Assembly of glycoprotein-80 adhesion complexes in Dictyostelium. Receptor compartmentalization and oligomerization in membrane rafts

The phospholipid-anchored membrane glycoprotein (gp)-80 mediates cell-cell adhesion through a homophilic trans-interaction mechanism during Dictyostelium development and is enriched in a Triton X-100-insoluble floating fraction. To elucidate how gp80 adhesion complexes assemble in the plasma membrane, gp80-gp80 and gp80-raft interactions were investigated. A low density raft-like membrane fraction was isolated using a detergent-free method. It was enriched in sterols, the phospholipid-anchored proteins gp80, gp138, and ponticulin, as well as DdCD36 and actin, corresponding to components found in the Triton X-100-insoluble floating fraction. Chemical cross-linking revealed that gp80 oligomers were enriched in the raft-like membrane fraction, implicating stable oligomer-raft interactions. However, gp80 oligomers resisted sterol sequestration and were partially dissociated with Triton X-100, suggesting that compartmentalization in rafts was not solely responsible for their formation. The trans-dimer known to mediate adhesion was identified, but cis-oligomerization predominated and displayed greater accumulation during development. In fact, oligomerization was dependent on the level of gp80 expression and occurred among isolated gp80 extracellular domains, indicating that it was mediated by direct gp80-gp80 interactions. Rafts existed in gp80-null cells and such pre-existent membrane domains may provide optimal microenvironments for gp80 cis-oligomerization and the assembly of adhesion complexes.     

Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand

Neutrophils release soluble Fas ligand (sFasL), which can induce apoptosis in certain Fas-bearing cell types (Liles WC, Kiener PA, Ledbetter JA, Aruffo A, and Klebanoff SJ. J Exp Med 184: 429-440, 1996). We hypothesized that neutrophils could induce alveolar epithelial apoptosis via release of sFasL. A549 pulmonary adenocarcinoma cells expressed surface Fas and underwent cell death (10 +/- 7% viability) and DNA fragmentation (354 +/- 98% of control cells) when incubated with agonistic CD95/Fas monoclonal antibody (P < 0.05). Coincubation with human neutrophils induced significant A549 cell death at 48 (51 +/- 9% viability; P < 0.05) and 72 h (25 +/- 10%; P < 0.05) and increased DNA fragmentation (178 +/- 42% of control cells; P < 0.05), with morphological characteristics of apoptosis. The addition of antioxidants did not inhibit apoptosis. sFasL concentrations were maximally increased in coculture medium at 24 h (4.9 +/- 0.7 ng/ml; P < 0.05). Neutrophil-induced A549 cell apoptosis was blocked by inhibitory anti-Fas (42 +/- 6% of control cells; P < 0.05) and anti-FasL monoclonal antibodies (29 +/- 3%; P < 0.05). Human neutrophils and Fas similarly affected murine primary alveolar epithelial cell bilayers, and caspase activation occurred in response to Fas exposure. We conclude that neutrophils undergoing spontaneous apoptosis induce A549 cell death and DNA fragmentation, independent of the oxidative burst, that is mediated by sFasL.     

Opposing Roles of the Holliday Junction Processing Systems of Escherichia Coli in Recombination-Dependent Adaptive Mutation

Aspects of the molecular mechanism of ``adaptive' mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.     

Increased cytosol Ca(2+) and type 1 programmed cell death in Bcl-2-positive U937 but not in Bcl-2-negative PC-3 and Panc-1 cells induced by the 5-lipoxygenase inhibitor MK 886

MK 886, an arachidonic acid-related analog which inhibits the enzyme, 5-lipoxygenase by an indirect mechanism involving the 5-lipoxygenase activating protein, rapidly increased U937 cytosol Ca(2+), much of which localized around the cell nuclei. Five-lipoxygenase activity was not directly involved since the direct redox-dependent 5-LPOx inhibitor, SC-41661A did not increase Ca(2+). U937 cells subsequently undergo classic type 1 programmed cell death. At least initially the ionized calcium originates from internal stores. Coincident with the rise in U937 ionized calcium, MK 886 rapidly increased reactive oxygen species and reduced mitochondrial membrane potential, as judged by several fluorescent probes. The Ca(2+) response of myeloid leukemia-derived HL-60 cells to MK 886 was similar and both cell lines express Bcl-2 protein. Bcl-2-negative Panc-1 and PC-3 cells did not respond to MK 886 with a Ca(2+) signal but did develop oxidative stress and a decline in mitochondrial membrane potential; these events are thought to contribute to the inhibition of cell proliferation and induction of a type 2 PCD. In addition to its marked inhibition of Bcl-2 mRNA synthesis, an interesting hypothesis is that MK 886, serving as a low molecular weight ligand, either by direct or indirect inhibition of U937 Bcl-2 protein function, possibly related to an ion channel activity, alters the distribution of intracellular, possibly nuclear Ca(2+), thereby promoting the development of type 1 programmed cell death.     

