Nucleocytoplasmic Recycling of the Nuclear Localization Signal Receptor α Subunit In Vivo Is Dependent on a Nuclear Export Signal, Energy, and RCC1

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The α subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the β subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207–217 or a heterologous nuclear export signal, but not a mutant form of residues 207–217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.     

Activation of G-Proteins with AlF4Induces LFA-1-Mediated Adhesion of T-Cell Hybridoma Cells to ICAM-1 by Signal Pathways That Differ from Phorbol Ester- and Manganese-Induced Adhesion

T-cell hybridomas metastasize widely, and the extent of dissemination correlates with invasiveness in fibroblast cultures. Previously, we provided evidence that both metastasis andin vitroinvasion require activation of LFA-1, induced by G-protein-transduced signals triggered by as yet unidentified factors. We show here that LFA-1-mediated adhesion of TAM2D2 T-cell hybridoma cells to ICAM-1 can in fact be induced by direct activation of G-proteins using AlF⁻₄, to the same extent as by using PMA or Mn²⁺. We assessed effects of protein kinase C (PKC), tyrosine kinase (TK), PI3-kinase (PI3K), and phospholipase C (PLC) inhibitors. Both AlF⁻₄-induced adhesion and invasion were completely blocked by the TK inhibitor genistein and partially blocked by the PI3K inhibitor wortmannin, but not influenced by PKC inhibitor GF109203X. Downregulation of PKC did not affect invasion or adhesion induced by AlF⁻₄either. In contrast, GF109203X and PKC downregulation blocked PMA-induced adhesion, but genistein and wortmannin had no effect. Invasion and both AlF⁻₄- and PMA-induced adhesion were completely blocked by the PLC inhibitor U73122. Mn²⁺-induced adhesion, which was not or was only partially blocked by the other inhibitors, was delayed by U73122, and spreading of Mn²⁺-treated cells was completely prevented by U73122. However, PLC activity during adhesion was not detected. We conclude that signals required for invasion and G-protein-induced adhesion are similar and are distinct from PKC-induced adhesion, and that in all cases PLC is likely to be activated, but is probably too local and/or transient to be detected.     

High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma

Berg, John T., Zhenxing Fu, Ellen C. Breen, Hung-Cuong Tran,Odile Mathieu-Costello, and John B. West. High lung inflation increases mRNA levels of ECM components and growth factors in lungparenchyma. J. Appl. Physiol. 83(1):120-128, 1997.Remodeling of pulmonary capillaries occurs afterchronic increases in capillary pressure (e.g., mitral stenosis). Also,remodeling of pulmonary arteries begins within 4 h of increased wallstress and is endothelium dependent. We have previously shown that highlung inflation increases wall stress in pulmonary capillaries. Thisstudy was designed to determine whether high lung inflation inducesremodeling of the extracellular matrix (ECM) in lung parenchyma.Open-chest rabbits were ventilated for 4 h with9-cmH₂O positive end-expiratory pressure (PEEP) on one lung and1-cmH₂O PEEP on the other(High-PEEP group), or with 2-cmH₂OPEEP on both lungs (Low-PEEP group). An additional untreated controlgroup was also included. We found increased levels of mRNA in bothlungs of High-PEEP rabbits (compared with both the Low-PEEP anduntreated groups) for ₁(III)and ₂(IV) procollagen,fibronectin, basic fibroblast growth factor, and transforming growthfactor-₁. In contrast,₂(I) procollagen and vascularendothelial growth factor mRNA levels were not changed. We concludethat high lung inflation for 4 h increases mRNA levels of ECMcomponents and growth factors in lung parenchyma.

     

In vivo validation of whole body composition estimates from dual-energy X-ray absorptiometry

