Role of N- and C-terminal residues of insulin-like growth factor (IGF)-binding protein-3 in regulating IGF complex formation and receptor activation

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu(77), Leu(80), and Leu(81) in the N terminus and Gly(217) and Gln(223) in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF.IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.     

Complete nucleotide sequence of the conjugative tetracycline resistance plasmid pFBAOT6, a member of a group of IncU plasmids with global ubiquity

This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.     

Protein kinase C{varepsilon} contributes to regulation of the sarcolemmal Na+-K+ pump

A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa⁺-K⁺ pump current (I_p)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured I_p of myocytes voltageclamped with wide-tipped patch pipettes. I_p ofmyocytes from captopril-treated rabbits was larger thanI_p of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedI_p to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on I_p that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa⁺-K⁺ pump.

     

Oxidative stress contributes to chronic leg vasoconstriction in estrogen-deficient postmenopausal women.

Basal whole leg blood flow and vascular conductance are reduced in estrogen-deficient postmenopausal compared with premenopausal women. The underlying mechanisms are unknown, but oxidative stress could be involved. We studied 9 premenopausal [23 +/- 1 yr (mean +/- SE)] and 20 estrogen-deficient postmenopausal (55 +/- 1 yr) healthy women. During baseline control, oxidized low-density lipoprotein (LDL), a marker of oxidative stress, was 50% greater in the postmenopausal women (P < 0.001). Basal whole leg blood flow (duplex ultrasound of femoral artery) was 34% lower in the postmenopausal women because of a 38% lower leg vascular conductance (P < 0.0001); mean arterial pressure was not different. Intravenous administration of a supraphysiological dose of the antioxidant ascorbic acid increased leg blood flow by 15% in the postmenopausal women as a result of an increase in leg vascular conductance (both P < 0.001), but it did not affect leg blood flow in premenopausal controls or mean arterial pressure in either group. In the pooled subjects, the changes in leg blood flow and leg vascular conductance with ascorbic acid were related to baseline plasma oxidized LDL (r = 0.46 and 0.53, P < 0.01) and waist-to-hip ratio and total body fat (r = 0.41-0.44, all P < 0.05). Our results are consistent with the hypothesis that oxidative stress contributes to chronic leg vasoconstriction and reduced basal whole leg blood flow in estrogen-deficient postmenopausal women. This oxidative stress-related suppression of leg vascular conductance and blood flow may be linked in part to increased total and abdominal adiposity.     

A new leaf rust resistance gene <Emphasis Type="Italic">Lr79</Emphasis> mapped in chromosome 3BL from the durum wheat landrace Aus26582

Key message

A new leaf rust resistance gene Lr79 has been mapped in the long arm of chromosome 3B and a linked marker was identified for marker-assisted selection.

Abstract

Aus26582, a durum wheat landrace from the A. E. Watkins Collection, showed seedling resistance against durum-specific and common wheat-specific Puccinia triticina (Pt) pathotypes. Genetic analysis using a recombinant inbred line (RIL) population developed from a cross between Aus26582 and the susceptible parent Bansi with Australian Pt pathotype showed digenic inheritance and the underlying loci were temporarily named LrAW2 and LrAW3. LrAW2 was located in chromosome 6BS and this study focused on characterisation of LrAW3 using RILs lacking LrAW2. LrAW3 was incorporated into the DArTseq map of Aus26582/Bansi and was located in chromosome 3BL. Markers linked with LrAW3 were developed from the chromosome survey sequence contig 3B_10474240 in which closely-linked DArTseq markers 1128708 and 3948563 were located. Although bulk segregant analysis (BSA) with the 90 K Infinium array identified 51 SNPs associated with LrAW3, only one SNP-derived KASP marker mapped close to the locus. Deletion bin mapping of LrAW3-linked markers located LrAW3 between bins 3BL11-0.85-0.90 and 3BL7-0.63. Since no other all stage leaf rust resistance gene is located in chromosome 3BL, LrAW3 represented a new locus and was designated Lr79. Marker sun786 mapped 1.8 cM distal to Lr79 and Aus26582 was null for this locus. However, the marker can be reliably scored as it also amplifies a monomorphic fragment that serves as an internal control to differentiate the null status of Aus26582 from reaction failure. This marker was validated among a set of durum and common wheat cultivars and was shown to be useful for marker-assisted selection of Lr79 at both ploidy levels.

     

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.     

Comparative Genome Structure,Secondary Metabolite,and Effector Coding Capacity across Cochliobolus Pathogens

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP–encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.     

The Microvesicle Component of HIV-1 Inocula Modulates Dendritic Cell Infection and Maturation and Enhances Adhesion to and Activation of T Lymphocytes

HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1_BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.     

It’s Not Just Conflict That Motivates Killing of Orangutans

We investigated why orangutans are being killed in Kalimantan, Indonesia, and the role of conflict in these killings. Based on an analysis of interview data from over 5,000 respondents in over 450 villages, we also assessed the socio-ecological factors associated with conflict and non-conflict killings. Most respondents never kill orangutans. Those who reported having personally killed an orangutan primarily did so for non-conflict reasons; for example, 56% of these respondents said that the reason they had killed an orangutan was to eat it. Of the conflict-related reasons for killing, the most common reasons orangutans were killed was fear of orangutans or in self-defence. A similar pattern was evident among reports of orangutan killing by other people in the villages. Regression analyses indicated that religion and the percentage of intact forest around villages were the strongest socio-ecological predictors of whether orangutans were killed for conflict or non-conflict related reasons. Our data indicate that between 44,170 and 66,570 orangutans were killed in Kalimantan within the respondents’ active hunting lifetimes: between 12,690 and 29,024 for conflict reasons (95%CI) and between 26,361 and 41,688 for non-conflict reasons (95% CI). These findings confirm that habitat protection alone will not ensure the survival of orangutans in Indonesian Borneo, and that effective reduction of orangutan killings is urgently needed.