A non-linear model of phosphorus flux in the phytoplankton of a temperate eutrophic reservoir

Phosphorus fluxes in phytoplankton, between intracellular andexternal compartments have been measured, in the epilimnion ofatemperate eutrophic reservoir, by means of phosphorusdepletionexperiments performed in situ. Flow rates betweencompartments were plotted against the phosphorus concentrationofthe compartments' origin of the flows and fitted to saturationfunctions.A value of phosphorus cell subsistence quota for the wholecommunity has been computed from the X-intercept values ofeveryfunction. The obtained figures are 10–100 times higher,dependingon the reference value, than those measured running theclassicalsingle species growth response experiments in chemostats.We propose a model using five compartments and a series offlowrates between them in order to simulate the general phosphoruscycling in freshwater phytoplankton. Short term simulation ofphosphorus content in every compartment shows a fluctuatingpatternaround rates of equilibrium, in agreement with the pattern ofvariation observed in situ for the selectedcompartments.     

Catecholamine and MHPG plasma levels,platelet MAO activity,and3H-imipramine binding in heroin and cocaine addicts

This work evaluated in a population of heroin and heroin plus cocaine human addicts:

1. Norepinephrine (NE), epinephrine (Epi), and 3-methoxy-4-hydroxyphenylglycol (MHPG) (the principal metabolite of brain NE) plasma levels;
2. Monoamine oxidase (MAO) activity; and
3. ³H-imipramine specific binding to the amine carrier in platelets.

NE plasma levels were significantly lower in the short-term heroin user groups (1–3 and 4–6 yr), a finding not observed in both the long-term heroin user (>6 yr) and heroin plus cocaine user (>6 yr) groups. Epi levels changed in a similar manner, except that a significant increase was noted in heroin plus cocaine abusers. Conversely, dopamine and MHPG plasma levels increased with the duration of heroin use, and even more with cocaine abuse. Platelet MAO activity increased in all groups. Specific³H-imipramine binding sites showed an increase after 3 yr of heroin abuse and in all heroin plus cocaine addicts. In conclusion, short-term use of heroin decreases NE or Epi release, but with prolonged use, a slow adpatation occurs. In contrast, cocaine inhibits the neuronal Epi uptake, even in a situation of long duration of abuse. Probably the amine levels additionally regulate the amine carrier, resulting in changes that show a different pattern from major depression. These drugs of abuse may also influence directly or indirectly related enzymatic systems.     

Crystallization,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.     

Immunochemistry of membrane F1-Adenosine triphosphatase fromMicrococcus lysodeikticus. Role of the alpha subunit

The membrane-bound ATPase activity from two substrains ofMicrococcus lysodeikticus, designated as A and B, was inhibited by antibodies raised against the two forms of purified F₁-ATPase. Form B of the enzyme, which behaved as a poorer immunogen than form A, also showed less reactivity as an antigen, independent of the physical state of the F₁-ATPase form. Antibodies were raised against the two major subunits ( and ) isolated fromM. lysodeikticus F₁-ATPase form A, which was the most stable form of the enzyme. Anti-(-subunit) serum strongly inhibited the ATPase activity of membrane-bound ATPase but showed little inhibition of the purified, soluble F₁-ATPase. The anti-(-subunit) serum inhibited the soluble F₁-ATPase, but to a lesser extent than the membrane-bound enzyme. In any event, the effect of anti- antibodies on the membrane-bound ATPase was smaller than that of anti- antibodies. It was postulated that the subunit ofM. lysodeikticus F₁-ATPase plays an essential and regulatory role in the expression of the immunochemical properties of the protein.     

Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation

An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ⁺ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.     

Phospholipid turnover during phagocytosis in human polymorphonuclear leucocytes

We have previously observed that the phagocytosis of zymosan particles coated with complement by human polymorphonuclear leucocytes is accompanied by a time- and dose-dependent inhibition of phosphatidylcholine synthesis by transmethylation [García Gil, Alonso, Sánchez Crespo & Mato (1981) Biochem. Biophys. Res. Commun. 101, 740–748]. The present studies show that phosphatidylcholine synthesis by a cholinephosphotransferase reaction is enhanced, up to 3-fold, during phagocytosis by polymorphonuclear cells. This effect was tested by both measuring the incorporation of radioactivity into phosphatidylcholine in cells labelled with [Me-¹⁴C]choline, and by assaying the activity of CDP-choline:diacylglycerol cholinephosphotransferase. The time course of CDP-choline:diacylglycerol cholinephosphotransferase activation by zymosan mirrors the inhibition of phospholipid methyltransferase activity previously reported. The extent of incorporation of radioactivity into phosphatidylcholine induced by various doses of zymosan correlates with the physiological response of the cells to this stimulus. This effect was specific for phosphatidylcholine, and phosphatidyl-ethanolamine turnover was not affected by zymosan. The purpose of this enhanced phosphatidylcholine synthesis is not to provide phospholipid molecules rich in arachidonic acid. The present studies show that about 80% of the arachidonic acid generated in response to zymosan derives from phosphatidylinositol. A transient accumulation of arachidonoyldiacylglycerol has also been observed, which indicates that a phospholipase C is responsible, at least in part, for the generation of arachidonic acid. Finally, isobutylmethylxanthine and quinacrine, inhibitors of phosphatidylinositol turnover, inhibit both arachidonic acid generation and phagocytosis, indicating a function for this pathway during this process.     

Catecholamine-Sensitive Guanylate Cyclase from Human Caudate Nucleus

Abstract: Partial purification of soluble guanylate cyclase on DEAE-Sephacel yields two separate peaks of guanylate cyclase activity. After 10-fold purification of the soluble enzyme, guanylate cyclase is markedly inhibited by micromolar concentrations of dopamine (I₅₀= 0.2 μm). Dopamine inhibition is observed whether the reaction is conducted with Mn²¹ or with Mg²⁺, under atmosphere or N₂(g), and using enzyme from either peak from the DEAESephacel column. Other catecholamines also inhibit partially purified guanylate cyclase with an order of potency at 1 μm of: dopamine =l -DOPA > norepinephrine = isoproterenol = adrenochrome > epinephrine. The structural requirements for inhibition are two free hydroxyl groups on the phenyl ring and an ethylamine side chain. Dopamine also inhibits the Triton X-100-solubilized microsomal guanylate cyclase after partial purification on DEAESephacel. Neither chlorpromazine, propranolol, nor phentolamine at 20 μm effectively block the dopamine inhibition of partially purified soluble guanylate cyclase. Micromolar concentrations of the reducing agents dithiothreitol and glutathione also inhibit partially purified guanylate cyclase, but unlike these agents, catecholamines can inhibit whether added in the reduced or the oxidized forms. Inhibition of enzyme activity by micromolar concentrations of dopamine, adrenochrome, or dithiothreitol is rapidly reversed by dilution and the dopamine inhibition is competitive with MgGTP. Inhibition does not appear to involve covalent binding or to result from the ability of catecholamines to reduce the concentrations of oxygen or free radicals in solution.     

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

The N¹-methyl-Adenosine (m¹A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m¹A58 modification is essential for viability because it is required for the stability of the initiator-tRNA^Met. However, m¹A58 modification is not required for the stability of several other tRNAs in yeast. This differential m¹A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m¹A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m¹A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA^Met is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m¹A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m¹A58 hypomodified tRNAs. Most m¹A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m¹A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.