ATP-dependent calcium transport in membrane vesicles of the cyanobacterium, Anabaena variabilis

Transport of Ca²⁺ in membrane vesicles of the cyanobacterium Anabaena variabilis has been investigated. The light membranes previously shown to carry a Mg²⁺-dependent, Ca²⁺-stimulated ATPase (Lockau, W. and Pfeffer, S. (1982) Z. Naturforsch. 37C, 658–664) accumulate Ca²⁺ upon addition of ATP, whereas the (heavier) thylakoids do not. A stoichiometry of 0.3 Ca²⁺ taken up per ATP hydrolyzed has been determined from initial rates, which is considered to be an underestimation of the true stoichiometry of the pump. Calcium transport and Ca²⁺-stimulated ATPase activity are both sensitive to Na₃VO₄ (an inhibitor of ATPases forming a phosphorylated intermediate), show the same pH optimum and a comparable dependence on ATP concentration. Calcium transport is also supported by nucleoside triphosphates other than ATP, although at lower rates. Accumulation of calcium is abolished by an ionophore of divalent cations, ionophore A23187, but is resistant to ionophores of monovalent cations and to the inhibitor of F₁-F₀-type ATPases, N,N′-dicyclohexylcarbodiimide. It is concluded that the ATPase is a primary calcium pump.     

Interferon effects on the growth and division of human fibroblasts.

The overall rate of proliferation of human fibroblasts in culture is reduced at interferon concentrations greater than 40 international reference units (U)/ml. Inhibition is near maximal at 640 U/ml, at which concentration the doubling time between 24 and 72 h after beginning of treatment is increased 2–3 times over the control value. Inhibition of cell proliferation was not readily reversible upon removal of interferon and refeeding of cultures. Study of the mitotic behavior of individual cells showed that the first intermitotic interval after beginning of treatment with interferon (640 U/ml) was prolonged in about two-thirds of the cells. In this fraction, many cells failed to divide again after the second post-treatment mitosis, while others exhibited a progressively increasing intermitotic interval with subsequent divisions. One-third of the interferon-treated fibroblasts initially divided at a rate similar to the rate of proliferation of control cells, but subsequently these cells also slowed down and finally stopped dividing. After treatment at 640 U/ml for 3 days, the rates of DNA, RNA, and protein synthesis were depressed to 86, 75, and 64% of control values, respectively. However, the interferon-treated fibroblasts had grown larger than control cells as indicated by the following parameters: cell attachment area, 165%; volume, 131%; DNA content, 130% and protein content, 150%. Thus, interferon does not prevent cell growth, but interferes with cell division.     

Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus.

Hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait, is associated with a predisposition to neoplasia. The present study describes the differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Primary cutaneous outgrowths were derived from normal appearing subepidermal biopsies of ACR phenotypes and appropriate controls. Exponentially growing cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny subjected to the viral probe were 100-1000 fold more susceptible to transformation than were normal skin fibroblast cultures. The virally transformed human skin fibroblasts showed a loss of anchorage dependency in carboxymethylcellulose suspension and formed tumors in athymic mice. The results suggest that skin fibroblasts obtained from individuals gine sarcoma virus.     

P relaxation responses associated with n(2)/o(2) diffusion in soybean nodule cortical cells and excised cortical tissue

N₂-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O₂ followed by N₂ or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (δ 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O₂ perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T₁), and spin-spin relaxation time (T₂), under perfusion with N₂ or Ar as opposed to O₂, indicated that loss of signal was due to T₁ saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T₁, values derived from biexponential relaxation processes under perfusing O₂ were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N₂ these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T₁ relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T₂ values for cortical tissue were also biexponential but were essentially the same under O₂ (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N₂ (39% 0.07 ± 0.01 s and 61% 0.37 ± 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T₁ decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T₁ experiments demonstrate that O₂ does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O₂ perfusion) is responsible for the observed relaxation changes.     

Biological diversity versus risk for mosquito nuisance and disease transmission in constructed wetlands in southern Sweden

In southern Sweden, many wetlands have been constructed, and maintaining or increasing biological diversity is often included in the aims. Some wetlands are constructed near human settlements, thus raising the problem of wetlands being associated with mosquitoes (Diptera: Culicidae). Increased biodiversity (including mosquito diversity) is considered desirable, whereas mosquito nuisance from a human point of view is not. Adult mosquito abundance, diversity and species assemblages of constructed wetlands were compared to natural wetlands. The potential of constructed wetlands for mosquito nuisance and transmission of mosquito-borne viruses was evaluated. The study areas included five constructed and four natural wetlands. Mosquito abundance and species richness were higher in the natural than in the constructed wetlands, and showed a positive correlation with wetland size. Mosquito species assemblages formed three clusters, which were not explained by origin, size and water permanence of wetlands. In a redundancy analysis, however, mosquito faunas showed significant relationships with these variables, and size and origin of wetlands were most important. Major nuisance species (multivoltine species feeding on mammals and laying eggs on soil) were found in all wetlands, although in relatively low numbers. Risk assessment for Sindbis virus transmission showed moderate risk for two constructed wetlands near human settlements. It is concluded that small size of constructed wetlands has the advantage of low mosquito numbers from a human point of view. The use of functional groups is recommended as a tool for presenting mosquito data to the public, and for helping communication between scientists and administrative decision makers.     

