A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants. In this paper, we derive a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7-positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. Therefore, P4M reports a wider cellular distribution of PtdIns4P than previous probes and therefore will be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments.     

Ultrastructure of type-I salivary-gland acini in four species of ticks and the influence of hydration states on the type-I acini ofAmblyomma americanum

We describe the ultrastructure of type-I salivary-gland acini in two argasid and two ixodid species. The basic cell types in the agranular or type-I acini, and their associations, are very similar in argasids and ixodids; therefore, we propose an anatomical nomenclature for cells in the type-I acinus based on the adult ixodidsAmblyomma americanum andDermacentor variabilis, and the argasid adultArgas (Persicargas) arboreus and on nymphalOrnithodoros moubata. Four cell types were present in all specimens: one central lamellate cell, a variable number of peripheral lamellate cells, a variable number of peritubular cells depending on the species, and one circumlumenal cell. The lamellate cells had infolded basal plasma membranes that presented an amplified surface area to the hemolymph. These cells most likely secreted the fluid involved in water vapor uptake by ticks. ForAmblyomma americanum females, abundant K⁺-dependent, ouabain-sensitive Na⁺, K⁺-ATPase complexes were located on the infolded basal plasma membranes of the lamellate cells. Apical membranes of the lamellate cells, and plasma membranes of other cell types in the acinus had little or no evidence of Na⁺, K⁺-ATPase activity. Only the central lamellate cell extended from the hemolymph of the acinus to its lumen; peripheral cells did not contact the lumen. Except when the ticks were rehydrating, lipid inclusions were common features in the lamellate cells of the ixodids. Lipid inclusions were not seen in argasid type I acini; however, glycogen deposits were common. To determine if acinar cells respond to the changing hydration state of the tick, unfed femaleA. americanum were subjected to dehydration/rehydrating conditions. During rehydration, mitochondria in the lamellate cells changed from a matrix of medium electron-density and intermembrane space (orthodox configuration) to a matrix of greater density and larger intermembrane space (condensed configuration). The orthodox configuration was consistently observed in control and dehydrating ticks. The condensed configuration was the norm for mitochondria in lamellate cells of rehydrating ticks. Lipid inclusions were depleted in the rehydrating ticks compared to control or dehydrating ticks. Acini appeared to be reverting to the control or desiccated state when ticks were returned to low humidity, suggesting that these changes were cyclical. Nymphs ofO. moubata subjected to the same dehydration/rehydrating conditions showed no obvious ultrastructural changes.     

Expression of renal cell protein markers is dependent on initial mechanical culture conditions.

The rotating wall vessel is optimized for suspension culture, with laminar flow and adequate nutrient delivery, but minimal shear. However, higher shears may occur in vivo. During rotating wall vessel cultivation of human renal cells, size and density of glass-coated microcarrier beads were changed to modulate initial shear. Renal-specific proteins were assayed after 2 days. Flow cytometry antibody binding analysis of vitamin D receptor demonstrated peak expression at intermediate shears, with 30% reduction outside this range. Activity of cathepsin C showed the inverse pattern, lowest at midshear, with twofold increases at either extreme. Dipeptidyl-peptidase IV had no shear dependence, suggesting that the other results are specific, not universal, changes in membrane trafficking or protein synthesis. On addition of dextran, which changes medium density and viscosity but not shear, vitamin D receptor assay showed no differences from controls. Neither cell cycle, apoptosis/necrosis indexes, nor lactate dehydrogenase release varied between experiments, confirming that the changes are primary, not secondary to cell cycling or membrane damage. This study provides direct evidence that mechanical culture conditions modulate protein expression in suspension culture.     

Ethylene production by axenic fruiting cultures of Agaricus bisporus

Axenic cultures of Agaricus bisporus were used to show that the rise in ethylene production during fruiting derives from its own metabolism.     

Mechanism of action of 2-hydroxyestradiol on steroidogenesis in ovarian granulosa cells: interactions with catecholamines and gonadotropins involve cyclic adenosine monophosphate

Previously we have shown that 2-hydroxyestradiol (2-OH-E2) synergizes with catecholamines to enhance progesterone production by porcine granulosa cells in vitro. The present studies were undertaken to determine if the synergistic effects of 2-OH-E2 and catecholamines were 1) modulated by gonadotropins, 2) unique to catecholamines, and 3) mediated by cyclic adenosine monophosphate (cAMP). Undifferentiated granulosa cells from 1- to 3-mm porcine follicles were cultured in serum-free medium for periods of 6-9 days. A 3-day pretreatment plus a 4-day cotreatment versus a 4-day cotreatment of granulosa cell cultures with follicle-stimulating hormone (FSH) did not significantly alter progesterone production stimulated by a saturating concentration of epinephrine (EPI; 2 micrograms/ml) but significantly reduced the effect of 4 micrograms/ml 2-OH-E2 on Day 7 of culture. Four-day cotreatment of either FSH or luteinizing hormone (LH) from Day 3 to 7 of culture dramatically enhanced progesterone production stimulated by 2-OH-E2 and estradiol (E2) but not by EPI when measured on Day 7 of culture. Progesterone production (expressed as "-fold of controls") stimulated by 4-day treatment of EPI, 2-OH-E2, or EPI-plus-2-OH-E2 was 1.4 +/- 0.2, 8.2 +/- 2.2, and 10.7 +/- 1.0, respectively, in the presence of LH (n = 5 experiments), and 1.9 +/- 0.1, 7.8 +/- 1.4, and 10.6 +/- 1.8, respectively, in the presence of FSH (n = 3 experiments). Similar to E2, 2-OH-E2 significantly enhanced the stimulating effect of the cAMP analog 8-bromo-cAMP (0.5 mM) on progesterone production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.