Degradation of 2-carboxyarabinitol 1-phosphate by a specific chloroplast phosphatase

The catalytic degradation of 2-carboxyarabinitol 1-phosphate (CA 1-P), a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), was investigated by chromatographic and spectroscopic analyses of the reaction products. Carboxy-labeled [¹⁴C]CA 1-P was incubated with a partially purified tobacco (Nicotiana rustica) chloroplast protein that has been shown previously to catalyze metabolism of CA 1-P to a form incapable of inhibiting Rubisco (ME Salvucci, GP Holbrook, JC Anderson, and G Bowes [1988] FEBS Lett 231: 197-201). In the presence and absence of NADPH, ion-exchange chromatography showed a progressive conversion of [2′-¹⁴C]CA 1-P to a labeled compound which coeluted with authentic carboxyarabinitol. Parallel assays with unlabeled CA 1-P showed a concomitant decrease in the ability of reaction samples to inhibit Rubisco activity. In separate experiments, a 1:1 stoichiometry was found between the release of inorganic phosphate from [2′-¹⁴C]CA 1-P and accumulation of the ¹⁴C-labeled product. Liberation of inorganic phosphate was not observed when the tobacco enzyme was incubated with ribulose-1,5-bisphosphate, fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, or 6-phosphogluconate. Proton nuclear magnetic resonance spectroscopy of the labeled CA 1-P reaction product established its identity as carboxyarabinitol. We therefore propose that light-stimulated degradation of CA 1-P is catalyzed in vivo by a specific phosphatase, 2-carboxyarabinitol 1-phosphatase. Carboxyarabinitol 1-phosphatase activity was detected in the absence of NADPH, but increased threefold when 2 millimolar NADPH was present. Thus, while not required for the reaction, NADPH may play an important role in the regulation of CA 1-P degradation.     

A Simple Purification of Indole-3-Acetic Acid and Abscisic Acid for GC-SIM-MS Analysis by Microfiltration of Aqueous Samples through Nylon

A simple procedure was developed for the partial purification of plant tissue samples to be analyzed simultaneously for indole-3-acetic acid (IAA) and abscisic acid (ABA). The procedure relies on removal of contaminants by filtration through nylon and partitioning into dichloromethane. This procedure successfully purified both IAA and ABA from muskmelon, cotton, and broccoli tissue. Twenty individual samples can be purified and methylated in 8 h for analysis of free IAA and ABA with gas chromatography-selected ion monitoring-mass spectrometry. The use of microfiltration of aqueous samples through nylon offers new opportunities for improving the efficiency of existing sample purification procedures.     

Cytochemical localization of calcium in renal sac nephrocytes of Helix aspersa

In renal sac nephrocytes of Helix aspersa, intracellular calcium has been localized using the oxalate-pyroantimonate (OPA) and phosphate-pyroantimonate (PPA) methods. Pyroantimonate precipitates are preferentially localized in the excretory spherule and in vesicles located in the basal and lateral regions of the nephrocyte. Such vesicles appear to release their content into the excretory vacuole. Calcium may interact with diverse types of molecules present in the excretory vacuole, thus favouring stabilization and packaging of the excretory spherule.     

Membranous tubes in pseudoscorpion spermiogenesis

The highly complicated differentiation of the spermatid in the pseudoscorpion Diplotemnus sp. is accomplished without the presence of microtubules. Instead membranous tubes measuring approximately 50 nm in diameter and closely associated with endoplasmic reticulum are found from early to mid spermatids. The lumen of the tube is devoid of electron dense contents but a fluffy material is attached to the cytoplasmic side. They run straight or slightly bent and are in open connection with the cell membrane. First appearing near the cell bridge of the interconnected spermatids they form a bundle in the longitudinal axis during a transitory phase of elongation. When the cell rounds off again the tubules together with the endoplasmic reticulum disappear. The arrangement of the tubes and their presence during abortive elongation of the spermatid suggest a supportive function commonly attributed to microtubules. Moreover, the open connection with the cell membrane and their close association with the endoplasmic reticulum may indicate their participation also in transport.     

Electrofusion parameters for mouse two-cell embryos

We studied electrofusion of mouse two-cell embryos in order to define parameters which would result in a high yield of fused embryos. Various cell alignment times (from <10 to >60 s) and alternating current percentages (2 to 100%) were examined. The fusion parameters tested were the number of fusion pulses (1-9), pulse length (30-90 mus) and pulse strength (0.50-1.79 kV/cm). Furthermore different combinations of these three parameters were tested. In addition the influence of several embryo culture media on the fusion rates was examined. The results show that the fusion rate of the embryos increases with shorter alignment and higher percentages of the alternating current. The highest fusion rate (95%) was obtained by use of one pulse with a duration of 70 mus and a field strength of 0.60-0.79 kV/cm. The survival rate of the embryos was best if Whitten Medium was used before and after the fusion pulses. The fusion of two-cell stages results in tetraploid embryos which can serve as models for studies in polyploid cells.     

Genetic and Biochemical Characterization of Mutations Affecting the Ability of the Yeast Pachysolen tannophilus To Metabolize d-Xylose

Induced mutants, selected for their defective growth on d-xylose while retaining the ability to grow normally on d-glucose, were studied in Pachysolen tannophilus, a yeast capable of converting d-xylose to ethanol. Fourteen of the mutations were found to occur at nine distinct loci, and data indicated that many more loci remain to be detected. Most of the mutations were pleiotropic in character, and the expression of some of them was much affected by nutritional conditions and by genetic background. Mutations at several loci resulted in poor growth on at least one compound that was either an intermediate of the tricarboxylic acid cycle, succinate or alpha-ketoglutarate, or on compounds metabolizable via this cycle, ethanol or glycerol. An initial biochemical characterization of the mutants was undertaken. Analysis for xylose reductase, xylitol dehydrogenase, and xylulose kinase activity showed that one or more of these activities was affected in 12 of 13 mutants. However, drastic reduction in activity of a single enzyme was confined to that of xylitol dehydrogenase by mutations at three different loci and to that of d-xylose reductase by mutation at another locus. Growth of these latter four mutants was normal on all carbon sources tested that were not five-carbon sugars.     

Influence of Water Potential on gamma-Decalactone Production by the Yeast Sporidiobolus salmonicolor

The influence of water potential on gamma-decalactone production by the yeast Sporidiobolus salmonicolor cultivated in a liquid medium was evaluated by gas-chromatographic analysis. Modifications in water potential led to a number of variations in the aroma production. Maximum extracellular production occurred at water activity (a(w)) with a value of 0.99. Further analyses revealed an important phenomenon of cellular accumulation of aroma for a(w) values between 0.97 and 0.99.     

Formal modeling of approximate relations in biochemical systems

Aiming to satisfy the need for the formalization of semiquantitative reasoning in the analysis of biochemical systems, the O[M] formalism for reasoning with orders of magnitude and approximate relations has been developed. It is based on seven primitive relations among quantities and compound relations which are formed as implicit disjunctions of primitives. O[M] can perform inferences by using formal approximate relations, algebraic equations, inequalities, if-then rules, assumptions, and goals. The applications discussed include Michaelis-Menten kinetics, different modes of inhibition of an enzymatic reaction, analysis of fluxes in biochemical networks, and identification of rate-limiting steps of biochemical pathways. In these applications, O[M] provides a medium for acquisition and formalization of previously informal concepts, analysis of systems at the order-of-magnitude level of detail, and automation of commonsense reasoning.