Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.     

Endothelial cell confluence regulates Weibel-Palade body formation

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.     

An in vitro model to evaluate cell adhesion to metals used in implantation shows significant differences between palladium and gold or platinum

Adhesion of tissue cells to metallic implants is a major factor that is important for proper tissue integration. Adhesion of Swiss mouse 3T3 fibroblasts to gold, platinum and palladium surfaces was investigated. Immunofluorescence staining for the integrin subunits alphav and beta1 and the focal contact protein vinculin revealed that cells growing on gold and platinum expressed many focal contacts. In contrast, cells on palladium surfaces had reduced numbers of focal contacts shown by vinculin staining and failed to demonstrate expression of alphav and beta1 in focal contacts. Spread cell area was also significantly reduced on palladium than on other surfaces suggesting that cells on palladium were more weakly attached. This may be due to either a different molecular composition of focal contacts in cells grown on palladium surfaces or unusual microstructural properties of the palladium surface. This model is useful to evaluate adhesion of cells to different metal surfaces.     

The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion

The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growing ends of cortical microtubules and was dependent on polymer growth state; the microtubule-related fluorescence dissipated upon polymer shortening. The protein has a repeated motif at both ends, separated by a predicted rod-like domain, suggesting that it may act as an intermolecular linker. These observations suggest that SPR1 is involved in microtubule polymerization dynamics and/or guidance, which in turn influences touch-induced directional cell expansion and axial twisting.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.     

A modular approach to the synthesis of new reagents useful in the chemical synthesis of modified DNA probes: derivatives of 3-(tert-butyldimethylsiloxy)glutaric anhydride as versatile building blocks in the synthesis of new phosphoramidites and modified solid supports

We present a flexible and cost-efficient synthetic strategy for the preparation of a new family of phosphoramidite and solid-support reagents that can introduce a broad range of modifications into DNA probes. The key intermediate material 3 is synthesized using the inexpensive and commercially available 3-(tert-butyldimethylsiloxy)glutaric anhydride 1 and can be used as common starting material for the preparation of new labeling reagents.     

Overview of interleukin-2 function, production and clinical applications

The existence of interleukin (IL)-2 has been recognized for over 25 years, and it remains one of the most extensively studied cytokines. Here we present a broad overview of IL-2 history, functional activities, biological sources, regulation and applications to disease treatment. IL-2 exerts a wide spectrum of effects on the immune system, and it plays crucial roles in regulating both immune activation and homeostasis. Both IL-2 and its multipartite receptor are prototypical of the Type I receptor superfamily, and both have been exploited in numerous ways in the clinic. Despite the wealth of information generated about IL-2 from in vitro culture systems, in vivo mouse knockout models, and clinical trials in humans, fascinating new aspects of its functions in the immune system continue to emerge.     

Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.     

Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.