Senescent phenotype can be reversed by reduction of caveolin status

Hyporesponsiveness to growth factors is one of the fundamental characteristics of senescent cells. We previously reported that the up-regulation of caveolin attenuates the growth factor response and the subsequent downstream signal cascades in senescent human diploid fibroblasts. Therefore, in the present experiment, we investigated the modulation of caveolin status in senescent cells to determine the effect of caveolin on mitogenic signaling efficiency and cell cycling. We reduced the level of caveolin-1 in senescent human diploid fibroblasts using its antisense oligonucleotides and small interfering RNA, and this resulted in the restoration of normal growth factor responses such as the increased phosphorylation of Erk, the nuclear translocation of p-Erk, and the subsequent activation of p-Elk upon epidermal growth factor stimulation. Moreover, DNA synthesis and the re-entry of senescent cells into cell cycle were resumed upon epidermal growth factor stimulation concomitantly with decreases in p53 and p21. Taken together, we conclude that the loss of mitogenic signaling in senescent cells is strongly related to their elevated levels of caveolin-1 and that the functional recovery of senescent cells at least in the terms of growth factor responsiveness and cell cycle entry might be achieved simply by lowering the caveolin level.     

Patterns of motor recruitment can be determined using surface EMG

Previous studies have reported how different populations of motor units (MUs) can be recruited during dynamic and locomotor tasks. It was hypothesised that the higher-threshold units would contribute higher-frequency components to the sEMG spectra due to their faster conduction velocities, and thus recruitment patterns that increase the proportion of high-threshold units active would lead to higher-frequency elements in the sEMG spectra. This idea was tested by using a model of varying recruitment coupled to a three-layer volume conductor model to generate a series of sEMG signals. The recruitment varied from (A) orderly recruitment where the lowest-threshold MUs were initially activated and higher-threshold MUs were sequentially recruited as the contraction progressed, (B) a recurrent inhibition model that started with orderly recruitment, but as the higher-threshold units were activated they inhibited the lower-threshold MUs (C) nine models with intermediate properties that were graded between these two extremes. The sEMG was processed using wavelet analysis and the spectral properties quantified by their mean frequency, and an angle θ that was determined from the principal components of the spectra. Recruitment strategies that resulted in a greater proportion of faster MUs being active had a significantly lower θ and higher mean frequency.     

The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein.

T-Even type bacteriophages recognize their cellular receptors with the distal ends of their long tail fibers. The distal part of these fibers consists of a dimer of gene product (gp) 37. The assembly of this gp to a functional dimer requires the action of two other proteins, gp57 and gp38. Genes (g) 38 have been cloned from five T-even type phages which use the Escherichia coli outer membrane protein OmpA as a receptor. The phages used differ in their ability to infect a series of ompA mutants producing altered OmpA proteins, i.e., each phage has a specific host range for these mutants. The cloned genes 38 complemented g38 amber mutants of phage T2, which uses the outer membrane protein OmpF as a receptor. The complemented phages had become phenotypically OmpA-dependent and, with one exception, OmpF-independent, but regained the host range of T2 upon growth in a host lacking the cloned g38. The host range of the complemented phages, as determined on the ompA mutants, was identical to, similar to, or different from that of the phage, from which the cloned g38 originated. The results presented show that gp38 from one phage can phenotypically 'imprint', in a finely-tuned manner, a host range onto gp37 of another phage with a different host specificity. In view of the extreme diversity of host ranges observed, it is suggested that gp38 of T2 and of the OmpA-specific phages may remain attached to gp37 in the phage particle and in cooperation with gp37 determine the host range.     

