Refined solution structure of human profilin I.

Profilin is a ubiquitous eukaryotic protein that binds to both cytosolic actin and the phospholipid phosphatidylinositol-4,5-bisphosphate. These dual competitive binding capabilities of profilin suggest that profilin serves as a link between the phosphatidyl inositol cycle and actin polymerization, and thus profilin may be an essential component in the signaling pathway leading to cytoskeletal rearrangement. The refined three-dimensional solution structure of human profilin I has been determined using multidimensional heteronuclear NMR spectroscopy. Twenty structures were selected to represent the solution conformational ensemble. This ensemble of structures has root-mean-square distance deviations from the mean structure of 0.58 A for the backbone atoms and 0.98 A for all non-hydrogen atoms. Comparison of the solution structure of human profilin to the crystal structure of bovine profilin reveals that, although profilin adopts essentially identical conformations in both states, the solution structure is more compact than the crystal structure. Interestingly, the regions that show the most structural diversity are located at or near the actin-binding site of profilin. We suggest that structural differences are reflective of dynamical properties of profilin that facilitate favorable interactions with actin. The global folding pattern of human profilin also closely resembles that of Acanthamoeba profilin I, reflective of the 22% sequence identity and approximately 45% sequence similarity between these two proteins.     

Detection and discrimination of individual viruses by flow cytometry.

A new flow cytometer with a very small observation volume has been developed to detect individual viruses with good resolution, and has been used to discriminate between two types of viral particles based on differences in their light scattering. Measurements of light scattering and fluorescence made with such an instrument can provide a basis for quantitative analysis and sorting of viruses and other particles in the micron and submicron size range.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.     

Degradation of HeLa S3 cell chromatin by auromomycin and its chromophore

The antitumor protein agent auromomycin was found to degrade chromatin structure primarily by inducing strand scissions in linker regions. The reaction was stimulated by dithiothreitol. The chromophore form of the drug caused similar effects on chromatin, but it appeared to function at a more rapid rate. There was no evidence that auromomycin could cause breakage in core regions of chromatin.     

Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells

Tyrosine phosphorylation of membrane-associated proteins is involved at two distinct sites of contact between cells and the extracellular matrix: adhesion plaques (cell adhesion and de-adhesion) and invadopodia (invasion into the extracellular matrix). Adhesion plaques from chicken embryonic fibroblasts or from cells transformed by Rous sarcoma virus contain low levels of tyrosine-phosphorylated proteins (YPPs) which were below the level of detection in 0.5-microns thin, frozen sections. In contrast, intense localization of YPPs was observed at invadopodia of transformed cells at sites of degradation and invasion into the fibronectin-coated gelatin substratum, but not in membrane extensions free of contact with the extracellular matrix. Local extracellular matrix degradation and formation of invadopodia were blocked by genistein, an inhibitor of tyrosine-specific kinases, but cells remained attached to the substratum and retained their free-membrane extensions. Invadopodia reduced or lost YPP labeling after treatment of the cells with genistein, but adhesion plaques retained YPP labeling. The plasma membrane contact fractions of normal and transformed cells have been isolated form cells grown on gelatin cross-linked substratum using a novel fractionation scheme, and analyzed by immunoblotting. Four major YPPs (150, 130, 81, and 77 kD) characterize invadopodial membranes in contact with the matrix, and are probably responsible for the intense YPP labeling associated with invadopodia extending into sites of matrix degradation. YPP150 may be an invadopodal-specific YPP since it is approximately 3.6-fold enriched in the invasive contact fraction relative to the cell body fraction and is not observed in normal contacts. YPP130 is enriched in transformed cell contacts but may also be present in normal contacts. The two major YPPs of normal contacts (130 and 71 kD) are much lower in abundance than the major tyrosine-phosphorylated bands associated with invadopodial membranes, and likely represent major adhesion plaque YPPs. YPP150, paxillin, and tensin appear to be enriched in the cell contact fractions containing adhesion plaques and invadopodia relative to the cell body fraction, but are also present in the soluble supernate fraction. However, vinculin, talin, and alpha-actinin that are localized at invadopodia, are equally concentrated in cell bodies and cell contacts as is the membrane-adhesion receptor beta 1 integrin. Thus, tyrosine phosphorylation of the membrane-bound proteins may contribute to the cytoskeletal and plasma membrane events leading to the formation and function of invadopodia that contact and proteolytically degrade the extracellular matrix; we have identified several candidate YPPs that may participate in the regulation of these processes.     

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis

The processes leading to bacterial colonization on solidwater interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 mum (for silicon) to 0.015 mum (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varried by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.     

Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463.

A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Br?nnstr?m et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH.