Co-packaging of sense and antisense RNAs: a novel strategy for blocking HIV-1 replication.

Retroviral vectors were engineered to express either sense (MoTiN-TRPsie+) or sense and antisense (MoTN-TRPsie+/-) RNAs containing the human immunodeficiency virus type-1 (HIV-1) trans -activation response (TAR) element and the extended packaging (Psie) signal. The Psie signal includes the dimer linkage structure (DLS) and the Rev response element (RRE). Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid (MT4) cell line. Stable transductants were then tested for sense and antisense RNA production and susceptibility to HIV-1 infection. HIV-1 production was significantly decreased in cells transduced with MoTiN-TRPsie+ and MoTN-TRPsie+/-vectors. Efficient packaging of sense and most remarkably of antisense RNA was observed within the virus progeny. Infectivity of this virus was significantly decreased in both cases, suggesting that the interfering RNAs were co-packaged with HIV-1 RNA. Vector transduction was not expected to occur and was not observed. Inhibition of HIV-1 replication was also demonstrated in human peripheral blood lymphocytes transduced with retroviral vectors expressing antisense RNA. These results suggest that (i) both sense and antisense RNAs were co-packaged with HIV-1 RNA, (ii) the co-packaged sense and antisense RNAs inhibited virus infectivity and (iii) the co-packaged sense and antisense RNAs were not transduced. Sense and antisense RNA-based strategies may also be used to co-package other interfering RNAs (e.g. ribozymes) to cleave HIV-1 virion RNA.     

Antisense peptide recognition of sense peptides: sequence simplification and evaluation of forces underlying the interaction

Structural principles were studied which underlie the recognition of sense peptides (sense DNA encoded) by synthetic peptides encoded in the corresponding antisense strand of DNA. The direct-readout antisense peptides corresponding to ribonuclease S-peptide bind to an affinity matrix containing immobilized S-peptide with significant selectivity and with dissociation constants in the range of 10(-6) M as judged by analytical affinity chromatography. Synthetic, sequence-modified forms of antisense peptides also exhibit substantial binding affinity, including a "scrambled" peptide in which the order of residue positions is changed while the overall residue composition is retained. The antisense mutants, as the original antisense peptides, bind at saturation with greater than 1:1 stoichiometry to immobilized S-peptide. The data suggest significant sequence degeneracy in the interaction of antisense with sense peptide. In contrast, selectivity was confirmed by the inability of several control peptides to bind to immobilized S-peptide. The idea was tested that the hydropathic pattern of the amino acid sequence serves to induce antisense peptide recognition. A hydropathically sequence-simplified mutant of antisense peptide was made in which all strongly hydrophilic (charged) residues were replaced by Lys, all strongly hydrophobic residues by Leu, and all weakly hydrophilic and hydrophobic residues by Ala, except Gly which was unchanged. This "KLAG" mutant also binds to immobilized S-peptide, with an affinity only an order of magnitude less than that with the original antisense peptide and with multiple stoichiometry. Mutants of the KLAG model, in which the hydropathic pattern was changed substantially, exhibited a lower binding affinity for S-peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small sense and antisense RNAs derived from a telomeric retroposon family in Giardia intestinalis

Sequencing of a library of small RNAs from Giardia intestinalis identified a novel class of small sense and antisense RNAs homologous to the retroposon family GilT/Genie1 that is located at certain telomeres. These small RNAs may contribute to silencing GilT expression via the RNA interference pathway.     

Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells.

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.     

Making sense of downward causation in manipulationism: illustrations from cancer research

Many researchers consider cancer to have molecular causes, namely mutated genes that result in abnormal cell proliferation (e.g. Weinberg 1998). For others, the causes of cancer are to be found not at the molecular level but at the tissue level where carcinogenesis consists of disrupted tissue organization with downward causation effects on cells and cellular components (e.g. Sonnenschein and Soto 2008). In this contribution, I ponder how to make sense of such downward causation claims. Adopting a manipulationist account of causation (Woodward 2003), I propose a formal definition of downward causation and discuss further requirements (in light of Baumgartner 2009). I then show that such an account cannot be mobilized in support of non-reductive physicalism (contrary to Raatikainen 2010). However, I also argue that such downward causation claims might point at particularly interesting dynamic properties of causal relationships that might prove salient in characterizing causal relationships (following Woodward 2010).     

