Reductions in I kappa B epsilon and changes in NF-kappa B activity during B lymphocyte differentiation

The levels and stability of IkappaBepsilon have been examined in unstimulated and stimulated splenic B cells and compared with that of IkappaBalpha and IkappaBbeta. Primary murine splenic B cells but not T cells were found to contain high levels of IkappaBepsilon protein, equivalent to levels of the abundant IkappaBalpha. Most agents that activate IkappaBalpha and IkappaBbeta degradation do not induce rapid degradation of IkappaBepsilon. Interestingly, however, the levels of IkappaBepsilon, but not of IkappaBalpha or IkappaBbeta, are dramatically reduced upon the stimulation of B cells both in vivo and in vitro. Since IkappaBepsilon exhibits substrate specificity for NF-kappaB Rel homodimers, this suggested the possibility that changes in NF-kappaB-responsive genes might also occur during this transition. Consistent with this hypothesis, we found that a NF-kappaB reporter construct sensitive to p65/RelA homodimers is activated at the time that IkappaBepsilon levels decline following B cell stimulation. In IgG(+) B cell lines, which contain low levels of IkappaBepsilon, this same reporter construct was inactive, suggesting that the increases in Rel homodimer activity that accompany B cell stimulation are transient. However, there are differences in the level of expression of NF-kappaB-responsive genes in these IgG(+) B cell lines compared with their IgM(+) counterparts. From these data, we conclude that there are transient changes in NF-kappaB activity due to reductions in IkappaBepsilon, which might contribute to long-term, persistent changes that accompany B cell differentiation. We propose an important role for IkappaBepsilon in the differential regulation of nuclear NF-kappaB activity in stimulated B cells.     

IL-13 regulates the immune response to inhaled antigens

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.     

A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.     

Diagnosis of the Earliest Strain-Specific Interactions between Tobacco Mosaic Virus and Chloroplasts of Tobacco Leaves in Vivo by Means of Chlorophyll Fluorescence Imaging

Fluorescence imaging was used to diagnose early stages of the strain-specific interactions between tobacco mosaic virus (strain PV230) and chloroplasts following infection of tobacco leaves (Nicotiana tabacum cv Xanthi). The earliest indication of interaction in tissues that ultimately become chlorotic was a reduction in chlorophyll fluorescence, and there was little fluorescence quenching compared with adjacent healthy tissues. Subsequently, fluorescence increased but remained unquenched. In the late stages fluorescence declined again in chlorotic regions as the chloroticmosaic symptoms developed. These in vivo data showing altered fluorescence yields confirm strain-specific interaction of virus coat protein with photosystem II (PSII) components in vitro, leading to photoinhibition and photooxidation of chlorophyll in infected cells and the development of visible chlorotic-mosaic symptoms. Although mechanisms leading to the low, unquenched fluorescence condition are not known, the intermediate high, unquenched fluorescence condition is consistent with impaired PSII electron transport as measured in vitro. Fluorescence lesions appear more rapidly and develop more extensively in high light, consistent with the faster and larger extent of symptom formation in high-light-grown leaves than in low-light-grown leaves.     

Statistical analysis of synaptic transmission: model discrimination and confidence limits.

Procedures for discriminating between competing statistical models of synaptic transmission, and for providing confidence limits on the parameters of these models, have been developed. These procedures were tested against simulated data and were used to analyze the fluctuations in synaptic currents evoked in hippocampal neurones. All models were fitted to data using the Expectation-Maximization algorithm and a maximum likelihood criterion. Competing models were evaluated using the log-likelihood ratio (Wilks statistic). When the competing models were not nested, Monte Carlo sampling of the model used as the null hypothesis (H0) provided density functions against which H0 and the alternate model (H1) were tested. The statistic for the log-likelihood ratio was determined from the fit of H0 and H1 to these probability densities. This statistic was used to determine the significance level at which H0 could be rejected for the original data. When the competing models were nested, log-likelihood ratios and the chi 2 statistic were used to determine the confidence level for rejection. Once the model that provided the best statistical fit to the data was identified, many estimates for the model parameters were calculated by resampling the original data. Bootstrap techniques were then used to obtain the confidence limits of these parameters.     

Recognising moulting behaviour in trilobites by examining morphology,development and preservation: Comment on Błażejowski et al. 2015

A 365 million year‐old trilobite moult‐carcass assemblage was described by B?a?ejowski et al. (2015) as the oldest direct evidence of moulting in the arthropod fossil record. Unfortunately, their suppositions are insufficiently supported by the data provided. Instead, the morphology, configuration and preservational context of the highly fossiliferous locality (Kowala Quarry, Poland) suggest that the specimen consists of two overlapping, queued carcasses. The wider fossil record of moulting actually extends back 520 million years, providing an unparalleled opportunity to study behaviour, ecology and development in early animals. Taking cues from modern analogues, it is possible to quantify precise details about moulting behaviour to determine broad‐scale evolutionary trends, ontogenetic sequences and morphological selection pressures. In this review, we argue that this rich source of data has been underused in evolutionary studies, though has great potential for investigating the life history and evolution of arthropods in deep time.

