Lithium Effects on Inositol Phospholipids and Inositol Phosphates: Evaluation of an In Vivo Model for Assessing Polyphosphoinositide Turnover in Brain

Administration of lithium chloride to rats injected intracerebrally with [3H]inositol led to time- and dose-dependent increases in levels of labeled inositol monophosphates in brain. Quantitative analysis of the inositol phosphates by ion chromatography revealed 37- and 20-fold increases in the mass of myo-inositol 1-phosphate and 4-phosphate, respectively, at 4 h intraperitoneal after injections of 6 mEq/kg of lithium chloride. Albeit to a much lesser extent, lithium administration also resulted in an increase in the level of myo-inositol, 1,4-bisphosphate in brain. The lithium-induced increase in content of labeled inositol monophosphates was marked by a concomitant decrease in content of labeled inositol, and after injections of high doses of lithium, e.g., 10 mEq/kg, this was followed by a general decrease in labeling of the inositol phospholipids. In general, animals injected with [3H]inositol but not lithium did not reveal obvious differences in labeling of inositol monophosphates on stimulation by mecamylamine or pilocarpine. However, when animals were injected with [3H]inositol and then lithium, there were large increases in the levels of labeled inositol monophosphates on administration of these compounds. Administration of atropine to the lithium-treated mice led to a partial reduction in the amount of labeled inositol monophosphates accumulated due to the administration of lithium alone. Furthermore, atropine was able to block the pilocarpine-induced increase in level of labeled inositol monophosphates. These results demonstrate the suitable use of the radiotracer technique together with lithium administration for assessing the effects of drugs and receptor agonists on the signaling system involving polyphosphoinositide turnover in brain.     

The rat mast cell antigen AD1 (homologue to human CD63 or melanoma antigen ME491) is expressed in other cells in culture.

Previously we reported that the mAb AD1 recognized a heavily glycosylated 50- to 60-kDa protein (AD1 Ag) sterically close to the high-affinity IgE receptor on rat basophilic leukemia (RBL-2H3) cells. The N-terminal amino acid sequence of the AD1 Ag was nearly identical to that of human CD63 (melanoma-associated Ag ME491). In this study we cloned the cDNA of AD1 Ag from a rat basophilic leukemia 2H3 cDNA library. An open reading frame of 238 amino acids was identified that contained the N-terminal 43 amino acid sequence. No evidence of a signal peptide was found. However, four predominantly hydrophobic stretches of sequence were predicted to form membrane-spanning helices, and three putative N-glycosylation sites were identified. The AD1 Ag and CD63 were highly conserved between rat and human, suggesting that the sequence of this protein is important for its function. By immunostaining various rat tissues, the AD1 Ag was found localized to mast cells. However, it was located to lysosomes, secretory granules and the plasma membrane of RBL-2H3 cells and to lysosomes and plasma membrane of many other cultured cell lines. The AD1 Ag could be induced by placing cells in culture. Fibroblasts and hepatocytes freshly isolated from rat embryos stained very weakly for AD1 Ag; however, after 24 to 48 h in culture they were strongly positive. This increase in the expression of the AD1 Ag was accompanied by an increase in detectable RNA message. Therefore, AD1/ME491/CD63 Ag is a mast cell marker in tissue, but is also associated with other cells in culture.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Isolation and characterization of the tricarboxylate transporter from pea mitochondria

The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-114, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles. The proteoliposomes exchanged external [¹⁴C]citrate for internal citrate or malate but not for preloaded d,l-isocitrate. Similarly, although external malate, succinate, and citrate competed with [¹⁴C]citrate in the exchange reaction, d,l-isocitrate and phosphoenolpyruvate did not. This tricarboxylate transporter differed from the equivalent activity from animal tissues in that it did not transport isocitrate and phosphoenolpyruvate. In addition, tricarboxylate transport in isolated plant mitochondria, as well as that measured with the partially purified and reconstituted transporter, was less active than the transporter isolated from animal tissues.     

The baboon model under anesthesia for in vivo cerebral blood flow studies using single photon emission computed tomographic (SPECT) techniques.

Single photon emission computed tomography of the brain can be useful in animal experimentation directed toward cerebral conditions. A well established and understood baboon model, necessarily under anesthesia, could be especially valuable in such investigations. Six normal baboons were studied under various anesthetic agents and their combinations: ketamine, thiopentone, pentobarbitone, and halothane. Cerebral blood flow (CBF) studies were performed with 99mTc-HMPAO. CBF effects from various anesthesia were detected, requiring careful choice of the anesthesia for cerebral investigations.     

Effect of organic anions on the crystallization of the Ca2(+)-ATPase of muscle sarcoplasmic reticulum

The effect of varying the solute species on the crystallization of the Ca2(+)-ATPase from rabbit muscle reticulum (SR) is reported. We have found that substitution of KCl with salts of organic acids in the crystallization protocol reported by Pikula et al. has a profound effect on the size of two-dimensional crystalline arrays. Crystalline arrays of up to 3 microns diameter have been obtained by incubating purified calcium ATPase in standard crystallization medium but with 0.8 M sodium propionate substituted for KCl. These two-dimensional (2-D) arrays display a reduced tendency to stack in addition to having larger planar dimensions. Increasing the KCl concentration does not have the same effect on stacking or crystal growth that sodium propionate has. The production of 2-D sheets has some dependence on the hydrocarbon chain length of the salt because crystals formed in propionate were larger and less stacked than those formed in acetate or formate. There seems to be no dependence on cation. These observations suggest that in addition to reducing the forces that lead to stacking of the sheets, propionate may facilitate incorporation of the detergent-solubilized protein into the 2-D sheet.     

Assay of β-galactosidase activity by Flow Injection Analysis (FIA)

Summary A novel method to analyze -galactosidase by Flow Injection Analysis is presented with a linear working range extended to at least 2150 U/mL, being the detection limit 25 U/mL with 55 samples per hour frecuency and a BSD of 0.954% versus 2.4% obtained by manual assay. The method was tested with optimal results with samples from Escherichia coli cultures producing -galactosidase.