Pterosaur dietary hypotheses: a review of ideas and approaches

Pterosaurs are an extinct group of Mesozoic flying reptiles, whose fossil record extends from approximately 210 to 66 million years ago. They were integral components of continental and marginal marine ecosystems, yet their diets remain poorly constrained. Numerous dietary hypotheses have been proposed for different pterosaur groups, including insectivory, piscivory, carnivory, durophagy, herbivory/frugivory, filter‐feeding and generalism. These hypotheses, and subsequent interpretations of pterosaur diet, are supported by qualitative (content fossils, associations, ichnology, comparative anatomy) and/or quantitative (functional morphology, stable isotope analysis) evidence. Pterosaur dietary interpretations are scattered throughout the literature with little attention paid to the supporting evidence. Reaching a robustly supported consensus on pterosaur diets is important for understanding their dietary evolution, and their roles in Mesozoic ecosystems. A comprehensive examination of the pterosaur literature identified 314 dietary interpretations (dietary statement plus supporting evidence) from 126 published studies. Multiple alternative diets have been hypothesised for most principal taxonomic pterosaur groups. Some groups exhibit a high degree of consensus, supported by multiple lines of evidence, while others exhibit less consensus. Qualitative evidence supports 87.3% of dietary interpretations, with comparative anatomy most common (62.1% of total). More speciose groups of pterosaur tend to have a greater range of hypothesised diets. Consideration of dietary interpretations within alternative phylogenetic contexts reveals high levels of consensus between equivalent monofenestratan groups, and lower levels of consensus between equivalent non‐monofenestratan groups. Evaluating the possible non‐biological controls on apparent patterns of dietary diversity reveals that numbers of dietary interpretations through time exhibit no correlation with patterns of publication (number of peer‐reviewed publications through time). 73.8% of dietary interpretations were published in the 21st century. Overall, consensus interpretations of pterosaur diets are better accounted for by non‐biological signals, such as the impact of the respective quality of the fossil record of different pterosaur groups on research levels. That many interpretations are based on qualitative, often untestable lines of evidence adds significant noise to the data. More experiment‐led pterosaur dietary research, with greater consideration of pterosaurs as organisms with independent evolutionary histories, will lead to more robust conclusions drawn from repeatable results. This will allow greater understanding of pterosaur dietary diversity, disparity and evolution and facilitate reconstructions of Mesozoic ecosystems.     

Statistics for proteomics: a review of tools for analyzing experimental data

Most proteomics experiments make use of 'high throughput' technologies such as 2-DE, MS or protein arrays to measure simultaneously the expression levels of thousands of proteins. Such experiments yield large, high-dimensional data sets which usually reflect not only the biological but also technical and experimental factors. Statistical tools are essential for evaluating these data and preventing false conclusions. Here, an overview is given of some typical statistical tools for proteomics experiments. In particular, we present methods for data preprocessing (e.g. calibration, missing values estimation and outlier detection), comparison of protein expression in different groups (e.g. detection of differentially expressed proteins or classification of new observations) as well as the detection of dependencies between proteins (e.g. protein clusters or networks). We also discuss questions of sample size planning for some of these methods.     

Aging of human skin: review of a mechanistic model and first experimental data

The physical, chemical, and biochemical factors that accelerate skin aging have been proposed to activate a self-maintained microinflammatory process, one of the expected end results of which is an imbalance in the turnover of macromolecules in the dermis. Surface peroxides are recognized as controllable factors of skin aging, and their accumulation is attributed to environmentally induced impairment of defense enzymes. Topical application of antioxidants decreases the rate at which skin elasticity and skin thickness are modified.     

Making drugs out of oligonucleotides: a brief review and perspective.

I provide a brief review and perspective thoughts concerning the antisense oligonucleotide, drug discovery paradigm.     

Capsaicin and the stomach. A review of experimental and clinical data

Capsaicin, the pungent principle of hot pepper, because of its ability to excite and later defunctionalize a subset of primary afferent neurons, has been extensively used as a probe to elucidate the function of these sensory neurons in a number of physiological processes. In the rat stomach, experimental data provided clear evidence that capsaicin-sensitive (CS) sensory nerves are involved in a local defense mechanism against gastric ulcer. Stimulation of CS sensory nerves with low intragastric concentrations of capsaicin protected the rat gastric mucosa against injury produced by different ulcerogenic agents. High local desensitizing concentrations of capsaicin or systemic neurotoxic doses of the agent markedly enhanced the susceptibility of the rat gastric mucosa to later noxious challenge. Resiniferatoxin, a potent analogue of capsaicin possesses an acute gastroprotective effect similar to that of capsaicin in the stomach. The gastroprotective effect of capsaicin-type agents involves an enhancement of the microcirculation effected through the release of mediator peptides from the sensory nerve terminals with calcitonin gene-related peptide being the most likely candidate implicated. They do not depend on vagal efferent or sympathetic neurons or involve prostanoids. The gastric mucosal protective effect of prostacyclin is retained after systemic or topical capsaicin desensitization. Capsaicin-sensitive fibers are involved in the repair mechanisms of the gastric mucosa. A protective role for CS sensory nerves has also been demonstrated in the colon. In most studies, capsaicin given into the stomach of rats or cats inhibited gastric acid secretion. In humans, although recent studies provide evidence in favor of a beneficial effect of capsaicin on the gastric mucosa, an exact concentration-related assessment of the effect of the agent is still lacking.     

Sodium-calcium exchange: derivation of a state diagram and rate constants from experimental data.