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

In this study, we explored whether SYBR Green-based quantitative real-time PCR (qPCR) could be used to determine the copy number of a plasmid and whether the method was broadly applicable to chromosomally encoded genetic elements often occurring in multiple copies, such as rRNA genes and insertion sequences (IS). Three different template sources (whole cells, total DNA, and restriction-enzyme digested total DNA) derived from the bacterium Comamonas sp. strain JS46 were analyzed by qPCR using primer-pairs targeting plasmid pJS46 and three chromosomally encoded sequences (16S rDNA, ISCsp1, and IS1071). The difference between threshold cycle values, C(T), of amplicons targeting these elements and of an amplicon targeting the single-copy reference element mnbA (chromosomally encoded) was used to establish DeltaC(T). DeltaC(T) values were then used to derive copy number. For pJS46, qPCR analyses of whole cells and total DNA underestimated the copy number of pJS46 approximately 7-fold and approximately 2.5-fold, respectively, whereas copy number values derived from qPCR analyses of digested total DNA were comparable to those derived from Southern blot (SB) analyses. In contrast, for the chromosomally encoded elements, qPCR analyses of all three template sources gave copy number values that were virtually identical to or differed by approximately 2 from copy number values derived by SB analysis. These data indicate that qPCR can be used to estimate the copy number of various genetic elements but that the accuracy of qPCR-derived values is affected by the template source.     

Trout coelomic fluid suitability as Goldfish oocyte extender can be determined by a simple turbidity test

Regeneration technologies such as androgenesis, intracytoplasmic sperm injection, and nuclear transfer require that handling conditions do not alter oocyte ability to sustain embryo development. One important parameter in the maintenance of oocyte quality in fish is the possibility to prevent oocytes activation during manipulation. In Cyprinid, such activation is known to be delayed when Salmonid coelomic fluid is used as incubation medium. Coelomic fluid however is a biological fluid whose ability to sustain oocyte quality during in vitro incubation may be variable. The purpose of the present work was to explore this variability using Rainbow Trout (Oncorhynchus mykiss) coelomic fluid (TCF) and Goldfish (Carassius auratus) oocytes, and to set up a test which would reflect TCF suitability for Goldfish oocyte incubation. We showed that different TCF induced very different development rates after oocyte incubation for 30 min at 20 °C: at 24h post fertilization (pf) and at hatching, rates ranged between 35% and 110% of the non-incubated controls. When TCF (1 volume) was mixed with tap water (9 volumes), a precipitate developed whose extent was measured by spectrophotometry. This turbidity test proved to be highly correlated to development rates after Goldfish oocyte incubation in TCF (r² = 0.83 at hatching, n = 150): TCF with the highest turbidity (> 1.5 absorbance unit at 400 nm) were the ones which altered the most the development rates after incubation (less than 50 % at hatching). This easy and rapid turbidity test can therefore be used as a reliable estimator of TCF suitability for Goldfish oocyte incubation and manipulation.     

Proportion of prey consumed can be determined from faecal DNA using real-time PCR

Reconstructing the diets of pinnipeds by visually identifying prey remains recovered in faecal samples is challenging because of differences in digestion and passage rates of hard parts. Analysing the soft-matrix of faecal material using DNA-based techniques is an alternative means to identify prey species consumed, but published techniques are largely nonquantitative, which limits their usefulness for some applications. We further developed and validated a real-time PCR technique using species-specific mitochondrial DNA primers to quantify the proportion of prey in the diets of Steller sea lions (Eumetopias jubatus), a pinniped species thought to be facing significant diet related challenges in the North Pacific. We first demonstrated that the proportions of prey tissue DNA in mixtures of DNA isolated from four prey species could be estimated within a margin of ～ 12% of the percent in the mix. These prey species included herring Clupea palasii, eulachon Thaleichthyes pacificus, squid Loligo opalescens and rosethorn rockfish Sebastes helvomaculatus. We then applied real-time PCR to DNA extracted from faecal samples obtained from Steller sea lions in captivity that were fed 11 different combinations of herring, eulachon, squid and Pacific ocean perch rockfish (Sebastes alutus), ranging from 7% to 75% contributions per meal (by wet weight). The difference between the average percentage estimated by real-time PCR and the percentage of prey consumed was generally <12% for all diets fed. Our findings indicate that real-time PCR of faecal DNA can detect the approximate relative quantity of prey consumed for complex diets and prey species, including cephalopods and fish.     

