Deficiency in integrin-mediated transmembrane signaling and microfilament stress fiber formation by aging dermal fibroblasts from normal and down''s syndrome patients

Previous evidence has shown a deficiency in microfilament stress fiber formation upon short-term cycloheximide treatment of cultured human dermal fibroblasts while cytoplasmic spreading appeared completely normal and other cytoskeletal networks organized normally. This deficiency applied to collagen substrata (not fibronectin substrata) and was specific for in vitro-aged normal fibroblasts and for fibroblasts from three different Down's syndrome patients at any passage level. To identify the mechanism(s) for matrix receptor deficiency in aging cells, cells were evaluated for amounts and distributions of several integrin subunits using specific monoclonal antibodies and two complementary experimental approaches. Flow cytometric analyses have shown that all these cells at all passage levels have large amounts of alpha 3 and beta 1 integrin subunits and smaller amounts of the alpha 5 subunit, directed to fibronectin, which are minimally affected in their cell surface availability by cycloheximide treatment. In contrast, cycloheximide treatment leads to the loss from surface availability of most of the alpha 2 subunit, directed to collagen, in late-passage papillary and reticular normal fibroblasts and in all three Down's patient cells at all passages. Prior growth of cells in ascorbate-supplemented medium, which overcomes the deficiency in stress fiber formation, conserves the large amounts of cell surface-available alpha 2 subunit detectable by flow cytometry. When amounts of integrin subunits were evaluated by immunoprecipitation of [35S]methionine-radiolabeled cells, there was no diminution of the alpha 2 subunit or any other subunit for any cells upon cycloheximide treatment; however, there was much less alpha 2 subunit complexed with beta 1 in aging normal and Down's cells. Therefore, cycloheximide treatment does not lead to loss in the amounts of the alpha 2 subunit but rather to its masking at the cell surface and inability to transmit signals across the plasma membrane to effect stress fiber formation. This aging-related deficiency in integrin-mediated signaling can now be studied mechanistically with a variety of approaches to determine the nature of cell-surface molecules interacting with integrins (cis- and/or trans-acting molecules) that discriminate functional from nonfunctional receptors.     

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.     

Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein.

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of "large offspring syndrome" were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.     

Subtelomeric expression regions of Borrelia hermsii linear plasmids are highly polymorphic

Borrelia hermsii, a relapsing fever agent, undergoes multiphasic antigenic variation to evade its host's immune response. Serotype specificity is determined by variable membrane lipoproteins, Vmps, which are expressed from genes located near the end of a linear plasmid. Using the polymerase chain reaction and primers representing the promoter of the active vmp and a conserved telomeric sequence, we characterized the subtelomeric expression regions of the 25 known serotypes of strain HS1. The distance from the promoter to the telomere fell into three size classes of approximately 1.0, 1.5, and 2.5 kilobases. In the sequenced serotypes the size differences were accounted for by variable lengths of the vmp genes and intervening sequences between 3' end of the vmp gene and the start of a downstream homology block. The degree of nucleotide identity between different vmp genes, or between the different 3' flanking DNA varied from 39-78%. Thus, there is length and sequence variability not only between vmp genes themselves but also between the 3' flanking regions of vmp genes.     

Gold mining in the Peruvian Amazon: global prices, deforestation, and mercury imports

Many factors such as poverty, ineffective institutions and environmental regulations may prevent developing countries from managing how natural resources are extracted to meet a strong market demand. Extraction for some resources has reached such proportions that evidence is measurable from space. We present recent evidence of the global demand for a single commodity and the ecosystem destruction resulting from commodity extraction, recorded by satellites for one of the most biodiverse areas of the world. We find that since 2003, recent mining deforestation in Madre de Dios, Peru is increasing nonlinearly alongside a constant annual rate of increase in international gold price (∼18%/yr). We detect that the new pattern of mining deforestation (1915 ha/year, 2006–2009) is outpacing that of nearby settlement deforestation. We show that gold price is linked with exponential increases in Peruvian national mercury imports over time (R² = 0.93, p = 0.04, 2003–2009). Given the past rates of increase we predict that mercury imports may more than double for 2011 (∼500 t/year). Virtually all of Peru''s mercury imports are used in artisanal gold mining. Much of the mining increase is unregulated/artisanal in nature, lacking environmental impact analysis or miner education. As a result, large quantities of mercury are being released into the atmosphere, sediments and waterways. Other developing countries endowed with gold deposits are likely experiencing similar environmental destruction in response to recent record high gold prices. The increasing availability of satellite imagery ought to evoke further studies linking economic variables with land use and cover changes on the ground.     

The mode of transmission of Tipula iridescent virus: II. Route of infection

Tipula iridescent virus (TIV) was inoculated into Tipula oleracea larvae via different routes. It was found to be much more infective when it was injected into the hemocoel than when it was ingested by the larvae.T. oleracea embryos did not become infected when eggs were laid in agar containing TIV, and there was no evidence that larvae can become infected via the spiracles or that transovum transmission occurs. It is suggested that TIV is transmitted principally by cannibalism, including killing and ingesting infected larvae, and finding dead infected larvae and ingesting them. It is proposed that a new generation becomes infected by first-instar larvae feeding upon infected fourth-instar larvae which have survived from the previous generation.     

Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component

Background

A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge.

Methodology/Principal Findings

NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP). It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1) that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al). Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m]) that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m). CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb) to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected.

Significance

The NMRC-MV-Ad-PfC vaccine expressing CSP was safe and well tolerated given as two doses, but did not provide sterile protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00392015","term_id":"NCT00392015"}}NCT00392015