Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early secretory pathway.

The small GTPase Rab2 immunolocalizes to vesicular tubular clusters (VTCs) that function as transport complexes carrying cargo between the endoplasmic reticulum and the Golgi complex. Our previous studies showed that Rab2 promotes vesicle formation from VTCs and that the released vesicles are enriched in beta-coat protein, protein kinase C iota/lambda (PKCiota/lambda), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the recycling protein p53/gp58. Because PKCiota/lambda kinase activity was necessary for vesicle formation, a search was initiated to identify the substrate(s) that potentiate Rab2 function within VTCs. In this study, we found that PKCiota/lambda phosphorylates GAPDH. Moreover, GAPDH interacts directly with the PKCiota/lambda regulatory domain. Based on numerous observations that show (beta-COP) GAPDH associates with cytoskeletal elements, we examined the role of phospho-GAPDH in promoting microtubule (MT) binding to membrane. Using a quantitative microsomal binding assay, we found that membrane association of beta-tubulin was dependent on phospho-GAPDH and was blocked by reagents that interfere with Rab2-dependent GAPDH membrane recruitment or with PKCiota/lambda kinase activity. Furthermore, normal rat kidney cells transfected with a constitutively activated form of Rab2 (Q65L) or with our anti-GAPDH polyclonal antibody displayed a dramatic change in MT organization. These combined results suggest that Rab2 stimulated PKCiota/lambda and GAPDH recruitment to VTCs, and the subsequent PKCiota/lambda phosphorylation of GAPDH ultimately influences MT dynamics in the early secretory pathway.     

Purification of ornithine aminotransferase by immunoadsorption

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.     

Consequences of increasing or decreasing plasma FSH concentrations during the preovulatory period in Romney ewes

Romney ewes were infused with ovine FSH (NIADDK-oFSH-16) for 48 h from the initiation of luteolysis with cloprostenol. Doses of 2.5 or 5 micrograms/h which partly or completely prevented the normal preovulatory decline in plasma FSH concentrations caused a significant increase in mean ovulation rates. Ovulation rates were not increased significantly if the FSH (5 micrograms/h) was infused for only 20 h starting from the initiation of luteolysis or 24 h later. Infusion of a less potent and relatively impure preparation of FSH (i.e. FSH-P) at 0.5 mg/h for 48 h after cloprostenol treatment also increased the mean ovulation rate significantly. However, if the FSH-P was given for only the first 24 h, or if the start of the infusion was delayed for more than 12 h, mean ovulation rates were not increased significantly. Infusion of LH (NIADDK-oLH-25, 5 micrograms/h) for 48 h from the initiation of luteolysis decreased the mean ovulation rate significantly. Administration of bovine follicular fluid to suppress plasma FSH concentrations below normal during the first 24 h after cloprostenol injection did not delay oestrus. However, oestrus was delayed by approximately 2 days if plasma FSH concentrations were reduced by bovine follicular fluid 24 h after the initiation of luteolysis. As ovulation rate increased, the mean weight of individual corpora lutea of each ewe decreased. In ewes with a single ovulation, most corpora lutea weighed greater than 600 mg, but as the ovulation rate increased the proportion of corpora lutea present weighing less than 400 mg rose steadily.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Simplified Geometric Model to Predict Nasal Spray Deposition in Children and Adults

A mathematical approach was developed to estimate spray deposition patterns in the nasal cavity based on the geometric relationships between the emitted spray plume and the anatomical dimensions of the nasal valve region of the nasal cavity. Spray plumes were assumed to be spherical cones and the nasal valve region was approximated as an ellipse. The effect of spray plume angle (15–85°) on the fraction of the spray able to pass through the nasal valve (deposition fraction) was tested for a variety of nasal valve (ellipse) shapes and cross-sectional areas based on measured dimensions from pediatric and adult nasal cavities. The effect of the distances between the tip of the nasal spray device and the nasal valve (0.2–1.9 cm) on the deposition fraction was also tested. Simulation results show that (1) decreasing spray plume angles resulted in higher deposition fractions, (2) deposition fraction was inversely proportional to the spray distance and the nasal valve (ellipse) major/minor axis ratio, and (3) for fixed major/minor axis ratios, improved deposition occurred with larger nasal valve cross-sectional areas. For a typical adult nasal valve, plume angles of less than 40° emitted from a distance of 1 cm resulted depositions greater than 90% within the main nasal cavity, whereas for a 12-year-old child, only the most narrow plume angles (<?20°) administered resulted in significant deposition beyond the nasal valve.