A mechanism is developed for Na(+)-Ca2+ exchange using a new approach made possible by the availability of computer software that allows the systematic search of a large parameter space for optimum sets of parameters to fit multiple sets of experimental data. The approach was to make the experimental data dictate the form of the mechanism: the qualitative features of the data dictating the number and nature of the states of the exchanger and their interrelationship, and the quantitative aspects of the data dictating the values of the rate constants that govern the amount of each state relative to the total amount of exchanger. A single set of experimental data served this initial purpose, namely, observations of equilibrium Ca(2+)-Ca2+ exchange in cardiac sarcolemmal vesicles (Slaughter et al., 1983, J. biol. Chem. 258, 3183-3190). From this data a minimum mechanism was induced having 56 states (SYM56), which gave satisfactory quantitative fits to the experimental data. With this set of parameters additional experimental data were fitted, from the same preparation, the single cardiac cell and the squid giant axon, with some changes in parameters, but none dramatic. In spite of the symmetric nature of the mechanism, i.e. binding constants for Na+ and Ca2+ do not depend on the orientation of the binding sites, the mechanism exhibits marked asymmetric behavior similar to that observed experimentally. Finally, in accounting for Ca(2+)-Ca2+ exchange in the absence of monovalent cations, Ca2+ influx becomes dependent on intracellular Ca(2+)--an unexpected outcome--exactly in keeping with the "essential activator" role of intracellular Ca2+ observed by DiPolo & Beaugé (1987, J. gen. Physiol. 90, 505-525). Observations of Na(+)-Ca2+ exchange in the retinal rod outer segment are well fitted with a simplified version of SYM56 comprising 25 states (namely, SYM25), supporting the notion that the exchanger in the retinal rod outer segment differs from that in cardiac sarcolemma and squid axon. Maximum turnover rate of 840 sec-1 for SYM56 and 20 sec-1 for SYM25 are comparable to those reported for the exchanger in cardiac muscle and retinal rod outer segment, respectively.     

Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide.

Recently several groups reported a dramatic improvement of reporter gene transfection efficiency using a fusogenic peptide, derived from the Influenza hemagglutinin envelop protein. This peptide changes conformation at acidic pH and destabilizes the endosomal membranes thus resulting in an increased cytoplasmic gene delivery. We describe the use of a similar fusogenic peptide in order to improve the antiviral potency of antisense oligodeoxynucleotides (anti TAT) and oligophosphorothioates (S-dC28) on de novo HIV infected CEM-SS lymphocytes in serum-free medium. We observed as 5 to 10 fold improvement of the anti HIV activities of the phosphodiester antisense oligonucleotides after chemical coupling to the peptide in a one to one ratio by a disulfide or thioether bond. No toxicities were observed at the effective doses (0.1-1 microM). No sequence specificity was obtained and the fusogenic peptide possessed some antiviral activities on its own (IC50: 6 microM). A S-dC28-peptide disulfide linked conjugate and a streptavidin-peptide-biotinylated S-dC28 adduct showed similar activities as the free S-dC28 oligonucleotide (IC50: 0.1-1 nM). As expected, all the compounds were less potent in the presence of serum but the relative contribution of peptide coupling was maintained.     

T cell recognition of distinct peptide:I-Au conformers in murine experimental autoimmune encephalomyelitis

We have used T cells bearing TCRs that are closely related in sequence as probes to detect conformational variants of peptide-MHC complexes in murine experimental autoimmune encephalomyelitis in H-2(u) mice. The N-terminal epitope of myelin basic protein (MBP) is immunodominant in this model. Our studies have primarily focused on T cell recognition of a position 4 analog of this peptide (MBP1-9[4Y]) complexed with I-A(u). Using site-directed mutagenesis, we have mapped the functionally important complementarity determining region residues of the 1934.4 TCR Valpha domain. One of the resulting mutants (Tyr(95) to alanine in CDR3alpha, Y95A) has interesting properties: relative to the parent wild-type TCR, this mutant poorly recognizes Ag complexes generated by pulsing professional APCs (PL-8 cells) with MBP1-9[4Y] while retaining recognition of MBP1-9[4Y]-pulsed unconventional APCs or insect cell-expressed complexes of I-A(u) containing tethered MBP1-9[4Y]. Insect cell expression of recombinant I-A(u) with covalently tethered class II-associated invariant chain peptide or other peptides which bind relatively weakly, followed by proteolytic cleavage of the peptide linker and replacement by MBP1-9[4Y] in vitro, results in complexes that resemble peptide-pulsed PL-8 cells. Therefore, the distinct conformers can be produced in recombinant form. T cells that can distinguish these two conformers can also be generated by the immunization of H-2(u) mice, indicating that differential recognition of the conformers is observed for responding T cells in vivo. These studies have relevance to understanding the molecular details of T cell recognition in murine experimental autoimmune encephalomyelitis. They are also of particular importance for the effective use of multimeric peptide-MHC complexes to characterize the properties of Ag-specific T cells.     

DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.