B1 cells contribute to serum IgM,but not to intestinal IgA,production in gnotobiotic Ig allotype chimeric mice

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.     

CD36 deficiency increases insulin sensitivity in muscle,but induces insulin resistance in the liver in mice

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.     

Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression

Two new 5'-untranslated region (5'UTR) exons were identified in the human gene for the lymphocyte-specific endonuclease recombination activating gene-1 (RAG1) required for the somatic recombination yielding functional Ag receptors. These 5'UTR exons were used in three different splice forms by jejunal lymphocytes of the T cell lineage. RAG1 mRNA containing the previously described 5'UTR exon was not expressed in these cells. Conversely, one of the new 5'UTR exons was not expressed in thymus. The new RAG1 mRNA splice forms were all expressed in immature T cells (CD2(+)CD7(+)CD3(-)). This cell population also expressed high levels of mRNA for the pre-T alpha-chain. In situ hybridization demonstrated jejunal cells expressing the new splice forms of RAG1 mRNA, both intraepithelially and in lamina propria. Pre-T alpha-chain mRNA-expressing cells were detected at the same sites. These results strongly suggest ongoing TCR gene rearrangement in human small intestinal mucosa, yielding T cells specially adapted for this environment. This seems to be achieved by two parallel processes, extrathymic T cell development and peripheral Ag-driven TCR editing.     

Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver

Scavenger receptor class B, type I (SRBI) is a key regulator of high density lipoprotein (HDL) metabolism. It facilitates the efflux of cholesterol from cells in peripheral tissues to HDL and mediates the selective uptake of cholesteryl esters from HDL in the liver. We investigated the effects of SRBI deficiency in the arterial wall and in the liver using SRBI-deficient mice and wild-type littermates fed a Western-type diet. The SRBI-deficient mice showed massive accumulation of cholesterol-rich HDL in the circulation, reflecting impaired delivery to the liver. Strikingly, SRBI deficiency did not alter hepatic cholesterol (ester) content nor did it affect the expression of key regulators of hepatic cholesterol homeostasis, including HMG-CoA reductase, the low density lipoprotein receptor, and cholesterol 7alpha-hydroxylase. However, a approximately 40% reduction in biliary cholesterol content was observed, and the expression of ABCG8 and ABCG5, ATP half-transporters implicated in the transport of sterols from the liver to the bile, was attenuated by 70 and 35%, respectively. In contrast to the situation in the liver, SRBI deficiency did result in lipid deposition in the aorta and atherosclerosis. Vascular mRNA analysis showed increased expression of inflammatory markers as well as of genes involved in cellular cholesterol homeostasis. Our data show that, although hepatic cholesterol homeostasis is maintained upon feeding a Western-type diet, SRBI deficiency is associated with de-regulation of cholesterol homeostasis in the arterial wall that results in an increased susceptibility to atherosclerosis.     

Bridging the gap between structural and lattice models: a parameterization of energy transfer and trapping in Photosystem I

In the absence of an accurate structural model, the excited state dynamics of energy-transferring systems are often modeled using lattice models. To demonstrate the validity and other potential merits of such an approach we present the results of the modeling of the energy transfer and trapping in Photosystem I based upon the 2.5 A structural model, and show that these results can be reproduced in terms of a lattice model with only a few parameters. It has recently been shown that at room temperature the dynamics of a hypothetical Photosystem I particle, not containing any red chlorophylls (chls), are characterized by a longest (trapping) lifetime of 18 ps. The structure-based modeling of the dynamics of this particle yields an almost linear relationship between the possible values of the intrinsic charge-separation time at P700, 1/gamma, and the average single-site lifetime in the antenna, tauss. Lattice-based modeling, using the approach of a perturbed two-level model, reproduces this linear relation between tauss and 1/gamma. Moreover, this approach results in a value of the (modified) structure-function corresponding to a structure exhibiting a mixture of the characteristics of both a square and a cubic lattice, consistent with the structural model. These findings demonstrate that the lattice model describes the dynamics of the system appropriately. In the lattice model, the total trapping time is the sum of the delivery time to the reaction center and the time needed to quench the excitation after delivery. For the literature value of tauss=150 fs, both these times contribute almost equally to the total trapping time of 18 ps, indicating that the system is neither transfer- nor trap-limited. The value of approximately 9 ps for the delivery time is basically equal to the excitation-transfer time from the bulk chls to the red chls in Synechococcus elongatus, indicating that energy transfer from the bulk to the reaction center and to the red chls are competing processes. These results are consistent with low-temperature time-resolved and steady-state fluorescence measurements. We conclude that lattice models can be used to describe the global energy-transfer properties in complex chromophore networks, with the advantage that such models deal with only a few global, intuitive parameters rather than the many microscopic parameters obtained in structure-based modeling.     

