Human immunodeficiency virus type 1-hepatitis C virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses

Both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lead to chronic infection in a high percentage of persons, and an expanding epidemic of HIV-1-HCV coinfection has recently been identified. These individuals provide an opportunity for simultaneous assessment of immune responses to two viral infections associated with chronic plasma viremia. In this study we analyzed the breadth and magnitude of the CD8(+)- and CD4(+)-T-lymphocyte responses in 22 individuals infected with both HIV-1 and HCV. A CD8(+)-T-lymphocyte response against HIV-1 was readily detected in all subjects over a broad range of viral loads. In marked contrast, HCV-specific CD8(+)-T-lymphocyte responses were rarely detected, despite viral loads in plasma that were on average 1,000-fold higher. The few HCV-specific responses that were observed were relatively weak and limited in breadth. CD4-proliferative responses against HIV-1 were detected in about half of the coinfected subjects tested, but no proliferative response against any HCV protein was found in these coinfected persons. These data demonstrate a major discordance in immune responses to two persistent RNA viruses. In addition, they show a consistent and profound impairment in cellular immune responses to HCV compared to HIV-1 in HIV-1-HCV-coinfected persons.     

Analysis of cholera toxin-ganglioside interactions by flow cytometry

Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit with the membrane surface. Interaction of cholera toxin B-subunit and whole cholera toxin with gangliosides other than GM(1) revealed specific binding only to GD1(b) and asialo-GM(1). These interactions, however, are marked by low avidity and require high receptor concentrations to be observed.     

Characterization of peptide-amphiphiles possessing cellular activation sequences

Numerous approaches have been described for modifying biomaterials to incorporate extracellular matrix components. "Peptide-amphiphiles", whereby monoalkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to (a) form specific molecular architecture with enhanced stability and (b) promote cell adhesion, spreading, and signaling. The present study has examined the use of chimeric peptide-amphiphiles for inducing protein-like structures and peptide-amphiphile mixtures for enhancing surface bioactivity. The alpha-helical propensity of a 21 residue peptide, incorporating the SPARC(119-122) angiogenesis-inducing sequence and either unmodified or acylated with a C(6), C(10), C(14), C(16), C(18), C(18:1), or C(18:1-OH) monoalkyl hydrocarbon chain, has been examined. Peptide and peptide-amphiphile structures were characterized by circular dichroism and one- and two-dimensional NMR spectroscopic techniques. The 21 residue peptide alone does not form a distinct structure in solution, whereas N-terminal acylation by monoalkyl hydrocarbon chains results in the 21 residue peptide-amphiphile adopting a predominantly alpha-helical structure in solution. The thermal stability of the alpha-helix increases with increasing hydrocarbon chain length. The SPARC(119-122) peptide-amphiphiles were then screened for promotion of endothelial cell adhesion and spreading. The greatest activity was achieved by using a mixture of the alpha-helical SPARC(119-122) peptide-amphiphile, a triple-helical peptide-amphiphile incorporating the alpha2beta1 integrin binding site from type I collagen, and a pseudolipid. The pseudolipid is most likely required for a spatial distribution of the peptide-amphiphiles that allows for optimal cellular interactions. Overall, we have found that incorporation of bioactive sequences within peptide-amphiphiles results in the induction of an ordered structure of the bioactive sequence and that mixtures of peptide-amphiphiles can be used to promote endothelial cell behaviors comparable to extracellular matrix components.     

Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals

Changes in plasma nitrite concentration in the human forearm circulation have recently been shown to reflect acute changes in endothelial nitric oxide synthase (eNOS)-activity. Whether basal plasma nitrite is a general marker of constitutive NOS-activity in vivo is yet unclear. Due to the rapid metabolism of nitrite in blood and the difficulties in its analytical determination literature data on levels of nitrite in mammals are largely inconsistent. We hypothesized that constitutive NOS-activity in the circulatory system is relatively uniform throughout the mammalian kingdom. If true, this should result in comparable systemic plasma nitrite levels in different species. Using three different analytical approaches we determined plasma nitrite concentration to be in a nanomolar range in a variety of species: humans (305 +/- 23 nmol/l), monkeys (367 +/- 62 nmol/l), minipigs (319 +/- 24 nmol/l), dogs (305 +/- 50 nmol/l), rabbits (502 +/- 21 nmol/l), guinea pigs (412 +/- 44 nmol/l), rats (191 +/- 43 nmol/l), and mice (457 +/- 51 nmol/l). Application of different NOS-inhibitors in humans, minipigs, and dogs decreased NOS-activity and thereby increased vascular resistance. This was accompanied by a significant, up to 80%, decrease in plasma nitrite concentration. A comparison of plasma nitrite concentrations between eNOS(-/-) and NOS-inhibited wild-type mice revealed that 70 +/- 5% of plasma nitrite is derived from eNOS. These results provide evidence for a uniform constitutive vascular NOS-activity across mammalian species.     

