Pattern and process in Amazon tree turnover, 1976-2001

Previous work has shown that tree turnover, tree biomass and large liana densities have increased in mature tropical forest plots in the late twentieth century. These results point to a concerted shift in forest ecological processes that may already be having significant impacts on terrestrial carbon stocks, fluxes and biodiversity. However, the findings have proved controversial, partly because a rather limited number of permanent plots have been monitored for rather short periods. The aim of this paper is to characterize regional-scale patterns of 'tree turnover' (the rate with which trees die and recruit into a population) by using improved datasets now available for Amazonia that span the past 25 years. Specifically, we assess whether concerted changes in turnover are occurring, and if so whether they are general throughout the Amazon or restricted to one region or environmental zone. In addition, we ask whether they are driven by changes in recruitment, mortality or both. We find that: (i) trees 10 cm or more in diameter recruit and die twice as fast on the richer soils of southern and western Amazonia than on the poorer soils of eastern and central Amazonia; (ii) turnover rates have increased throughout Amazonia over the past two decades; (iii) mortality and recruitment rates have both increased significantly in every region and environmental zone, with the exception of mortality in eastern Amazonia; (iv) recruitment rates have consistently exceeded mortality rates; (v) absolute increases in recruitment and mortality rates are greatest in western Amazonian sites; and (vi) mortality appears to be lagging recruitment at regional scales. These spatial patterns and temporal trends are not caused by obvious artefacts in the data or the analyses. The trends cannot be directly driven by a mortality driver (such as increased drought or fragmentation-related death) because the biomass in these forests has simultaneously increased. Our findings therefore indicate that long-acting and widespread environmental changes are stimulating the growth and productivity of Amazon forests.     

Effect of abietane diterpenes from Plectranthus grandidentatus on T- and B-lymphocyte proliferation

Five known abietane diterpenes of the royleanone and coleon type, namely, fatty acid esters of 7alpha-acyloxy-6beta-hydroxyroyleanone (1), grandidone A (2), 7alpha-acetoxy-6beta-hydroxyroyleanone (3), 6beta,7alpha-dihydroxyroyleanone (4) and coleon U (5), isolated from Plectranthus grandidentatus, were evaluated for their effect on the proliferation of human lymphocytes induced by the mitogen PHA. All except 4, showed a dose-dependent suppressor effect, with 3 yielding the most potent antiproliferative activity, followed by 5. These two compounds, that represent diterpenes of the royleanone and coleon type respectively, were also shown to be potent inhibitors of mouse splenocyte proliferation induced by ConA or LPS mitogens. However, the sensitivity of ConA-stimulated splenocytes to their suppressive effect was higher, suggesting a preferential inhibition of T-lymphocyte proliferation. The antiproliferative activity of 3 seemed to be exerted without affecting the expression of the lymphocyte activation marker CD69. On the contrary, 5 was shown to reduce the expression of CD69 of TCD8(+) and B-cells, suggesting a relationship between its antiproliferative effect and the expression of this early marker of activation on these cell populations. The capacity of 5 to induce apoptosis on ConA-stimulated splenocytes could also be related with its antiproliferative activity.     

Rapid population decline in red knots: fitness consequences of decreased refuelling rates and late arrival in Delaware Bay

Most populations of migrant shorebirds around the world are in serious decline, suggesting that vital condition-dependent rates such as fecundity and annual survival are being affected globally. A striking example is the red knot (Calidris canutus rufa) population wintering in Tierra del Fuego, which undertakes marathon 30,000 km hemispheric migrations annually. In spring, migrant birds forage voraciously on horseshoe crab eggs in Delaware Bay in the eastern USA before departing to breed in Arctic polar deserts. From 1997 to 2002 an increasing proportion of knots failed to reach threshold departure masses of 180-200 g, possibly because of later arrival in the Bay and food shortage from concurrent over-harvesting of crabs. Reduced nutrient storage, especially in late-arriving birds, possibly combined with reduced sizes of intestine and liver during refuelling, had severe fitness consequences for adult survival and recruitment of young in 2000-2002. From 1997 to 2002 known survivors in Delaware Bay were heavier at initial capture than birds never seen again, annual survival of adults decreased by 37% between May 2000 and May 2001, and the number of second-year birds in wintering flocks declined by 47%. Population size in Tierra del Fuego declined alarmingly from 51,000 to 27,000 in 2000-2002, seriously threatening the viability of this subspecies. Demographic modelling predicts imminent endangerment and an increased risk of extinction of the subspecies without urgent risk-averse management.     

