A CYTOCHEMICAL STUDY OF CYTOPLASMIC BASIC PROTEINS IN THE ASCIDIAN OOCYTE

The cytoplasm of young oocytes of the ascidians contains high concentrations of proteins which are stainable with alkaline fast green at pH 8.1 and above. These proteins cannot be stained even with acid dyes at low pH unless RNA is removed. Deamination and formalin blockage of amino groups is incapable of destroying the net positive charge on these protein molecules in the presence of RNA, but these treatments destroy the charge if RNA is removed. It is therefore concluded that basic proteins and RNA exist as a nucleoprotein complex in the ribosomes of these young oocytes. The detectable RNA of the mature oocytes and unfertilized eggs shows no evidence of being associated with basic proteins.     

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of timing of inspiratory occlusion on cerebral evoked potentials in humans

Previous studies from these laboratories have shown that airway occlusion applied from the onset of inspiration or during midinspiration is associated with cerebral evoked potentials in human subjects. The hypothesis tested in the present study was that the more abrupt decrease in mouth pressure produced by midinspiratory occlusion will be associated with evoked potentials that have shorter peak latencies and greater peak amplitudes than those produced by occlusions from the onset of inspiration. The second objective of the present study was to determine whether there is bilateral projection of inputs from the respiratory system to the somatosensory cortex. Random presentation of 64 midinspiratory occlusions and 64 occlusions from the onset of inspiration was performed in eight subjects. The inspirations preceding the occlusions served as control. Evoked potentials were recorded from the scalp with electrode pairs Cz-C3 and Cz-C4. Reaction time to each type of occlusion was measured from the burst in electromyogram activity produced by contraction of the muscles encircling the eye. Each type of inspiratory occlusion was associated with evoked potentials that could be recorded bilaterally. The peak amplitudes of the evoked potentials recorded over the right cerebral hemisphere were significantly greater than those recorded from the left side. The peak amplitude was greater and the peak latency shorter for the evoked potentials produced by the midinspiratory occlusions. The results are consistent with the hypothesis that afferents mediating these potentials are stimulated by added loads to breathing and project bilaterally to the somatosensory cortex in humans.     

Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe.

The use of the long-lived fluorescence probe coronene (mean value of tau(FL) approximately 200 ns) is described for investigating submicrosecond lipid dynamics in DPPC model bilayer systems occurring below the lipid phase transition. Time-resolved fluorescence emission anisotropy decay profiles, measures as a function of increasing temperature toward the lipid-phase transition temperature (T(C)), for coronene-labeled DPPC small unilamellar vesicles (SUVs), are best described in most cases by three rotational decay components (phi(i = 3)). We have interpreted these data using two dynamic lipid bilayer models. In the first, a compartmental model, the long correlation time (phi(N)) is assigned to immobilized coronene molecules located in "gel-like" or highly ordered lipid phases (S-->1) of the bilayer, whereas a second fast rotational time (phi(F) approximately 2-5 ns) is associated with probes residing in more "fluid-like" regions (with corresponding lower ordering, S-->0). Interests here have focused on the origins of an intermediate correlation time (50-100 ns), the associated amplitude (beta(G)) of which increases with increasing temperature. Such behavior suggests a changing rotational environment surrounding the coronene molecules, arising from fluidization of gel lipid. The observed effective correlation time (phi(EFF)) thus reflects a discrete gel-fluid lipid exchange rate (k(FG)). A refinement of the compartmental model invokes a distribution of gel-fluid exchange rates (d(S,T)) corresponding to a distribution of lipid order parameters and is based on an adapted Landau expression for describing "gated" packing fluctuations. A total of seven parameters (five thermodynamic quantities, defined by the free energy versus temperature expansion; one gating parameter (gamma) defining a cooperative "melting" requirement; one limiting diffusion rate (or frequency factor: d(infinity))) suffice to predict complete anisotropy decay curves measured for coronene at several temperatures below the phospholipid T(C). The thermodynamic quantities are associated with the particular lipid of interest (in this case DPPC) and have been determined previously from ultrasound studies, thus representing fixed constants. Hence resolved variables are r(O), temperature-dependent gate parameters (gamma), and limiting diffusion rates (d(infinity)). This alternative distribution model is attractive because it provides a general probe-independent expression for distributed lipid fluctuation-induced probe rotational rates occurring within bilayer membranes below the phospholipid phase transition on the submicrosecond time scale.     