Prior, Barry M., Kirk J. Cureton, Christopher M. Modlesky,Ellen M. Evans, Mark A. Sloniger, Michael Saunders, and Richard D. Lewis. In vivo validation of whole body composition estimates fromdual-energy X-ray absorptiometry. J. Appl.Physiol. 83(2): 623-630, 1997.We validated wholebody composition estimates from dual-energy X-ray absorptiometry (DEXA)against estimates from a four-component model to determine whetheraccuracy is affected by gender, race, athletic status, ormusculoskeletal development in young adults. Measurements of bodydensity by hydrostatic weighing, body water by deuterium dilution, andbone mineral by whole body DEXA were obtained in 172 young men(n = 91) and women(n = 81). Estimates of body fat(%Fat) from DEXA (%Fat_DEXA)were highly correlated with estimates of body fat from thefour-component model [body density, total body water, and totalbody mineral (%Fat_d,w,m);r = 0.94, standard error of theestimante (SEE) = 2.8% body mass (BM)] with no significantdifference between methods [mean of the difference ± SD ofthe difference = 0.4 ± 2.9 (SD) % BM,P = 0.10] in women and men. Onthe basis of the comparison with%Fat_d,w,m, estimates of%Fat_DEXA were slightly moreaccurate than those from body density(r = 0.91, SEE = 3.4%; mean of the difference ± SD of the difference = 1.2 ± 3.4% BM).Differences between %Fat_DEXA and%Fat_d,w,m were weakly related tobody thickness, as reflected by BMI (r = 0.34), and to the percentage of water in the fat-free mass(r = 0.51), but were notaffected by race, athletic status, or musculoskeletal development. Weconclude that body composition estimates from DEXA are accuratecompared with those from a four-component model in young adults whovary in gender, race, athletic status, body size, musculoskeletaldevelopment, and body fatness.

     

Individual constancy of local search strategies in the giant tropical ant,Paraponera clavata (Hymenoptera: Formicidae)

Paraponera clavata workers engage in a period of local search after encountering a small amount of artificial nectar. Giving-up times from local search are not distributed normally; there is a strong skew to longer times. There is no statistically significant relationship between the amount of time required to collect the food and the subsequent search time. Giving-up time in response to the first reward presented to an ant is positively correlated with that ant's response to a second such reward. However, giving-up times diminish when an ant is presented with a series of rewards. Local search is a function of individual strategies, which remain relatively constant in the short term.     

Bifurcations in haploid and diploid sequence space models

 Deterministic models of mutation and selection in the space of (binary) nucleotide-type sequences have been investigated for haploid populations during the past 25 years, and, recently, for diploid populations as well. These models, in particular their ‘error thresholds’, have mainly been analyzed by numerical methods and perturbation techniques. We consider them here by means of bifurcation theory, which improves our understanding of both equilibrium and dynamical properties. In a caricature obtained from the original model by neglecting back mutation to the favourable allele, the familiar error threshold of the haploid two-class model turns out to be a simple transcritical bifurcation, whereas its diploid counterpart exhibits an additional saddle node. This corresponds to a second error threshold. Three-class models with neutral spaces of unequal size introduce further features. Such are a global bifurcation in haploid populations, and simple examples of Hopf bifurcations (as predicted by Akin’s theorem) in the diploid case. Received 13 June 1995; received in revised form 26 July 1996     

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network

Site-directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a transfer-derepressed ( finO  ⁻) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1-16 was 10⁷-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ , traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.     

The activity of dung beetles increases foliar nutrient concentration in tropical seedlings

Dung beetles are extensively used as a focal taxon in tropical forests. Yet, information for most of their ecological functions comes from other systems. We present results from a field experiment in a tropical rain forest showing that dung beetle activity increases foliar phosphorus concentration in seedlings of the tree Brosimum lactescens. Our results open new lines of research to assess the multiple effects that dung beetles may have on rain forest plants.     

Evolutionary potential of the extrinsic incubation period of dengue virus in Aedes aegypti

Dengue fever is the most common arboviral disease worldwide. It is caused by dengue viruses (DENV) and the mosquito Aedes aegypti is its primary vector. One of the most powerful determinants of a mosquito's ability to transmit DENV is the length of the extrinsic incubation period (EIP), the time it takes for a virus to be transmitted by a mosquito after consuming an infected blood meal. Here, we repeatedly measured DENV load in the saliva of individual mosquitoes over their lifetime and used this in combination with a breeding design to determine the extent to which EIP might respond to the evolutionary forces of drift and selection. We demonstrated that genetic variation among mosquitoes contributes significantly to transmission potential and length of EIP. We reveal that shorter EIP is genetically correlated with reduced mosquito lifespan, highlighting negative life‐history consequences for virus‐infected mosquitoes. This work highlights the capacity for local genetic variation in mosquito populations to evolve and to dramatically affect the nature of human outbreaks. It also provides the impetus for isolating mosquito genes that determine EIP. More broadly, our dual experimental approach offers new opportunities for studying the evolutionary potential of transmission traits in other vector/pathogen systems.