes lacking upper metalimnetic P gradients (Green, Fish), higher windpower had little or no effect on epilimnetic phosphorus.In each of the four lakes epilimnetic P declined in early summer until a quasi steady-state was achieved between P influxes and P sedimentation. In Mendota and Delavan steady-state featured higher concentrations of total-P, and much higher epilimnetic concentrations of particulate-P and chlorophyll, than in Green and Fish Lakes — mainly on account of the high fluxes of molybdate-reactive, biologically-available P through the seasonal thermocline. The flux analysis illustrates why mean lake TP concentration after ice-out is an inconsistent measure (not sufficient statistic) for estimating nutrient potential and chlorophyll standing crops during the following summer in stratified lakes.     

Impact d'une pollution urbaine sur la partie zooplanctonique d'un systeme neitique (Marseille - Cortiou)

Sewage of Marseilles' main outfall permanently pollutes a large coastal area centered around Cortiou, south of the city. In order to study the impact of that urban pollution on the zooplankton, more than 200 samples were collected between 1977 and 1981, according to several sampling strategies.Quantitatively, the study area showed a rather poor zooplankton. The more important populations were encountered near Cortiou, the non-perturbated reference point with lowest abundance of organisms. Sampling sites located near the outfall are sometimes azoic. Qualitatively, the observed communities are not characteristic of a heavily polluted environment, but correspond to an impoverished neritic community. In the more polluted area, the community is organized around the copepods Clausocalanus sp., Paracalanus sp. and Oithona helgolandica, and a group of less important species (Oithona nana and the cladoceran Evadne spinifera). Centropagidae, Coryceidae, Onceidae, but also Chaetognathians, Fritilliarins and the meroplanktonic larvae are more frequently encountered in clean water. Community structure is higher during the cold months than summer. The latter period frequently shows a disorganized zooplankton. In most situations, the copepod Acartia clausi plays a minor role in the structural definition of the communities.The variations observed seem largely independent of the parameters reflecting pollution intensity. Stress integration, in relation with the anterior community history (intensity of contact with polluted water, trophic potential of the area) seem to be the main regulator factors.

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Impact d'une pollution urbaine sur la partie zooplanctonique d'un systeme neitique (Marseille - Cortiou)

Resumé Le rejet permanent du grand émissaire de Marseille (5 m³, sec^–1) perturbe considérablement, par son importance, le système néritique du secteur de Cortiou. Afin d'approcher l'impact de cette pollution sur la partie zooplanctonique du système, plus de 200 prélèvements, concernant l'hydrologie et le plancton, ont of é\'t é réalises entre 1977 et 1981, selon différentes strategiés d'échantillonnage (suivi de masse d'eau, radiales, réseaux).Quantitativement on observe, sur l'ensemble de la zone étudiée, une abondance générale en zooplancton moyenne, voire faible. Les effectifs les plus importants se rencontrent cependant dans la cuvette de Cortiou, alors que le point de référence considéré comme non perturbé présente les effectifs les plus faibles. Les stations situées face à l'égout sont parfois azoïques.Qualitativement les peuplements ne paraissent pas trés caractéristiques d'un secteur pollué mais correspondent plutôt à un appauvrissement du peuplement néritique. Dans le secteur le plus pollué, la composition spécifique varie au cours du temps autour d'une communauté composée d'un groupe important avec Clausocalanus sp., Paracalanus sp., Oithona helgolandica et d'un groups de taxons moins fréquents représentés par des larves méroplanctoniques, Oithona nana et Evadne spinifera, tandis que les coryceidés, onceidés, Centropages typicus, chaetognathes et fritillaires se retrouvent plus fréquemment en eaux propres. La structure des populations est plus importante en période froide qu'en période chaude, période durant laquelle la communauté planctonique est fortement désorganisée. Paradoxalement Acartia clausi joue un rôle assez secondaire dans la définition structurelle de la communauté.Les fluctuations observées paraissent cependant peu liées à des paramètres reflétant l'intensité de la pollution. L'intégration du stress, en relation avec l'histoire antérieure de la communauté (intensité et durée du contact avec la nappe de dilution, potentialités trophiques) semblent ainsi prépondérantes.

     

Calorimetric studies of oxygen and carbon monoxide binding to human hemoglobin. Sequential binding heats for oxygen

Two high precision techniques, titration microcalorimetry and thin-layer optical binding measurements, have made possible the evaluation of enthalpy changes for the overall oxygenation reactions for human hemoglobin (HbAo). Although the heat of adding three oxygen molecules could not be evaluated due to the indeterminate contribution of this species to the oxygen binding curve of the protein (Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., and Robert, C. H. (1987) Biochemistry, 26, 3995-4002), the heats for binding two and four oxygen molecules were found to be simple multiples of the first binding heat. A direct consequence of equal stepwise heats is invariance of the shape of the binding curve with temperature, as pointed out by Wyman (Wyman, J. (1939) J. Biol. Chem. 127, 581-599). Titration microcalorimetry was also performed for the binding of carbon monoxide to hemoglobin. While the tight binding of CO precludes high-precision binding measurements, it does allow one to accurately determine the heat of ligation as a function of the CO bound. In these titrations a uniform heat of reaction is not observed, but the heat of binding increases markedly near the end point. This implies that the stepwise binding enthalpy for adding the third CO molecule is anomalously endothermic and for adding the fourth strongly exothermic. A similar phenomenon cannot be ruled out in the case of oxygen because of imprecision intrinsic in the analysis of the weaker ligand binding.