The critical role of LIGHT in promoting intestinal inflammation and Crohn's disease

Crohn's disease (CD) is a type of inflammatory bowel disease associated with increased Th1 cytokines and unique pathological features. However, its pathogenesis has not been fully understood. Previous studies showed that homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for HVEM on T cells (LIGHT) transgenic (Tg) mice develop autoimmunity including intestinal inflammation with a variable time course. In this study, we establish an experimental model for CD by adoptive transfer of Tg mesenteric lymph node cells into RAG(-/-) mice. The recipients of Tg lymphocytes rapidly develop a disease strikingly similar to the key pathologic features and cytokine characterization observed in CD. We demonstrate that, as a costimulatory molecule, LIGHT preferentially drives Th1 responses. LIGHT-mediated intestinal disease is dependent on both of its identified signaling receptors, lymphotoxin beta receptor and herpes virus entry mediator, because LIGHT Tg mesenteric lymph node cells do not cause intestinal inflammation when transferred into the lymphotoxin beta receptor-deficient mice, and herpes virus entry mediator on donor T cells is required for the full development of disease. Furthermore, we demonstrated that up-regulation of LIGHT is associated with active CD. These data establish a new mouse model resembling CD and suggest that up-regulation of LIGHT may be an important mediator of CD pathogenesis.     

Molecular mechanisms of action of angiopreventive anti-oxidants on endothelial cells: microarray gene expression analyses

The anti-oxidants N-acetyl-l-cysteine (NAC) and (-)-epigallocatechin-3-gallate (EGCG) inhibit tumor vascularization by reducing endothelial cell migration and invasion in a similar, non additive and non synergistic manner but do not alter the growth of human umbilical vein endothelial cells. Here we address the effects of the two chemopreventive drugs on endothelial cell signaling by means of expression profiling and real-time PCR validation. We identify a series of angiogenesis related genes that are similarly regulated by the two drugs. Anti-oxidant treated endothelial cells show gene expression profiles compatible with a less activated, less apoptosis prone and less migratory phenotype. The anti-oxidants affect expression of several components of the TNFalpha response pathway including downstream genes that are regulated in the opposite direction in the absence of the inflammatory cytokine. The interference with the TNFalpha pathway is reflected by reduced NFkappaB activation in anti-oxidants treated cells but the compounds are not able to contrast TNFalpha mediated activation of NFkappaB. The chemopreventive action of these compounds thus relies on a reduction of basal levels of endothelial cell activation. Down-regulation of the TNFalpha responsive pro-metastatic, pro-inflammatory genes, urokinase plasminogen activator and selectin E, further implies anti-metastatic effects for these drugs.     

The uptake, metabolism, transport and transfer of nitrogen in an arbuscular mycorrhizal symbiosis

Nitrogen (N) is known to be transferred from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis, yet its metabolism, storage and transport are poorly understood. In vitro mycorrhizas of Glomus intra-radices and Ri T-DNA-transformed carrot roots were grown in two-compartment Petri dishes. (15)N- and/or (13)C-labeled substrates were supplied to either the fungal compartment or to separate dishes containing uncolonized roots. The levels and labeling of free amino acids (AAs) in the extra-radical mycelium (ERM) in mycorrhizal roots and in uncolonized roots were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Arginine (Arg) was the predominant free AA in the ERM, and almost all Arg molecules became labeled within 3 wk of supplying (15)NH(4) (+) to the fungal compartment. Labeling in Arg represented > 90% of the total (15)N in the free AAs of the ERM. [Guanido-2-(15)N]Arg taken up by the ERM and transported to the intra-radical mycelium (IRM) gave rise to (15)N-labeled AAs. [U-(13)C]Arg added to the fungal compartment did not produce any (13)C labeling of other AAs in the mycorrhizal root. Arg is the major form of N synthesized and stored in the ERM and transported to the IRM. However, NH(4) (+) is the most likely form of N transferred to host cells following its generation from Arg breakdown.     

Boron compartmentation in roots of sunflower plants of different boron status: A study using the stable isotopes 10B and 11B adopting two independent approaches

The intracellular compartmentation of boron (B) in roots of sunflower plants precultured with 100 μ M B (high B) or 1 μ M B (low B) was studied using two independent approaches. In the first approach, short-term efflux studies using the stable isotopes ¹¹B and ¹⁰B were carried out. In roots of high B plants, the calculated concentrations of B (nmol g_FW ⁻¹) were 52.6 in the cell wall, 7.5 in the vacuole, 27.1 in the cytosol and 48.0 in the free space. In roots of low B plants, the concentrations of B (nmol g_FW ⁻¹) were 43.4 in the cell wall, 2.8 in the vacuole, 17.9 in the cytosol and almost zero in the free space. Although the B supply differed by a factor 100, the B concentrations in the cytosol and the vacuole of low B plants were 66 and 37% of the respective concentrations in high B plants. This suggests an additional role for B in plant metabolism, besides its function in the cell wall. In the second approach, root B pools (cell sap and water-insoluble residue) were determined for comparison, and found to be in good agreement with the results from the efflux study.     

Targeting Rab GTPases to distinct membrane compartments

Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments. The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery. The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.