Deanol Affects Choline Metabolism in Peripheral Tissues of Mice

Abstract: The enzymatic hydrolysis of UDP-galactose in rat and calf brain was studied. The hydrolysis occurs in two steps: The first is the conversion of UDP-galactose to galactose-1-phosphate catalyzed by nucleotide pyrophosphatase (EC 3.6.1.9), and the second is the conversion of the latter to free galactose by alkaline phosphatase (EC 3.1.3.1). The overall conversion has a pH optimum of 9.0, but there is considerable activity at pH 7.4, which is the optimum for UDP-galactose:ceramide galactosyltransferase in the synthesis of cerebrosides. Preparations from cytosol from calf brain cerebellum or stem that were enriched in UDP-galactose hydrolytic activity inhibit cerebroside synthesis under conditions optimal for the synthesis. Microsome-rich and nuclear debris fractions contain the highest apparent specific activity among the subcellular fractions studied. Hydrolysis of UDP-galactose occurs in all areas of brain, brainstem having the highest activity. The apparent specific activity in jimpy mouse brain homogenate is nearly twice as high as in the control brain homogenate.     

Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea

Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation.     

CARBONIC ANHYDRASE IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

The zinc metalloenzyme carbonic anhydrase plays a critical role in inorganic carbon acquisition in marine diatoms, thus conferring on zinc a key role in oceanic carbon cycling. As a first step in determining the location and function of carbonic anhydrase (CA) in Bacillariophyceae, we purified and partially sequenced CA from T. weissflogii (Gru) Fryxell et Hasle (TWCA1) and cloned the corresponding cDNA (twca1). The twca1 sequence is different from other known algal carbonic anhydrase genes, and encodes a protein of roughly 34 kDa. The amino terminal amino acids sequenced from purified TWCA1 are 72 residues downstream of the putative starting methionine predicted by twca1. This difference may be due to the presence of a short-lived signal sequence designed to guide the enzyme to the correct cellular location. The absence of any homology between TWCA1 and previously sequenced CAs from Chlorophyceae may indicate either convergent evolution or that carbon acquisition represents a fundamental physiological difference among algal phyla.     

A study of the relative efficiency of mustard oil cake and urea and the effect of salinity in plankton production in indian fishponds

Augmentation of plankton production in Indian fishponds by application of manure and inorganic fertilisers was studied in laboratory experiments. The effects of the salinity of the medium were also examined.Laboratory trials were made with mustard oil cake and urea on equivalent nutrient bases in media with varying degrees of salinity (0–30 ppt at 2 ppt. intervals). It was observed that Closterium, Fragilaria, Pinnularia and Gyrosigma grew well in the higher salinity range between 24 to 30 ppt. whereas lower salinities ranging from a trace to 8 ppt. are suitable for improved production of Anabaena, Synedra, Navicula, Amphipleura, Amphora and Nitzschia. Comparatively better production of plankton was recorded with urea than with mustard oil cake.     

Chemical and biological mobilization of Fe(III) in marsh sediments

Iron reduction in marine sulfitic environments may occur via a mechanism involving direct bacterial reduction with the use of hydrogen as an electron donor, direct bacterial reduction involving carbon turnover, or by indirect reduction where sulfide acts to reduce iron. In the presented experiments, the relative importance of direct and indirect mechanisms of iron reduction, and the contribution of these two mechanisms to overall carbon turnover has been evaluated in two marsh environments. Sediments collected from two Northeastern US salt marshes each having different Fe (III) histories were incubated with the addition of reactive iron (as amorphous oxyhydroxide). These sediments were either incubated alone or in conjunction with sodium molybdate. Production of both inorganic and organic pore water constituents and a calculation of net carbon production were used as measures to compare the relative importance of direct bacterial reduction and indirect bacterial reduction. Results indicate that in the environments tested, the majority of the reduced iron found results from indirect reduction mediated by hydrogen sulfide, a result of dissolution and precipitation phenomena, or is a result of direct bacterial reduction using hydrogen as an electron donor. Direct iron reduction plays a minor role in carbon turnover in these environments.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast-like cells: Effect of Pi,Na+ and K+

Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106–01 and UMR 106–06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106–01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [³²Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular ³²P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106–01 and DET, but the decrease was smaller than that in [³²Pi]-uptake. Ouabain decreased [³²Pi]-uptake and cellular [Pi] in DET, but not in UMR 106–01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts.     

Inorganic phosphate determination: Colorimetric assay based on the formation of a rhodamine B-phosphomolybdate complex

A sensitive technique for inorganic phosphate determination was developed. It is based on the formation of an insoluble rhodamine B-phosphomolybdate complex. After it is washed with 1 HCl the precipitate is dissolved in acetone and rhodamine B is measured spectrophotometrically at 555 nm. In 1 HCl, the complex is composed of three molecules rhodamine B and one molecule phosphomolybdate. Due to the high molar absorbance of rhodamine B in acetone and to the threefold amplification of dye concentration compared to P_i concentration in the precipitated complex, a molar absorption coefficient of 330,000 ± 5000 ⁻¹ cm⁻¹ (SD) is obtained. This allows the determination of quantities as low as 1.5 nmol P_i with good precision, while quantities as low as 0.5 nmol P_i are detectable. The effect of anions and buffers was studied. Some possible applications of the method are illustrated, as, e.g., enzyme activity measurement at very low substrate concentration and determination of small quantities of P_i and total phosphate in (biological) samples.     

Revealing Key Interactions in a Metabolic Network: Model of Primary Phosphate Transport in Bacteria

One of the main problems of metabolic engineering is to determine the genetically controlled limiting links of a metabolic network. We have built a model of the primary transport of inorganic phosphates (P _i ), analyzed the P _i metabolic network in Gram-negative bacteria, and determined the factors controlling the phosphate exchange. The model explains why the P _i primary transport is not observed at the release stage. The nonlinearity of primary transport and the differences in its parameters in the membrane and within the cell give rise to transport asymmetry, i.e., the P _i release rate is low as compared with the uptake rate, and is small at the background of secondary transport. Discussed is a general scheme of coordination between primary and secondary transport, which are interconnected through the substrate–product relation.