A human monoclonal antibody to a complex epitope in the V3 region of gp120 of human immunodeficiency virus type 1 has broad reactivity within and outside clade B.

We have used virus neutralization and antibody-binding techniques to define the epitope for a human monoclonal antibody, designated 19b, within the V3 region of the gp120 surface glycoprotein of human immunodeficiency virus type 1. Unusually, the 19b epitope encompasses residues on both flanks of the V3 loop. However, 19b binding to gp120 is independent of sequences at the crown of the V3 loop, provided that they are compatible with the formation of a type II beta turn that is presumably necessary to juxtapose the antigenic residues on the V3 flanks. By comparing the V3 sequences of virus gp120s able and unable to bind 19b, we were able to define the canonical 19b epitope as -I----G--FY-T, where residues at the positions indicated by the gaps do not contribute directly to the 19b-binding site. A few conservative substitutions at the more critical residues are also compatible with 19b binding. Inspection of V3 sequences in the human immunodeficiency virus database indicated that the canonical 19b epitope is well conserved among isolates from the North American-European clade B and also among clade E isolates from Thailand and clade F isolates from Brazil. A minority of gp120s from clades A and C also possess the 19b epitope. Consistent with the theoretical predictions of its cross-clade reactivity, 19b was found to bind to gp120s from clades A, B, C, E, and F in immunoassays. However, 19b was not able to reduce the infectivity of primary viruses from clades A, E, and F that were predicted to possess the 19b epitope and only modestly reduced the infectivity of a clade C virus at low input virus concentrations. Cross-clade neutralization via V3-directed antibodies may, therefore, be difficult, even if the antibodies show broad reactivities in binding assays and the viruses theoretically possess the relevant binding site.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

Bootlegging on a desert mountain: The political ecology of Agave (Agave spp.) demographic change in the Sonora river valley,Sonora, Mexico

Recent studies suggest that wild agave (Agavespp.) plants in Sonora, Mexico, are being over-harvested by mescal makers on communal lands. Using the conceptual framework of regional political ecology (Blaikie and Brookfield, 1987), I discuss the ecological processes of agave depletion, and investigate the social, economic, and political contexts in which unsustainable harvest practices arise. Whereas all the mescal makers have knowledge of sustainable harvest methods, population growth, expansion of agriculture onto ecologically marginal lands, and increasing dependence on wild harvested products from communal lands created the socioeconomic context for increased demand for mescal income. The ideology of household autonomy, and the belief that the village has no right to internally regulate use of the commons, created the political context for rapid, unsustainable harvesting—a tragedy of the commons. However, recent cultural changes have caused a reversal of this trend, and some wild agave populations may be recovering.     

Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A

Winder, W. W., H. A. Wilson, D. G. Hardie, B. B. Rasmussen,C. A. Hutber, G. B. Call, R. D. Clayton, L. M. Conley, S. Yoon, and B. Zhou. Phosphorylation of rat muscle acetyl-CoA carboxylase byAMP-activated protein kinase and protein kinase A. J. Appl. Physiol. 82(1): 219-225, 1997This studywas designed to compare functional effects of phosphorylation of muscleacetyl-CoA carboxylase (ACC) by adenosine 3,5-cyclicmonophosphate-dependent protein kinase (PKA) and by AMP-activatedprotein kinase (AMPK). Muscle ACC (272 kDa) was phosphorylated and thensubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresisfollowed by autoradiography. Functional effects of phosphorylation weredetermined by measuring ACC activity at different concentrations ofeach of the substrates and of citrate, an activator of the enzyme. Themaximal velocity(V_max) and theMichaelis constants(K_m) for ATP,acetyl-CoA, and bicarbonate were unaffected by phosphorylation by PKA.Phosphorylation by AMPK increased theK_m for ATP andacetyl-CoA. Sequential phosphorylation by PKA and AMPK, first withoutlabel and second with label, appeared to reduce the extent of label incorporation, regardless of the order. The activation constant (K_a) forcitrate activation was increased to the same extent by AMPKphosphorylation, regardless of previous or subsequent phosphorylation by PKA. Thus muscle ACC can be phosphorylated by PKA but with noapparent functional effects on the enzyme. AMPK appears to be the moreimportant regulator of muscle ACC.

