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.     

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.     

15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate

The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5-15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex [(1982) Biochemistry 21, 5831-5838]. The 15N spectra of the binary enzyme-folate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7-proton of bound folate, located by two-dimensional exchange spectroscopy.     

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.     

Sequence diversity of pistil S-proteins associated with gametophytic self-incompatibility in Nicotiana alata

Summary In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the S_z-, S_F11-, S₁-, and S_a-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published S₂-, S₃-, and S₆-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with S_z and S_F11 in one class and S₁, S₂, S₃, and S₆ in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.