Forms and distribution of inorganic phosphorus in sediments of two shallow eutrophic lakes in Florida

Physicochemical attributes were measured and aquatic macroinvertebrates were collected from six wetlands near Perth, Western Australia at three weekly intervals over a 13 month period from August 1988 to September 1989. The six wetlands encompassed a range of depths, pH, concuctivities, nutrient concentrations and colours. Temporal changes in the macroinvertebrate communities appeared to be related to seasonal changes in the physical and chemical characteristics of the wetlands. Community composition differed more between the less enriched wetlands then the higly enriched wetlands where communities were generally similar. High species richness was associated with seasonal drying. High macro invertebrate abundance appeared to be associated with the presence of either green algal or cyanobacterial blooms in the enriched wetlands. The highest macroinvertebrate biomass was recorded in wetlands with both cyanobacterial blooms and abundant macrophytes present.     

Systematics and randomly amplified polymorphic DNA in the genusLeucaena (Leguminosae,Mimosoideae)

Randomly amplified polymorphic DNA (RAPD) was used to examine genomic diversity in taxa of the neotropical legume genusLeucaena. Data were analysed using both similarity- and parsimony-based approaches and the data compared to a parsimonybased analysis of chloroplast DNA restriction fragment length polymorphisms (RFLP). Distance-based methods of RAPD analysis produced groups inconsistent with those identified by RFLP analysis. Parsimony-based analysis of the data produced groupings largely consistent with those identified using RFLPs. The major differences were grouping of the two subspecies ofLeucaena diversifolia (subsp.diversifolia and subsp.stenocarpa) in the RAPD tree, but their separation in the RFLP tree. The value of RAPD data in systematics as a result of these data and our understanding of the molecular basis of RAPDs are discussed.     

Glutathione transferase P1‐1 as an arsenic drug‐sequestering enzyme

Arsenic‐based compounds are paradoxically both poisons and drugs. Glutathione transferase (GSTP1‐1) is a major factor in resistance to such drugs. Here we describe using crystallography, X‐ray absorption spectroscopy, mutagenesis, mass spectrometry, and kinetic studies how GSTP1‐1 recognizes the drug phenylarsine oxide (PAO). In conditions of cellular stress where glutathione (GSH) levels are low, PAO crosslinks C47 to C101 of the opposing monomer, a distance of 19.9 Å, and causes a dramatic widening of the dimer interface by approximately 10 Å. The GSH conjugate of PAO, which forms rapidly in cancerous cells, is a potent inhibitor (K_i = 90 nM) and binds as a di‐GSH complex in the active site forming part of a continuous network of interactions from one active site to the other. In summary, GSTP1‐1 can detoxify arsenic‐based drugs by sequestration at the active site and at the dimer interface, in situations where there is a plentiful supply of GSH, and at the reactive cysteines in conditions of low GSH.     

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.     

Reaction of lysyl oxidase with soluble protein substrates: effect of neutral salts.

Soluble proteins released into the medium of aortic tissues in culture behave as substrates for the enzyme lysyl oxidase. The reaction shows an unusual dependence on the concentration of neutral salts in the assay medium. Practically no enzyme activity was observed in Tris-HCl, 0.005 m, pH 7.6 buffer. However, supplementing the buffer with high concentrations of KCl, KBr, NaCl, and (NH₄)₂SO₄ (in decreasing order of effectiveness) accelerated velocities as much as 10-fold. CaCl₂, KSCN, and KI at increasing concentrations became strongly inhibitory. β-Aminopropionitrile, a specific inhibitor of lysyl oxidase, effectively blocked the catalysis in low and high KCl. The salt-stimulated effects on lysyl oxidase activity were not as noticeable when insoluble proteins were used as substrates. Kinetic studies employing double reciprocal plots revealed that high KCl concentrations (2.0 m) raised the maximum velocity of the reaction but did not alter the apparent K_m. Thus high salt concentrations did not affect the binding of the soluble substrate to the enzyme. In high salts, however, more radioactive substrate proteins appeared to bind to the enzyme, suggesting that the high salt environment increases the fraction of the total enzyme potentially capable of binding to and catalyzing a reaction with the substrate.