Differential distribution of endothelin peptides and receptors in human adrenal gland

Summary Sub-type selective ligands revealed a differential distribution of endothelin (ET) receptors within human adrenal glands. High densities of ET_A receptors were localized, using [¹²⁵I]-PD151242, to the smooth muscle layer of the arteries, smaller vessels within the capsular plexus and to the secretory cells of zona glomerulosa (K _D=139.8±39.7,B _max=69.7±9.1 fmol mg⁻¹ protein, mean of 3 individuals±sem). ET_B receptors were present in the medulla (K _D=145.2±16.4,B _max=75.5±12.3), zona glomerulosa (K_D=100.6±35.1,B _max=63.1±10.0), fasiculata (K _D 145.1±162.,B _max=67.9±6.9) and reticularis (K_D=118.2±18.6,B _max=71.9±6.5). ET_B receptors were not detected within the smooth muscle of the vasculature. Messenger RNA encoding both sub-types was present in adrenals. ET-like immunoreactivity was localized to the cytoplasm of the endothelial cells from arteries supplying the gland and resistance vessels within the capsular plexus. Staining was also detected in these cells using anti-big ET-1 and less intensely with anti-big ET-2 antisera but not within cells within the cortex or medulla. Big ET-3-like immunoreactivity was localized to secretory cells of the medulla. Staining was not found using antiserum that could detect ET-3, suggesting further processing of big ET-3 may occur within the plasma, and that the cdrenals could be a source of ET-3. The presence of ET-1 was confirmed by high performance liquid chromatography and radioimmunoassay although ET-3 was not detected. The results suggest that ET-1 is the predominant mature isoform, which is localized mainly to adrenal vasculature, particularly the capsular plexus, and may contribute to blood flow regulation in the gland.     

Edge effects influence the composition and density of reef residents on subtidal restored oyster reefs

Within estuarine and coastal ecosystems globally, extensive habitat degradation and loss threaten critical ecosystem functions and necessitate widescale restoration efforts. There is abundant evidence that ecological processes and species interactions can vary with habitat characteristics, which has important implications for the design and implementation of restoration efforts aimed at enhancing specific ecosystem functions and services. We conducted an experiment examining how habitat characteristics (presence; edge vs. interior) influence the communities of resident fish and mobile invertebrates on restored oyster (Crassostrea virginica) reefs. Similar to previous studies, we found that restored reefs altered community composition and augmented total abundance and biomass relative to unstructured sand habitat. Community composition and biomass also differed between the edge and interior of individual reefs as a result of species-specific patterns over small spatial scales. These patterns were only weakly linked to oyster density, suggesting that other factors that vary between edge and interior (e.g. predator access or species interactions) are likely more important for community structure on oyster reefs. Fine-scale information on resident species' use of oyster reefs will help facilitate restoration by allowing decision makers to optimize the amount of edge versus interior habitat. To improve the prediction of faunal use and benefits from habitat restoration, we recommend investigations into the mechanisms shaping edge and interior preferences on oyster reefs.     

Actinomycete diversity associated with foaming in activated sludge plants

Large numbers of mycolic acid-containing actinomycetes were isolated from foam and scum samples taken from three activated-sludge sewage-treatment plants using several selective isolation media. Organisms presumptively identified as gordonae formed the dominant population in all of the samples. A representative set of these strains have chemical properties consistent with their classification in the genusGordona. Forty-eight of theGordona strains were compared through 165 unit characters with the type strains of validly described species ofGordona. The resultant data were examined using the Jaccard and simple matching coefficients and clustering achieved using the unweighted pair group method with arithmetic averages algorithm. The numerical classification was only marginally affected by the statistics used or by test error, estimated as 3.92%. The isolates were assigned to five multi-membered and 28 single-membered clusters defined by the simple matching coefficient at the 89% similarity level. With few exceptions, the isolates were sharply separated from theGordona marker strains. Essentially the same classification was obtained when the test strains were examined using a Curie-point pyrolysis mass spectrometric procedure. It can be concluded that the gordonae form a heterogeneous taxonomic group, the members of which can be distinguished from representatives of validly described species ofGordona.