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Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

A method is outlined that completely separates intracellular and extracellular information in NMR spectra of perfused cells. The technique uses diffusion weighting to exploit differences in motional properties between intra- and extracellular constituents. This allows monitoring of intracellular metabolism, and of transport of small drugs and nutrients through the cell membrane, under controlled physiological conditions. As a first example, proton spectra of drug-resistant MCF-7 human breast cancer cells are studied, and uptake of phenylalanine is monitored.

In this report, we demonstrate the sequence-specific suppression of viral expression in T cells chronically infected with human immunodeficiency virus 1 (HIV-1), using antisense phosphorothioate oligodeoxynucleotides. As a target for antisense intervention, we used the HIV-1 gene rev, which is essential for viral replication and regulates the expression of virion proteins, in part, by affecting the splicing of the viral mRNA. A phosphorothioate oligomer complementary to the initiation sequence of HIV-1 rev had a significant and selective inhibitory effect on the production of several viral proteins in chronically HIV-1-infected T cells and drastically reduced the unspliced (genomic) viral mRNA transcripts, with relative sparing of smaller (spliced) transcripts. By contrast, the antisense sequence with unmodified normal phosphodiester linkages as well as phosphorothioate oligomers containing sense, random, homopolymeric sequences, or antisense sequence with N3-methylthymidine residues did not have an inhibitory effect on viral expression. Thus, sequence specificity and nuclease resistance were critical for the anti-viral-gene regulatory effect of the antisense molecules. The altered HIV-1 mRNA profile induced by the antisense phosphorothioate oligomer suggests that the mechanism for the inhibition of viral expression is due to an interference with the regulatory gene, rev, by translation arrest.

Nuclease-resistant phosphorothioate analogs of certain oligodeoxynucleotides have been tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Phosphorothioate analogs complementary to HIV sequences, as well as noncomplementary analogs including homooligomers, exhibited potent antiviral activity. The antiviral activity was related to the base composition of the analogs, and longer phosphorothioates were more effective than shorter ones. A 28-mer phosphorothioate oligodeoxycytidine (S-dC28) at a concentration of 1 microM exhibited potent antiviral activity and inhibited de novo viral DNA synthesis as shown by Southern blot analysis. However, S-dC28 failed to inhibit gag expression in chronically infected T cells assessed by immunofluorescent assay at concentrations up to 25 microM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanism of action is likely to be complex.

Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptor-mediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.

Growth of germinal cells at different angular positions within the posterior portion of the embryonic frog eye has been examined by orthotopically transplanting small groups of germinal cells from pigmented (stage 30-38) donor embryos into albino (stage 28-36) hosts and then serially photographing the polyclonal-cell progeny domain (typically a black sector) in the pigmented retinal epithelium of the living, growing eye. Far-ventral (6 o'clock) germinal cells formed a narrow sector along the ventral fissure, but ventral germinal cells at a position just posterior to the fissure (7 o'clock on a right eye) were seen to expand rapidly their angular territory on the germinal zone and formed huge sectors that widened toward the front of the older larval eye. Posterior (8, 9, and 10 o'clock) germinal cells were seen to shift their angular positions gradually toward dorsal and formed sectors that appeared to veer dorsalward nearing the front of the older eye. Dorsal (11 o'clock) germinal cells showed attenuative growth, forming sectors that narrowed approaching the front of the older eye. A simulation model of the growth dynamic was used to examine how expansive growth ventrally drives the positional variations in growth. When far-ventral germinal cells were programmed to retain the 6 o'clock position and ventral (7 o'clock) germinal cells were programmed to divide symmetrically at a high probability to produce two daughter germinal cells, not only were the observed ventral chimeric patterns simulated, but also simulated were the attenuative growth of dorsal transplants and the dorsal displacement and veering seen in the growth of posterior transplants.