     

Ventilatory response to helium-oxygen breathing during exercise: effect of airway anesthesia

Krishnan, Bharath S., Ron E. Clemens, Trevor A. Zintel,Martin J. Stockwell, and Charles G. Gallagher. Ventilatory response to helium-oxygen breathing during exercise: effect of airwayanesthesia. J. Appl. Physiol. 83(1):82-88, 1997.The substitution of a normoxic helium mixture(HeO₂) for room air (Air) during exercise results in a sustained hyperventilation, which is present evenin the first breath. We hypothesized that this response is dependent onintact airway afferents; if so, airway anesthesia (Anesthesia) shouldaffect this response. Anesthesia was administered to the upper airwaysby topical application and to lower central airways by aerosolinhalation and was confirmed to be effective for over 15 min. Subjectsperformed constant work-rate exercise (CWE) at 69 ± 2 (SE) % maximal work rate on a cycle ergometer on three separate days: twiceafter saline inhalation (days 1 and3) and once after Anesthesia(day 2). CWE commenced after a briefwarm-up, with subjects breathing Air for the first 5 min (Air-1),HeO₂ for the next 3 min, and Airagain until the end of CWE (Air-2). The resistance of the breathingcircuit was matched for Air andHeO₂. BreathingHeO₂ resulted in a small butsignificant increase in minute ventilation(I) anddecrease in alveolar PCO₂ in both theSaline (average of 2 saline tests; not significant) and Anesthesiatests. Although Anesthesia had no effect on the sustainedhyperventilatory response to HeO₂breathing, theI transientswithin the first six breaths ofHeO₂ were significantly attenuatedwith Anesthesia. We conclude that theI response to HeO₂ is not simply due to areduction in external tubing resistance and that, in humans, airwayafferents mediate the transient but not the sustained hyperventilatoryresponse to HeO₂ breathing duringexercise.

     

Effects of the pSC101 partition (par) locus on in vivo DNA supercoiling near the plasmid replication origin.

Previous work has shown that deletion of the partition (par) locus of plasmid pSC101 results in decreased overall superhelical density of plasmid DNA and concommitant inability of the plasmid to be stably inherited in populations of dividing cells. We report here that the biological effects of par correlate specifically with its ability to generate supercoils in vivo near the origin of pSC101 DNA replication. Using OsO4 reactivity of nucleotides adjoining 20 bp (G-C) tracts introduced into pSC101 DNA to measure local DNA supercoiling, we found that the wild type par locus generates supercoiling near the plasmid's replication origin adequate to convert a (G-C) tract in the region to Z form DNA. A 4 bp deletion that decreases par function, but produces no change in the overall superhelicity of pSC101 DNA as determined by chloroquine/agarose gel analysis, nevertheless reduced (G-C) tract supercoiling sufficiently to eliminate OsO4 reactivity. Mutation of the bacterial topA gene, which results in stabilized inheritance of par-deleted plasmids, restored supercoiling of (G-C) tracts in these plasmids and increased OsO4 reactivity in par+ replicons. Removal of par to a site more distant from the origin decreased supercoiling in a (G-C) tract adjacent to the origin and diminished par function. Collectively, these findings indicate that par activity is dependent on its ability to produce supercoiling at the replication origin rather than on the overall superhelical density of the plasmid DNA.     

QUANTITATIVE BEHAVIORAL STUDY OF BOTTLENOSE DOLPHINS IN SWIM-WITH-DOLPHIN PROGRAMS IN THE UNITED STATES

The behavior of dolphins in four Swim-With-Dolphin programs was compared by type of Swim encounter, defined by the presence ("Controlled") or absence ("Not-Controlled") of explicit trainer regulation of interactions between dolphins and human swimmers. Dolphin-swimmer interactions involving aggressive, submissive, or sexual behavior were designated as "high-risk" in the Swim context; sexual behavior was included as high-risk based on analyses that demonstrated co-occurrence of sexual and agonistic behaviors. High-risk activity comprised a substantial proportion of dolphin-swimmer social activity during Not-Controlled Swims. In contrast, high-risk activity rarely occurred during Controlled Swims, even though agonistic and sexual behaviors were normal components of the same dolphins' free-time social repertoire. These results indicated that direct trainer control of dolphin-swimmer interactions virtually eliminated high-risk activity from the Swim context, and thereby diminished the potential for dolphin distress, swimmer injury, and rejection of dolphins from Swim programs due to swimmer injury. This study illustrates effective use of quantitative behavioral sampling techniques for evaluation of captive management concerns and promotes broader use of these techniques for a better understanding of cetacean behavior.     

A temperate bacteriophage specific for strains ofAeromonas salmonicida possessing A-layer,a cell surface virulence factor

A temperate bacteriophage designated TP446 was isolated from culture supernatants ofAeromonas salmonicida strain A446. Phage TP446 adsorbed to all of the typical and atypical strains ofA. salmonicida tested that possessed A-layer, the surface protein array that represents the primary virulence factor of this fish pathogen. In contrast, TP446 failed to adsorb to mutants lacking A-layer. These results indicate that the A-layer is a component of the receptor for phage TP446.