Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

The development of a population of spinal cord neurons and their axonal projections revealed by GABA immunocytochemistry in frog embryos

The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.     

Depolarized fluorescence photobleaching recovery

The effects of the fact that the laser sources typically used in fluorescence photobleaching recovery (FPR) experiments in the most commonly employed in-line microscope imaging geometries, are highly linearly polarized, are examined in some detail. The implications of the results, in particular for the interpretation of FPR data in complex cell membrane systems in terms of laterally mobile and immobile sub-populations of the labelled molecular species of concern, are discussed. Methods of experimentally eliminating the potentially major rotational diffusion-based artifacts, different from those appropriate to three-dimensional (solution or suspension) systems which require other than in-line geometries, are delineated.Abbreviations FPR fluorescence photobleaching recovery - FRAP fluorescence recovery after photobleaching - 2- and 3-D two- and three-dimensional     

Ion-Exchange Chromatography Separates Activities Synthesizing and Degrading Fructose 2,6-Bisphosphate from C3 and C4 Leaves but Not from Rat Liver

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C₃ (spinach, lettuce, and pea) and C₄ (sorghum and amaranthus) plants but not from rat liver—a tissue known to contain a bifunctional enzyme with both activities. [2-³²P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.     

Effect of cleft palate repair and pharyngeal flap surgery on upper airway obstruction during sleep

The effect of von Langenbeck palatoplasty and pharyngeal flap surgery on upper airway obstruction during sleep was studied by obtaining polysomnographic sleep studies on 10 patients undergoing each procedure at 1 to 2 days prior to surgery, 2 to 3 days postoperatively, and approximately 3 months postoperatively. The effects of von Langenbeck palatoplasty on sleep-related upper airway obstruction were usually minimal and clinically insignificant, whereas severe obstructive sleep apnea was present in all but one of the patients undergoing pharyngeal flap surgery at 2 to 3 days postoperatively. In most patients the upper airway obstruction was resolved at the 3-month postoperative sleep study. These data suggest that palatoplasty carries with it a very slight risk of upper airway obstruction, whereas pharyngeal flap surgery has as a very frequent concomitant the occurrence of severe obstructive sleep apnea in the immediate postoperative period.     

A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver.

1. A rapid extraction and purification scheme was designed for the recovery of [3H]diacylglycerol formed during the assay of phosphatidate phosphohydrolase. 2. The importance of removing polyvalent cations, particularly Ca2+, from the phosphatidate and other reagents used in the assay of the phosphohydrolase activity was demonstrated. This was achieved mainly by treating the phosphatidate with a chelating resin and by adding 1 mM-EGTA and 1 mM-EDTA to the assays. 3. The activity of the phosphohydrolase in dialysed samples of the soluble and microsomal fractions of rat liver was very low. 4. Addition of optimum concentrations of MgCl2 resulted in a 110-167-fold stimulation in activity. 5. CaCl2 was also able to stimulate phosphohydrolase activity, but to a much smaller extent than MgCl2. 6. Chlorpromazine, an amphiphilic cation, inhibited the reaction when it was measured in these experiments by using a mixed emulsion of phosphatidylcholine and phosphatidate at pH 7.4. 7. Microsomal fractions that were preincubated with albumin contained very low activities of the Mg2+-dependent phosphohydrolase. When these were then incubated with the soluble fraction in the presence of oleate, the soluble phosphohydrolase attached to the microsomal membranes, and it retained its high dependency on Mg2+.     

Testosterone inhibits cAMP-induced de Novo synthesis of Leydig cell cytochrome P-450(17 alpha) by an androgen receptor-mediated mechanism

The regulation of the novo synthesis of the microsomal cytochrome P-450 enzyme, P-450(17 alpha), was studied in mouse Leydig cell cultures. Chronic treatment with 0.05 mM 8-Br-cAMP (cAMP) caused a time-dependent increase in 17 alpha-hydroxylase activity and in the amount of P-450(17 alpha), quantitated by immunoblotting. This increase in both activity and amount was enhanced by inhibiting testosterone production with aminoglutethimide, an inhibitor of cholesterol side-chain cleavage or SU 10603, an inhibitor of 17 alpha-hydroxylase. To examine the mechanism by which cAMP or cAMP plus inhibitors of testosterone production increased the activity and amount of P-450(17 alpha), changes in the rate of de novo synthesis were studied by measuring [35S]methionine incorporation into newly synthesized protein. Treatment with cAMP plus aminoglutethimide or SU 10603 caused a 2-fold or greater increase in the rate of de novo synthesis of P-450(17 alpha) compared to treatment with cAMP only. The addition of exogenous testosterone reversed this increase in the rate of synthesis, indicating that testosterone modulates the extent of cAMP-stimulated induction of P-450(17 alpha). This negative effect of testosterone could be mimicked by the addition of the androgen agonist, mibolerone, and prevented by the addition of the antiandrogen, hydroxyflutamide. Neither estradiol nor dexamethasone had any effect on the synthesis of P-450(17 alpha). Studies on the degradation of newly synthesized P-450(17 alpha) demonstrated that testosterone had no effect on the decay of P-450(17 alpha) during the first 24 h but caused a significant increase in the rate of decay between 24 and 48 h. These data indicate that testosterone produced during cAMP induction of P-450(17 alpha) negatively regulates the amount of this cytochrome P-450 enzyme by two distinct mechanisms: by repressing cAMP-induced synthesis of P-450(17 alpha) by an androgen receptor-mediated mechanism and by increasing the rate of degradation of P-450(17 alpha). A model is proposed for the regulation of P-450(17 alpha) in Leydig cells.