Simultaneous selection on two fitness-related traits in the butterfly Bicyclus anynana

Abstract. Theory about the role of constraints in evolution is abundant, but few empirical data exist to describe the consequences a bias in phenotypic variation has for micro evolution. Responses to natural selection can be severely hampered by a genetic correlation among a suite of traits. Constraints can be studied using antagonistic selection experiments, that is, two-trait selection in opposition to this correlation. The two traits studied here were development time and wing pattern (eyespot size) in the butterfly Bicyclus anynana , both of which have a clear adaptive significance. Rates of response were higher for eyespot size than for development time, but were independent of the concurrent selection (either in the same direction as the correlation or perpendicular to it). Regimes differed in both traits in all directions after 11 generations of selection. The uncoupling lines had higher relative responses than the synergistic lines in development time and equal relative responses in eyespot size. The patterns for eyespot size (reaction norms) were consistent across different rearing temperatures. Differences in lines selected for fast and slow development time were more pronounced at lower temperatures, irrespective of the direction of joint wing pattern selection. Furthermore, correlated responses in pupal weight and growth rate were observed; lines selected for a slower development had higher pupal weights, especially at lower temperatures. The response of the uncoupling lines was not hampered by a lack of selectable genetic variation, and the relative response in the development time was larger than expected based on response in the coupled direction and quantitative genetic predictions. This suggests that the structure of the genetic architecture does not constrain the short-term, independent evolution of both wing pattern and development time.     

The small GTPase Rap1 is activated by turbulence and is involved in integrin [alpha]IIb[beta]3-mediated cell adhesion in human megakaryocytes

The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrin-mediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin alphaIIbbeta3, in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by alpha-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls alphaIIbbeta3-mediated cell adhesion.     

Demonstration of spontaneous and stretch induced urinary bladder EMG in the living rabbit

Spontaneous bladder EMG was recorded in the living rabbit from an isovolumetric bladder without chemical or electrical stimulation. Mechanical intervention, either by lifting the bladder out of the abdomen or by rapid filling, resulted in stretch induced bladder EMG. A self made epoxy resin electrode device that embedded 32 EMG recording electrodes in a matrix like pattern, each electrode Ag/AgCl, d = 0.6 mm with an interdistance of 2.3 mm, was used for registration. The recorder used a common average reference technique and a sample frequency of 400 Hz. A signal bandwidth of 0.05 to 108 Hz was available for analysis. Spontaneous EMG consisted of single spikes and bursts (2-20 spikes), but not of continuous activity. The shape of spikes was triphasic. Single spikes appeared with and without burst activity. Small (2-5 spikes) and large bursts (6-20 spikes) were discerned; small bursts not necessarily propagated across electrodes, large bursts did and were able to organize, suggesting that they were under short neuron system control. Spontaneous EMG was probably related to both contraction and relaxation. Stretch induced EMG was characterised by continuous activity on all electrodes, spikes that followed each other immediately, slowly fading away. The spikes had an elongated third phase when compared to the shape of spontaneous activity. Highest activity and amplitudes were found after lifting the bladder out of the abdomen and placing it on the electrode device. A concept is put forward in which the continuous activity is not unequivocally related to muscle shortening, but where the current stress and strain situation of the bladder tissue can cause a muscle fibre elongation upon the appearance of electrical activity. The EMG activity found was in many aspects similar to results of a previous study using mortalized rabbits. Artifact sources like the heart, respiration, or local movement between electrode and bladder could easily be identified due to the new experimental methodology used.     

Rap1 signalling: adhering to new models

Ras-like GTPases are ubiquitously expressed, evolutionarily conserved molecular switches that couple extracellular signals to various cellular responses. Rap1, the closest relative of Ras, has attracted much attention because of the possibility that it regulates Ras-mediated signalling. Rap1 is activated by extracellular signals through several regulatory proteins, and it might function in diverse processes, ranging from modulation of growth and differentiation to secretion, integrin-mediated cell adhesion and morphogenesis.     

Stenurus globicephalae Baylis et Daubney, 1925 (Nematoda: Pseudaliidae) from a false killer whale,Pseudorca crassidens (Cetacea: Delphinidae), stranded on the coast of Uruguay

Stenurus globicephalae Baylis et Daubney, 1925 (Nematoda: Pseudaliidae) was found in the cranial air sinuses of a false killer whale, Pseudorca crassidens (Owen), stranded on the coast of Uruguay in 1999. Although this species has been reported once in P. crassidens from the North Atlantic, this is the first record for South America. A total of 920 specimens were obtained, of which 663 were females (body length: 4.34 +/- 0.45 cm) and 257 were males (2.99 +/- 0.18 cm). Morphometric details are presented for S. globicephalae in this host, which do not show significant differences from those parasitizing Globicephala melas (Traill), but are distinct from those parasitizing Peponocephala electra (Gray). The host's skull revealed loss of osseous mass with the disappearance of the left zygomatic arch, and the left jaw had three osseous fenestrations in the region related to the organ of acoustic reception. These lesions support the hypothesis that this infection, known as stenurosis, was related to the stranding.