Between 2.5 and 4 days of development, cell proliferation in the Xenopus eye becomes confined to a narrow ring of germinal cells at the front rim of the eye cup. Continued growth of the eye (which lasts until well beyond metamorphosis) is by the continued proliferation of cells in this germinal zone. To determine what factor(s) promotes cell division in this region of the eye long after it ceases at the back of the eye (near the optic nerve), we have transplanted small groups of eye cells from pigmented donor embryos into the eyes of albino hosts, transposing cells from the mitotically quiescent back of the eye to the germinal zone and vice versa. Regardless of their position of origin in the donor eye, only implants into the host germinal zone behaved like germinal cells--as assayed in the living growing eye by the addition of black tissue to the pigment retinal epithelium. Conversely when donor germinal cells were implanted into the back of the host eye, they ceased dividing once they became integrated into the eye and remained as a tiny black spot on the back of the host eye. This suggests that local environmental cues, rather than intrinsic cellular determinants, specify the fates of eye cells ensuring that cells on the eye rim will continue to function as germinal cells while others will withdraw from the cell cycle.

Several of the carboxyl carbon atom resonances of hen egg-white lysozyme (mucopeptide N-acetylmuramoyl hydrolase, EC 3.2.1.17) have been resolved by 13C-nuclear magnetic resonance (NMR) at 68 MHz. The change in chemical shift of the carboxyl carbon atom resonances, as a function of pH, has enabled the distinction of these resonances against the background of many nontitrating carbonyl group resonances. Several apparent microscopic ionization constants have been determined from the carboxyl group NMR titration curves, and possible assignments are discussed. Preliminary experiments were carried out in the presence of cobaltous ion, and selective shifts of several resonances were observed. Our results indicate the possibility of the direct observation of a wide range of single functional groups of proteins in solution by NMR techniques.

We have examined the process by which small groups of pigmented germinal cells transplanted orthotopically from stage 30-38 donor embryos into stage 28-38 albino hosts contribute new postmitotic cells to the pigmented retinal epithelium of the growing larval eye in Xenopus. In the great majority of chimeric eyes, the transplant healed to occupy a small arc-territory at the intended dorsal or anterior position on the host germinal zone. Over the course of subsequent weeks, the transplanted germinal cells added new mitotically quiescent cells to the distal rim of the pigmented retinal epithelium and so gave rise to an elongating black sector on the growing larval eye. Cellular details at the boundaries of the graft-derived sector were stable over time; the accumulation of such landmarks provided a summary record--in the proximodistal axis of the older eye--of the growth history of the transplant. Case-to-case variation among both groups of transplants suggested a measure of indeterminancy in the details of germinal cell growth.

The reversible unfolding and folding of staphylococcal nuclease in the acid transition has been studied by 220 MHz proton magnetic resonance spectroscopy. The values of area, line-width, and chemical shift of each of the imidazole C2 proton resonances of the four histidine residues have been measured in this transition. The change of areas of three histidine resonances and the change of fluorescence of the single tryptophan residue, as a function of pH, appear to follow a single equilibrium. In contrast, a fourth histidine resonance follows a biphasic transition. These findings indicate that local conformational changes can be detected by magnetic resonance spectroscopy in the cooperative transition of the overall structure.

The possible mechanisms of action of bovine pancreatic ribonuclease are discussed in the light of the detailed knowledge of the geometry of the active site that has been derived from studies of inhibitor binding by X-ray diffraction and nuclear magnetic resonance. When combined with a knowledge of the mechanism of phosphate ester hydrolysis, this information imposes severe geometric constraints on possible mechanisms of action of the enzyme. Two types of mechanism can be distinguished, the linear and the pseudorotation. The linear mechanism includes a catalytic role for both histidine residues at the active site and does not involve pseudorotation of the intermediate. In contrast, in the pseudorotation mechanism one histidine residue performs all the catalytic functions, while the other serves only to bind the phosphate anion; this necessarily involves pseudorotation of the intermediate and specific protonation of the leaving group by the enzyme. The mode of binding of the product of the reaction, cytidine-3'-monophosphate, has been elucidated by X-ray diffraction and nuclear magnetic resonance. If the substrate binds in an analogous way, only the linear mechanism is possible. This mechanism is described in detail.