     

Identification and isolation of a T4+T8+ cell with high T3 expression in human thymus: a possible late intermediate in thymocyte differentiation

By using sensitive three-color fluorescence flow cytometric techniques, we were able to identify a T4+T8+ thymocyte with high T3 surface density (T3H) representing 4 to 9% of thymocytes. To characterize the T3HT4+T8+ cell, thymic subpopulations with high T3 surface density (T3H) and lower T3 density (T3L/T3-) were compared with regard to T6 expression. The T3H subpopulation was characterized by lower numbers of T6+ cells and reduced levels of T6 antigen density, whereas the T3L/T3- population was greater than 90% T6+ and expressed this antigen at high cell surface density. In addition, T3H fractions appeared to possess higher levels of nuclear activation with respect to the T3L/T3- population as indicated by increased log 90 degrees scatter profiles. These results suggest that thymocytes with high T3 surface expression are not only more differentiated, but also more activated than the majority of the thymic population. The T3HT4+T8+ fraction could be distinguished from T4+T8+ thymocytes with lower T3 density not only by an increased log 90 degrees scatter profile, but also by the presence of T4+T8+ cells with reduced levels of T8 surface antigen. Our results indicate that T4+T8+ thymocytes with high T3 surface density are a distinct subpopulation and may represent the immediate precursors of the phenotypically more mature T3HT4+T8- and T3HT8+T4- subpopulations found in human thymus.     

Activation of immature cortical thymocytes through the T11 sheep erythrocyte binding protein

Two major pathways, the T cell receptor and the T11 alternate pathway, allow for T cell activation. In the human thymus, the T cell antigen receptor complex is reduced or absent on immature thymocytes, whereas the T11 glycoprotein is present at high cell surface density on all thymocytes. To determine whether activation through the T11 pathway induces similar or different changes in mature and immature thymocytes, we fractionated thymocytes according to their surface expression of the T3-T cell receptor (T3/Ti) complex. We report that two populations, one with high and one with low T3/Ti expression, can be activated through the T11 pathway to undergo nuclear activation and express IL 2 receptors. Moreover, in the absence of accessory cells, only the most mature population, expressing high T3 density, could be induced to proliferate, whereas the subset representing immature cortical thymocytes required accessory cells for proliferation. These findings suggest that the cellular microenvironment may have a critical role in regulating the activation of immature cortical thymocytes and that this cell population may not represent "nonfunctional" dead end cells, but rather a valid intermediate in human thymic differentiation.     

B7, a B-cell-restricted antigen that identifies preactivated B cells

After activation with antigen or mitogen, a number of cell surface proteins appear that are not expressed on resting B cells. To date, a number of B lineage restricted and associated activation antigens have been reported that appear at distinct intervals after in vitro activation. In this report, we describe a new B lineage restricted activation antigen (B7) that appears within 24 hr of in vitro stimulation. The expression of B7 antigen, which is detected on a minor subpopulation of B cells isolated from peripheral blood and lymphoid tissues, is strongly induced following stimulation with either anti-immunoglobulin or Epstein-Barr virus. In contrast, B7 was not detected on resting or activated T cells or monocytes. The B7 antigen was expressed on a subset of B cell lines and B cell neoplasms, but was not detected on leukemias and lymphomas of T cell or myeloid origin. B7 was distinguished from other B cell restricted and associated activation antigens by its unique pattern of expression on a variety of hemopoietic cell lines. The biochemical characterization of B7, that it is a single chain protein of 60 kDa, further distinguishes it from other B cell activation antigens. The functional importance of the B7 antigen was demonstrated when splenic B cells were fractionated into the B7+ and B7- populations. The peak of proliferation in response to anti-Ig, appeared earlier within the B7+ population. These studies suggest that B7 antigen identifies a subpopulation of B cells that are preactivated or primed in vivo, and have an accelerated response to subsequent activation via cross-linking of surface Ig.     

Alternative 5' exons in c-abl mRNA

The cellular abl proto-oncogene encodes a protein-tyrosine kinase and is expressed in many cell types in two or three mRNA size species. Four types of mouse c-abl cDNAs have been cloned from 70Z/3 lymphoid cells that have different 5' sequences encoding predicted N-terminal regions of 20-45 amino acids. One of the four cDNAs has a predicted N-terminal sequence of met-gly-gln in common with the gag N terminus of v-abl. The 5' heterogeneity appears to be generated by alternative addition of 5' exons onto a common set of 3' exons. Alternative splicing occurs at the same site at which bcr sequences join to abl sequences in the Philadelphia chromosome translocation.