Homologies in Cambrian Onychophora

Marine animals related to Recent onychophorans form a significant component in Cambrian faunas. Twelve characters are analysed for homologies in the seven best known Cambrian onychophorans. New morphological evidence and homology analyses for several characters indicate an anteroposterior reversal of Hallucigenia and Microdictyon . Proposed expansion of the trunk in Microdietyon during compaction is rejected. A jaw is tentatively identified in Onychodictyon . The shape of the annulations and the disposition of the tenth leg pair in Aysheaia are reinterpreted, and the suggestion of two somites to the first appendage pair is rejected. A suggested morphocline may mirror the phylogeny of the group. The taxonomic confusion surrounding the supposed radiolarian family Eoconchariidae is cleared     

Characterization of the overall and internal dynamics of short oligonucleotides by depolarized dynamic light scattering and NMR relaxation measurements

The dynamics of three synthetic oligonucleotides d(CG)4, d(CG)6, and d(CGCGTTGTTCGCG) of different length and shape were studied in solution by depolarized dynamic light scattering (DDLS) and time-resolved nuclear Overhauser effect cross-relaxation measurements. For cylindrically symmetric molecules the DDLS spectrum is dominated by the rotation of the main symmetry axis of the cylinder. The experimental correlation times describe the rotation of the oligonucleotides under hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre gives good predictions of the rotational diffusion coefficients of cylindrically symmetric molecules of the small axial ratios studied here. These relations are used to calculate the solution dimensions of the DNA fragments from measured correlation times. The hydrodynamic diameter of the octamer and dodecamer is 20.5 +/- 1.0 A, assuming a rise per base of 3.4 A. The tridecamer, d(CGCGTTGTTCGCG), adopts a hairpin structure with nearly spherical dimensions and a diameter of 23.0 +/- 2.0 A. The DDLS relaxation measurements provide a powerful method for distinguishing between different conformations of the oligonucleotides (e.g., DNA double-helix versus hairpin structure). Furthermore, the rotational correlation times are a very sensitive probe of the length of different fragments. The NMR results reflect the anisotropic motion of the molecules as well as the amount of local internal motion present. The experimental correlation time from NMR is determined by the rotation of both the short and long axes of the oligonucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen bonding modulates binding of exogenous ligands in a myoglobin proximal cavity mutant.

In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. (1)H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.     

Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA

COS-1 cells bearing FcgammaRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-delta (PKCdelta) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcgammaRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcgammaRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.     

Deposition of reactive oxygen metabolites onto and within living tumor cells during neutrophil-mediated antibody-dependent cellular cytotoxicity

In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5′ (or 6′) carboxyl-2′,7′-dichlorodihy-drofluorescein (H₂-CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H₂-CDCF-conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H₂-CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H₂-CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethyl-rosamine within tumor cells. Using two-color automated epifluorescence micros-copy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (<12) sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant-mediated damage. © 1993 Wiley-Liss, Inc.