Physical map of the chromosome of the apple proliferation phytoplasma

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.     

Having a Lot of a Good Thing: Multiple Important Group Memberships as a Source of Self-Esteem

Membership in important social groups can promote a positive identity. We propose and test an identity resource model in which personal self-esteem is boosted by membership in additional important social groups. Belonging to multiple important group memberships predicts personal self-esteem in children (Study 1a), older adults (Study 1b), and former residents of a homeless shelter (Study 1c). Study 2 shows that the effects of multiple important group memberships on personal self-esteem are not reducible to number of interpersonal ties. Studies 3a and 3b provide longitudinal evidence that multiple important group memberships predict personal self-esteem over time. Studies 4 and 5 show that collective self-esteem mediates this effect, suggesting that membership in multiple important groups boosts personal self-esteem because people take pride in, and derive meaning from, important group memberships. Discussion focuses on when and why important group memberships act as a social resource that fuels personal self-esteem.     

Embryonic Lethality in Homozygous Human Her-2 Transgenic Mice Due to Disruption of the Pds5b Gene

The development of antigen-targeted therapeutics is dependent on the preferential expression of tumor-associated antigens (TAA) at targetable levels on the tumor. Tumor-associated antigens can be generated de novo or can arise from altered expression of normal basal proteins, such as the up-regulation of human epidermal growth factor receptor 2 (Her2/ErbB2). To properly assess the development of Her2 therapeutics in an immune tolerant model, we previously generated a transgenic mouse model in which expression of the human Her2 protein was present in both the brain and mammary tissue. This mouse model has facilitated the development of Her2 targeted therapies in a clinically relevant and suitable model. While heterozygous Her2^+/- mice appear to develop in a similar manner to wild type mice (Her2^-/-), it has proven difficult to generate homozygous Her2^+/+ mice, potentially due to embryonic lethality. In this study, we performed whole genome sequencing to determine if the integration site of the Her2 transgene was responsible for this lethality. Indeed, we report that the Her2 transgene had integrated into the Pds5b (precocious dissociation of sisters) gene on chromosome 5, as a 162 copy concatemer. Furthermore, our findings demonstrate that Her2^+/+ mice, similar to Pds5b^-/- mice, are embryonic lethal and confirm the necessity for Pds5b in embryonic development. This study confirms the value of whole genome sequencing in determining the integration site of transgenes to gain insight into associated phenotypes.     

Programmed anuclear cell death delimits platelet life span

Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.     

Effect of summer drought relief on the impact of the root weevil Cyphocleonus achates on spotted knapweed

A recent decline in spotted knapweed, Centaurea stoebe L. subsp. micranthos (Asteraceae), has been observed in parts of western Montana. The release of the biological control agent Cyphocleonus achates (Fahraeus) is thought to contribute to the decline, but persistent drought since at least 1999 may be an additional factor. We conducted outdoor plot experiments to test the relative impacts of C. achates weevils and summer drought relief on spotted knapweed survival and growth. Groups of spotted knapweed transplants were assigned to one of four weekly water addition treatments (no added water, and 0.25, 0.5 or full recovery of plant water deficit, where "deficit" refers to potential evapotranspiration minus rainfall) in May to August 2004 and June to August 2005 and to either exposure to or protection from C. achates. In June of each subsequent year (2005 and 2006), plants were harvested and growth attributes that reflect plant vigor were measured. Drought indices showed that throughout the time of the study until January 2006, western Montana was in drought alert or severe drought. Summer drought relief had no effect on aboveground biomass and plant height of knapweed plants in subsequent years, but feeding by C. achates larvae reduced these two measures of plant vigor. Knapweed plants resuming growth after the drought ended in spring 2006 were significantly larger than those resuming growth under drought conditions in spring 2005. Spring drought may reduce knapweed growth, but C. achates reduced knapweed growth regardless of drought conditions.