Territorial aggression can be sensitive to the status of heterospecific intruders

Territorial animals are known to be able to differentiate between intruding individuals posing a low or high threat and adjust their aggressive response accordingly. However, plastic territorial aggression based on recognising individuals with different attributes is typically assumed to be relevant only in the context of conspecific interactions. In this study, we investigated territorial aggression of neotropical cichlid fish in their natural habitat to assess whether responses to different types of individuals of another species can also be plastic. We show that arrow cichlids (Amphilophus zaliosus) adjusted their territorial aggression regarding the status of heterospecific intruders: breeding individuals of Amphilophus astorquii received a lower level of aggression than non-breeders. The same pattern was also found for the two different types of A. astorquii individuals intruding into conspecific territories. These results suggest that heterospecific individuals should not be ignored when considering selection pressures shaping plasticity of aggressive behaviour in territorial animals.     

Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands

Thymic expression of self-Ags results in the deletion of high-avidity self-specific T cells, but, at least for certain Ags, a residual population of self-specific T cells with low-affinity TCRs remains after negative selection. Such self-specific T cells are thought to play a role in the induction of T cell-mediated autoimmunity, but may also be used for the induction of antitumor immunity against self-Ags. In this study, we examine the functional competence of a polyclonal population of self-specific CD8+ T cells. We show that low-affinity interactions between TCR and peptide are associated with selective loss of critical T cell functions. Triggering of low levels of IFN-gamma production and cytolytic activity through low-affinity TCRs readily occurs provided high Ag doses are used, but IL-2 production and clonal expansion are severely reduced at all Ag doses. Remarkably, a single peptide variant can form an improved ligand for the highly diverse population of low-avidity self-specific T cells and can improve their proliferative capacity. These data provide insight into the inherent limitations of self-specific T cell responses through low-avidity TCR signals and the effect of modified peptide ligands on self-specific T cell immunity.     

The type of human papillomavirus present in cervical infections can be determined by the occurrence of specific marker proteins

Four different proteins have been identified on high resolution two dimensional gels of [35S] methionine labelled human cervical biopsies whose expression correlates with the presence of papillomavirus. They are all basic proteins having molecular weights in the region of 48 to 50 kd and are normally expressed individually in different lesions unless the lesion results from a co-infection of two virus types. Comparison of the occurrence of these marker proteins with the actual HPV type present, determined by in situ filter hybridisation, has shown that two are found exclusively with HPV types 6/11 while the other two are found with types 16/18.     

Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be determined by receptor specificity: apoptosis induction by TRAIL

Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity.     

T cell tolerance induced by cross-reactive TCR ligands can be broken by superagonist resulting in anti-inflammatory T cell cytokine production

Cross-reactive activation of potentially autoreactive T cells by high-affinity nonself ligands may be important in breaking self-tolerance in autoimmunity. In a mouse transgenic for a cross-reactive TCR, we have previously shown that a hyper-stimulating altered peptide ligand, L144, induced unresponsiveness to the self peptide, proteolipid protein 139-151. In this study, we demonstrate that a superagonist ligand can break T cell tolerance induced by the lower affinity cognate Ag. T cells tolerant to the cognate ligand, Q144, responded to superagonist, L144, by proliferation and the production of mainly IL-4 and IL-10 in vitro. In contrast, T cells that were tolerized to the superagonist were unable to respond to any peptide that cross-reacted with the transgenic TCR. Low-dose immunization with the superagonist L144 was able to break tolerance to the cognate ligand in vivo and resulted in a blunted proliferative response with production of Th2 cytokines.     

