Mice with Spontaneous Mammary Tumors Develop Type-Specific Neutralizing and Cytotoxic Antibodies Against the Mouse Mammary Tumor Virus Envelope Protein gp52

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.     

Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA.

All retroviruses contain, in the nucleocapsid domain of the Gag protein, one or two copies of the sequence Cys-X2-Cys-X4-His-X4-Cys. We have generated a series of mutants in the two copies of this motif present in human immunodeficiency virus type 1. These mutants encoded virus particles that were apparently composed of the normal complement of viral proteins but contained only 2 to 20% of the normal level of genomic RNA. No infectivity could be detected in the mutant particles, while 10(5) infectious U were present in an equivalent amount of wild-type particles. Thus, the mutants have another defect in addition to the inefficiency with which they encapsidate genomic RNA. Our results show that both copies of the motif are required for normal RNA packaging and for infectivity. Mutants of this type may have important applications, including nonhazardous materials for research, immunogens in vaccine and immunotherapy studies, and diagnostic reagents.     

Loss of the Plasma Membrane-Bound Protein Gas1p in Saccharomyces cerevisiae Results in the Release of β1,3-Glucan into the Medium and Induces a Compensation Mechanism To Ensure Cell Wall Integrity

Deletion of GAS1/GGP1/CWH52 results in a lower β-glucan content of the cell wall and swollen, more spherical cells (L. Popolo, M. Vai, E. Gatti, S. Porello, P. Bonfante, R. Balestrini, and L. Alberghina, J. Bacteriol. 175:1879–1885, 1993; A. F. J. Ram, S. S. C. Brekelmans, L. J. W. M. Oehlen, and F. M. Klis, FEBS Lett. 358:165–170, 1995). We show here that gas1Δ cells release β1,3-glucan into the medium. Western analysis of the medium proteins with β1,3-glucan- and β1,6-glucan-specific antibodies showed further that at least some of the released β1,3-glucan was linked to protein as part of a β1,3-glucan–β1,6-glucan–protein complex. These data indicate that Gas1p might play a role in the retention of β1,3-glucan and/or β-glucosylated proteins. Interestingly, the defective incorporation of β1,3-glucan in the cell wall was accompanied by an increase in chitin and mannan content in the cell wall, an enhanced expression of cell wall protein 1 (Cwp1p), and an increase in β1,3-glucan synthase activity, probably caused by the induced expression of Fks2p. It is proposed that the cell wall weakening caused by the loss of Gas1p induces a set of compensatory reactions to ensure cell integrity.     

Combining bulk segregation analysis and microarrays for mapping of the pH trait in melon

The availability of sequence information for many plants has opened the way to advanced genetic analysis in many non-model plants. Nevertheless, exploration of genetic variation on a large scale and its use as a tool for the identification of traits of interest are still rare. In this study, we combined a bulk segregation approach with our own-designed microarrays to map the pH locus that influences fruit pH in melon. Using these technologies, we identified a set of markers that are genetically linked to the pH trait. Further analysis using a set of melon cultivars demonstrated that some of these markers are tightly linked to the pH trait throughout our germplasm collection. These results validate the utility of combining microarray technology with a bulk segregation approach in mapping traits of interest in non-model plants.     

Two-temperature LATE-PCR endpoint genotyping

Background  

In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.     

Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.     

Measurement of imino <Superscript>1</Superscript>H–<Superscript>1</Superscript>H residual dipolar couplings in RNA

Imino ¹H–¹⁵N residual dipolar couplings (RDCs) provide additional structural information that complements standard ¹H–¹H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino ¹H–¹H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA^Val using a BEST-Jcomp-HMQC2 experiment. ¹H–¹H RDCs are observed between the imino protons in G–U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino ¹H–¹H RDCs complement standard ¹H–¹⁵N RDCs because the ¹H–¹H vectors generally point along the helical axis, roughly perpendicular to ¹H–¹⁵N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino ¹H–¹H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.     

Sensitivity of tibio-menisco-femoral joint contact behavior to variations in knee kinematics

Use of computational models with kinematic boundary conditions to study the knee joint contact behavior for normal and pathologic knee joints depends on an understanding of the impacts of kinematic uncertainty. We studied the sensitivities of tibio-menisco-femoral joint contact behavior to variations in knee kinematics using a finite element model (FEM) with geometry and kinematic boundary conditions derived from sequences of magnetic resonance (MR) images. The MR images were taken before and after axial compression was applied to the knee joint of a healthy subject. A design of experiments approach was used to study the impact of the variation in knee kinematics on the contact outputs. We also explored the feasibility of using supplementary hip images to improve the accuracy of knee kinematics. Variations in knee kinematics (0.25mm in medial-lateral, 0.1mm in anterior-posterior and superior-inferior translations, and 0.1 degrees in flexion-extension and varus-valgus, 0.25 degrees in external-internal rotations) caused large variations in joint contact behavior. When kinematic boundary conditions resulted in close approximations of the model-predicted joint contact force to the applied force, variations in predictions of contact parameters were also reduced. The combination of inferior-superior and medial-lateral translations accounted for over 70% of variations for all the contact parameters examined. The inclusion of hip images in kinematic calculations improved knee kinematics by matching the femoral head position. Our findings demonstrate the importance of improving the accuracy and precision of knee kinematic measurements, especially when utilized as an input for finite element models.     

Th17 cells undergo Fas-mediated activation-induced cell death independent of IFN-gamma

IL-17-secreting CD4+ T cells (Th17 cells) play a critical role in immune responses to certain infections and in the development of many autoimmune disorders. The mechanisms controlling homeostasis in this cell population are largely unknown. In this study, we show that murine Th17 cells undergo rapid apoptosis in vitro upon restimulation through the TCR. This activation-induced cell death (AICD), a common mechanism for elimination of activated T cells, required the Fas and FasL interaction: Fas was stably expressed, while FasL was up-regulated upon TCR reactivation of Th17 cells; Ab ligation of Fas induced Th17 cell death; and AICD was completely absent in Th17 cells differentiated from gld/gld CD4+ T cells. Thus, the Fas/FasL pathway is essential in regulating the AICD of Th17 cells. Interestingly, IFN-gamma, a cytokine previously found to be important for the AICD of T cells, did not affect Th17 cell apoptosis. Furthermore, Th17 cells derived from mice deficient in IFN-gamma receptor 1 (IFN-gammaR1-/-) underwent AICD similar to wild-type cells. Thus, AICD of Th17 cells occurs via the Fas pathway, but is independent of IFN-gamma.