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.     

Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.     

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.     

Systemic and regional hemodynamic effects of angiotensin-(1-7) in rats

The systemic and regional hemodynamics effects of ANG-(1-7) were examined in urethane-anesthetized rats. The blood flow distribution (kidneys, skin, mesentery, lungs, spleen, brain, muscle, and adrenals), cardiac output, and total peripheral resistance were investigated by using fluorescent microspheres. Blood pressure and heart rate were recorded from the brachial artery. ANG-(1-7) infusion (110 fmol x min(-1) x 10 min(-1) iv) significantly increased blood flow to the kidney (5.10 +/- 1.07 to 8.30 +/- 0.97 ml x min(-1) x g(-1)), mesentery (0.73 +/- 0.16 to 1.17 +/- 0.49 ml x min(-1) x g(-1)), brain (1.32 +/- 0.44 to 2.18 +/- 0.85 ml x min(-1) x g(-1)), and skin (0.07 +/- 0.02 to 0.18 +/- 0.07 ml x min(-1) x g(-1)) and the vascular conductance in these organs. ANG-(1-7) also produced a significant increase in cardiac index (30%) and a decrease in total peripheral resistance (2.90 +/- 0.55 to 2.15 +/- 0.28 mmHg x ml(-1) x min x 100 g). Blood flow to the spleen, muscle, lungs, and adrenals, as well as the blood pressure and heart rate, were not altered by the ANG-(1-7) infusion. The selective ANG-(1-7) antagonist A-779 reduced the blood flow in renal, cerebral, mesenteric, and cutaneous beds and blocked the ANG-(1-7)-induced vasodilatation in the kidney, mesentery, and skin, suggesting a significant role of endogenous ANG-(1-7) in these territories. The effects of ANG-(1-7) on the cerebral blood flow, cardiac index, systolic volume, and total peripheral resistance were partially attenuated by A-779. A high dose of ANG-(1-7) (11 pmol x min(-1) x 10 min(-1)) caused an opposite effect of that produced by the low dose. Our results show for the first time that ANG-(1-7) has a previously unsuspected potent effect in the blood flow distribution and systemic hemodynamics.     

Activation of TGF-beta by Leishmania chagasi: importance for parasite survival in macrophages

TGF-beta is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-gamma, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-beta is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-beta levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-beta was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-beta in human macrophage cultures without changing the total TGF-beta. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-beta. Incubation of latent TGF-beta with Leishmania sp. promastigotes caused active TGF-beta to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-beta. TGF-beta activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-beta inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-beta had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-beta stores at extracellular sites during infection, and the ability of the parasite to activate TGF-beta in its local environment, leads to high levels of active TGF-beta in the vicinity of the infected macrophage. Locally activated TGF-beta could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.     

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.     

Induction of antigen-specific regulatory T cells following overexpression of a Notch ligand by human B lymphocytes

In mice, activation of the Notch pathway in T cells by antigen-presenting cells overexpressing Notch ligands favors differentiation of regulatory T lymphocytes responsible for antigen-specific tolerance. To determine whether this mechanism operates in human T cells, we used Epstein-Barr virus-positive lymphoblastoid cell lines (EBV-LCL) as our (viral) antigen-presenting cells and overexpressed the Notch ligand Jagged-1 (EBV-LCL J1) by adenoviral transduction. The EBV-LCL J1s were cocultured with autologous T cells, and the proliferative and cytotoxic responses to EBV antigens were measured. Transduction had no effect on EBV-LCL expression of major histocompatibility complex (MHC) antigens or of costimulatory molecules CD80, CD86, and CD40. However, we observed a 35% inhibition of proliferation and a >65% reduction in cytotoxic-T-cell activity, and interleukin 10 production was increased ninefold. These EBV-LCL J1-stimulated T lymphocytes act as antigen-specific regulatory cells, since their addition to fresh autologous T cells cultured with autologous nontransduced EBV-LCL cells significantly inhibited both proliferation and cytotoxic effector function. Within the inhibitory population, CD4(+)CD25(+) and CD8(+)CD25(-) T cells had the greatest activity. This inhibition appears to be antigen-specific, since responses to Candida and cytomegalovirus antigens were unaffected. Hence, transgenic expression of Jagged-1 by antigen-presenting cells can induce antigen-specific regulatory T cells in humans and modify immune responses to viral antigens.