Protein synthesis rates of human PBMC and PMN can be determined simultaneously in vivo by using small blood samples

Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-¹³C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [¹³C]leucine or -[¹³C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [¹³C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [¹³C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [¹³C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [¹³C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors. peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine     

Natural ligands of the B cell adhesion molecule CD22 beta can be masked by 9-O-acetylation of sialic acids

CD22 beta is a B cell-restricted phosphoprotein expressed on the surface of mature resting B cells. It mediates interactions with other cells partly or exclusively via recognition of alpha 2-6-linked sialic acids on glycoconjugates. The sialylated N-linked oligosaccharides recognized best by CD22 beta are common to many glycoproteins, suggesting that additional regulatory mechanisms may exist. Since the exocyclic side chain of sialic acid is required for recognition, we explored the effects of a naturally occurring modification of the side chain, 9-O-acetylation. Semisynthetic N-linked oligosaccharides terminating with 9-O-acetylated, alpha 2-6-linked sialic acids showed markedly reduced binding to CD22 beta relative to their non-O- acetylated counterparts. Murine lymphoid cells were probed for natural CD22 beta ligands that might be O-acetylated using recombinant soluble forms of CD22 beta (CD22 beta Rg) and influenza C esterase (CHE-Fc, which specifically removes 9-O-acetyl esters from sialic acids). By flow cytometry analysis, CD22 beta Rg binding to splenic B cells and a subset of T cells was increased by pretreatment with CHE-Fc, indicating that some potential CD22 beta ligands are naturally "masked" by 9-O- acetylation. Unmasking of these CD22 beta ligands by removal of 9-O- acetyl esters from intact splenocytes substantially increases their CD22 beta-dependent adhesion in an in vitro adhesion assay. Probing of murine lymphoid tissue sections by CD22 beta Rg and CHE-Fc treatment demonstrates regionally restricted and differentially expressed patterns of distribution between masked and unmasked ligands. For example, lymph node-associated follicular B cells express high levels of CD22 beta ligands, none of which are masked by 9-O-acetylation. In contrast, the ligands on lymph node-associated dendritic cells are almost completely masked by 9-O-acetylation, suggesting that masking may regulate interactions between CD22 beta-positive B cells and dendritic cells. In the thymus, only medullary cells express CD22 beta ligands, and a significant portion of these are masked by 9-O- acetylation, particularly at the cortical-medullary junction. Thus, 9-O- acetylation of sialic acids on immune cells is in a position to negatively regulate CD22 beta adhesion events in a manner depending on both cell type and tissue localization.     

Aging by telomere loss can be reversed

Recently in Nature, Jaskelioff et al. (2010) demonstrated that multiple aging phenotypes in a mouse model of accelerated telomere loss can be reversed within 4 weeks of reactivating telomerase. This raises the major question of whether physiological aging, likely caused by a combination of molecular defects, may also be reversible.     

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.     

Collagen binding site in collagenase can be determined using the concept of sense-antisense peptide interactions.

Tissue degradation and invasion are hallmarks of the metastatic phenotype. While several extracellular matrix components can be digested by proteases, degradation of interstitial collagen is selectively initiated by collagenase. It is obvious that inhibitors of collagenase activity would be extremely useful in preventing tissue destruction and tumor cell invasion and thus prove invaluable therapeutic agents. We describe here the possible development of such inhibitors through the use of the principle of complementary hydropathy. A peptide was deduced from the nucleotide sequence complementary to that coding for the region in interstitial collagen surrounding the bond between Gly775 and Ile776 which is cleaved by the enzyme. Labeled collagen binds specifically and quantitatively to this peptide. A polyclonal mouse serum raised against this peptide recognized purified human collagenase, was able to immunoprecipitate collagenase from cultured human keratinocyte supernatants and was effective in inhibiting collagenolytic activity with a K(iapp) = 0.3 microM.     

Structure of Plasmodium vivax dihydrofolate reductase determined by homology modeling and molecular dynamics refinement

The structure of Plasmodium vivax dihydrofolate reductase (PvDHFR), a potentially important target for antimalarial chemotherapy, was determined by means of homology modeling and molecular dynamics refinement. The structure proved to be consistent with DHFRs of known crystal structure. The comparison of the complexes of the antifolate inhibitor pyrimethamine bound at the active sites of PvDHFR and PfDHFR, the related enzyme from Plasmodium falciparum, prospected the possibility of using structure-based drug design to develop inhibitors that are effective against both malarial enzymes. This study constitutes a first step toward understanding of the antifolate-PvDHFR molecular interactions and possible rationalization of resistance in vivax malaria.