Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca²⁺-transport and storage pathways that include Ca²⁺ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca²⁺ binding characteristics of the C-domain of calreticulin to selectively increase Ca²⁺ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20–50 moles of Ca²⁺ per mole of protein and has been shown to be the major site of Ca²⁺ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9–35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca²⁺ stores, and that this Ca²⁺ reserve can be used by the plant in times of stress.     

Temporal changes in the distribution of thoracic duct lymphoblasts to synovium and other tissues of rats with adjuvant-induced arthritis

The distribution of lymphoblasts(lymphocytes in cell cycle) obtained from the central lymph of donor rats and transferred adoptively to syngeneic recipients has been shown previously to be influenced by the presence of arthritis in either donor or recipient rats. The intent of the present study was to examine patterns of distribution of lymphoblasts in the early period after transfer, when extravasation of donor lymphoblasts was expected to occur. Thoracic duct lymphoblasts labelled in vitro with [125I]-iododeoxyuridine were detected in recipient rats by external radiometry and autoradiography.Irrespective of donor status, fewer donor lymphoblasts accumulated in the feet of normal recipients when compared to arthritic recipients at 15 min, 2 h and 24 h after cell transfer.When recipients of similar disease status were compared, the percentages of injected lymphoblasts from normal and arthritic donors recovered in the feet were similar at 15 min and 2 h after transfer. The proportions of lymphoblasts recovered in the feetat 24 h after injection declined in normal recipients and arthritic recipients of cells from normal donor rats. Importantly,this decline did not occur when both the donor and the recipient were arthritic. In the hindpaws, donor lymphoblasts were located predominantly in the bone marrow, except in transfers between arthriticrats, when at 24 h they were predominantly in the synovium.At 15 min, lymphoblasts were detected within the lumen of vessels within synovium, whereas by 2 h extravasation of these cells was evident. In conclusion, lymphoblasts accumulate more readily in hindfeet that are inflamed. In the early hours after injection, lymphoblasts from normal and arthritic donors are recruited equally, but these early levels are only maintained for 24 hin the combination of arthritic donor and arthritic recipient. Adramatic change in the proportion of lymphoblasts located in synoviumat this later time suggests that a dynamic process of relocation,retention and/or local cell division maintains the numbers of arthritic donor cells in the latter combination.     

The gene for juvenile hyaline fibromatosis maps to chromosome 4q21

Juvenile hyaline fibromatosis (JHF) is an autosomal recessive condition characterized by multiple subcutaneous nodular tumors, gingival fibromatosis, flexion contractures of the joints, and an accumulation of hyaline in the dermis. We performed a genomewide linkage search in two families with JHF from the same region of the Indian state of Gujarat and identified a region of homozygosity on chromosome 4q21. Dense microsatellite analyses within this interval in five families with JHF who were from diverse origins demonstrate that all are compatible with linkage to chromosome 4q21 (multipoint LOD score 5.5). Meiotic recombinants place the gene for JHF within a 7-cM interval bounded by D4S2393 and D4S395.     

Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4-yellow fluorescent protein (YFP) move along tubulin-cyan fluorescent protein-labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long-range movements of GLUT4-YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co-localized with perinuclear GLUT4. Dominant-negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc-tagged GLUT4-green fluorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4-containing membranes on microtubules via conventional kinesin through a PI3-kinase-independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.     

Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation,yet promotes growth and survival independent of G protein-coupled receptors

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.     

Nitric oxide and Fenton/Haber-Weiss chemistry: nitric oxide is a potent antioxidant at physiological concentrations

We have examined the action of nitric oxide (NO) on the ability of Fenton's reagent (ferrous iron and hydrogen peroxide), to oxidize a number of organic optical probes. We found that NO is able to arrest the oxidation of organic compounds at concentrations of NO found in brain, in vivo. We present evidence that Fenton's reagent proceeds via a ferryl intermediate ([Fe[double bond]O]2+), before the generation of hydroxyl radical *OH. NO reacts rapidly with this ferryl, blocking the production of *OH. We propose that NO has an important role in protecting biological tissues, and the brain in particular, from Fenton chemistry.     

Altered Antibody Profiles against Common Infectious Agents in Chronic Disease

Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons.  We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-γ autoantibodies (IFN-γ AAB), HIV and Sjögren’s syndrome (SjS) to determine if their antibody profiles differed from control subjects.  The IFN-γ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005),   and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-γ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls.  The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.     

Genetic structure of the xerophilous bromeliad Pitcairnia geyskesii on inselbergs in French Guiana – a test of the forest refuge hypothesis

Inselbergs are isolated granitic rock outcrops that provide distinctive ecological conditions. In northern South America they rise above the surrounding rainforest. Among inselberg specialists, Pitcairnia geyskesii (Bromeliaceae) is restricted to these habitats in French Guiana. We studied populations from 12 inselbergs using 7 microsatellite loci to give a "reverse image" of the reduction-expansion of the rainforest in the context of the refuge hypothesis. Our analyses showed that populations are fragmented with dispersal occurring only over very short distances. Genetic diversity was higher in northern French Guiana, whereas specific alleles were observed in the south. The results point to the occurrence of a dry corridor in the north, as hypothesized by Tardy (1998) based on charcoal analyses, whereas de Granville's (1982) hypothesis of a unique past refuge is not confirmed. Moreover, our data suggests the importance of Oyapock River as a pathway for range expansion, arguing against the potential role of the Inini-Camopi Mountains as a physical barrier. Finally, in spite of a strongly argued scenario in favour of a north-to-south migration history, a clear genetic isolation of P. geyskesii populations living on inselbergs of the Mitaraka archipelago suggests a distinct ancestry of the most southern populations.