Comparative immunogenicity of HIV-1 clade C envelope proteins for prime/boost studies.

BACKGROUND: Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses. METHODOLOGY/PRINCIPAL FINDINGS: A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested. CONCLUSIONS/SIGNIFICANCE: While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies.

The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.

-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.

Regulation of G protein-coupled receptor signaling by scaffold proteins.

The actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.

A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.

Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

Evolution of networks and sequences in eukaryotic cell cycle control.

The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.

Chromatin: bind at your own RSC.

Recent work has identified a novel RSC-nucleosome complex that both strongly phases flanking nucleosomes and presents regulatory sites for ready access. These results challenge several widely held views.

Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast.

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.

Consensus nomenclature for the human ArfGAP domain-containing proteins.

At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi.

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.