Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells.

A new property of ursolic acid, lipoxygenase and cyclooxygenase inhibition, has been described in an acetone-extract of heather flowers (Calluna vulgaris) which could help explain the anti-inflammatory characteristics of this plant. In mouse peritoneal macrophages, human platelets and differentiated HL60 leukemic cells, ursolic acid, at 1 microM, blocks arachidonate metabolism.     

Mitochondrial DNA Length Variation and Heteroplasmy in Populations of White Sturgeon (Acipenser Transmontanus)

Southern blot analysis was used to quantify the extent of mtDNA D-loop length variation in two populations of white sturgeon, Acipenser transmontanus. Over 42% of individuals were heteroplasmic for up to six different mtDNA length variants attributable to varying copy numbers of an 82-bp repeat sequence. Chi-square analyses revealed that the frequencies of length genotypes and the incidence of heteroplasmy were significantly different between Fraser and Columbia River sturgeon populations but not between restriction site haplotypes. Heteroplasmic fish have, on average, higher copy number than homoplasmic fish. Forty-five of 101 homoplasmic individuals carry only a single copy of the repeat, while none of the 73 heteroplasmic fish has the single repeat as the predominant variant. On the basis of differences in frequency distributions of copy number within and between fish, we suggest that (1) heteroplasmy is maintained by high recurrent mutation of multiple copy genomes, favoring increased copy number and (2) the mutation pressure toward higher copy number heteroplasmy is partially offset by selection to reduced genome size and segregation to the homoplasmic condition.     

A Novel Mitochondrial Genome Organization for the Blue Mussel, Mytilus Edulis

The sequence of 13.9 kilobases (kb) of the 17.1-kb mitochondrial genome of Mytilus edulis has been determined, and the arrangement of all genes has been deduced. Mytilus mitochondrial DNA (mtDNA) contains 37 genes, all of which are transcribed from the same DNA strand. The gene content of Mytilus is typically metazoan in that it includes genes for large and small ribosomal RNAs, for a complete set of transfer RNAs and for 12 proteins. The protein genes encode the cytochrome b apoenzyme, cytochrome c oxidase (CO) subunits I-III, NADH dehydrogenase (ND) subunits 1-6 and 4L, and ATP synthetase (ATPase) subunit 6. No gene for ATPase subunit 8 could be found. The reading frames for the ND1, COI, and COIII genes contain long extensions relative to those genes in other metazoan mtDNAs. There are 23 tRNA genes, one more than previously found in any metazoan mtDNA. The additional tRNA appears to specify methionine, making Mytilus mtDNA unique in having two tRNA(Met) genes. Five lengthy unassigned intergenic sequences are present, four of which vary in length from 79 to 119 nucleotides and the largest of which is 1.2 kb. The base compositions of these are unremarkable and do not differ significantly from that of the remainder of the mtDNA. The arrangement of genes in Mytilus mtDNA is remarkably unlike that found in any other known metazoan mtDNA.     

Characterization of a cytosolic fucosylation pathway in Dictyostelium.

FP21 is a 21-kDa fucoprotein which fractionates with the cytosol after high-speed centrifugation of gently lysed Dictyostelium cells. Less than 0.7% of FP21 is associated with vesicles. In proliferating cells, 4 x 10(5) fucosyl moieties/cell are associated with FP21 as anionic, possibly O-linked oligosaccharides equal in size to 4.8 glucose units. FP21 is underfucosylated in a mutant strain (HL250) that depends on extracellular fucose for synthesis of GDP-fucose. To determine the cellular site of FP21 fucosylation, cytosolic and vesicular preparations from strain HL250 were compared for their ability to transfer fucose from GDP-fucose to FP21. Cytosolic preparations fucosylate endogenous FP21 in a time-, concentration-, and divalent cation-dependent fashion, with a Km for GDP-fucose of 1.4 microM. Activity in normal cell cytosol is dependent on exogenous mutant FP21, demonstrating that FP21 is normally fully fucosylated. Both mutant and normal cytosols are also able to alpha-fucosylate a type 1 glycolipid substrate (8-methoxycarbonyloctyl-Gal beta 1-3 beta GlcNAc), but not related substrates, with Km values for the type 1 glycolipid of 0.99 mM and for GDP-fucose of 1.6 microM. Competitive inhibition between FP21 and the type 1 glycolipid shows that the same enzyme fucosylates both substrates. Intact and permeabilized vesicle preparations from wild-type cells are unable to fucosylate FP21 or the type 1 glycolipid by a divalent cation-dependent mechanism, and thus are devoid of FP21-fucosyltransferase. Since control experiments showed that vesicle leakage is minimal during cytosol preparation, these results indicate that FP21 is synthesized and fucosylated in the cytosolic compartment, by an unusual soluble fucosyltransferase.     

The morphology but not the function of endosomes and lysosomes is altered by brefeldin A

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.     

Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice

When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.     

γ-Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Equilibrium binding interactions at the gamma-aminobutyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor-Cl- ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cl- flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (KD) values for [3H]muscimol and [3H]diazepam bindings are 1.9 microM and 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36Cl- flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.     

Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.     

A single nonpolar residue in the deep pore of related K+ channels acts as a K+:Rb+ conductance switch.

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.