Global trends of cropland phosphorus use and sustainability challenges.

To meet the growing food demand while addressing the multiple challenges of exacerbating phosphorus (P) pollution and depleting P rock reserves1-15, P use efficiency (PUE, the ratio of productive P output to P input in a defined system) in crop production needs to be improved. Although many efforts have been devoted to improving nutrient management practices on farms, few studies have examined the historical trajectories of PUE and their socioeconomic and agronomic drivers on a national scale1,2,6,7,11,16,17. Here we present a database of the P budget (the input and output of the crop production system) and PUE by country and by crop type for 1961-2019, and examine the substantial contribution of several drivers for PUE, such as economic development stages and crop portfolios. To address the P management challenges, we found that global PUE in crop production must increase to 68-81%, and recent trends indicate some meaningful progress towards this goal. However, P management challenges and opportunities in croplands vary widely among countries.

Seasonality of temperate forest photosynthesis and daytime respiration.

Terrestrial ecosystems currently offset one-quarter of anthropogenic carbon dioxide (CO2) emissions because of a slight imbalance between global terrestrial photosynthesis and respiration. Understanding what controls these two biological fluxes is therefore crucial to predicting climate change. Yet there is no way of directly measuring the photosynthesis or daytime respiration of a whole ecosystem of interacting organisms; instead, these fluxes are generally inferred from measurements of net ecosystem-atmosphere CO2 exchange (NEE), in a way that is based on assumed ecosystem-scale responses to the environment. The consequent view of temperate deciduous forests (an important CO2 sink) is that, first, ecosystem respiration is greater during the day than at night; and second, ecosystem photosynthetic light-use efficiency peaks after leaf expansion in spring and then declines, presumably because of leaf ageing or water stress. This view has underlain the development of terrestrial biosphere models used in climate prediction and of remote sensing indices of global biosphere productivity. Here, we use new isotopic instrumentation to determine ecosystem photosynthesis and daytime respiration in a temperate deciduous forest over a three-year period. We find that ecosystem respiration is lower during the day than at night-the first robust evidence of the inhibition of leaf respiration by light at the ecosystem scale. Because they do not capture this effect, standard approaches overestimate ecosystem photosynthesis and daytime respiration in the first half of the growing season at our site, and inaccurately portray ecosystem photosynthetic light-use efficiency. These findings revise our understanding of forest-atmosphere carbon exchange, and provide a basis for investigating how leaf-level physiological dynamics manifest at the canopy scale in other ecosystems.

Evidence for multiple sex-determining loci in Tasmanian Atlantic salmon (Salmo salar).

Phenotypic sex in salmonids is determined primarily by a genetic male heterogametic system; yet, sex reversal can be accomplished via hormonal treatment. In Tasmanian Atlantic salmon aquaculture, to overcome problems associated with early sexual maturation in males, sex-reversed females are crossed with normal females to produce all female stock. However, phenotypic distinction of sex-reversed females (neo-males) from true males is problematic. We set out to identify genetic markers that could make this distinction. Microsatellite markers from chromosome 2 (Ssa02), to which the sex-determining locus (SEX) has been mapped in two Scottish Atlantic salmon families, did not predict sex in a pilot study of seven families. A TaqMan 64 SNP genome-wide scan suggested SEX was on Ssa06 in these families, and this was confirmed by microsatellite markers. A survey of 58 families in total representing 38 male lineages in the SALTAS breeding program found that 34 of the families had SEX on Ssa02, in 22 of the families SEX was on Ssa06, and two of the families had a third SEX locus, on Ssa03. A PCR test using primers designed from the recently published sdY gene is consistent with Tasmanian Atlantic salmon having a single sex-determining gene that may be located on at least three linkage groups.

Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid.

The initial phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F(ab) fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Glycosaminoglycans of normal and malignant cultured human mammary cells.

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.

Chemical and biological properties of B16 murine melanoma cells grown in defined medium containing bovine serum albumin.

The addition of 1 percent (w/v) bovine serum albumin (BSA) to the F12 medium utilized for the growth of the B16 melanoma cells significantly stimulated the growth of this cell line. The synthesis of mucopolysaccharides and sialoglycopeptides in this medium is identical with that in Eagle's minimal essential medium with Earle's balanced salt solution supplemented with 2 mM L-glutamine, twice the recommended concentration of vitamins, nonessential amino acids, sodium pyruvate, and 10 percent (v/v) fetal calf serum. Cell volume and morphology did not change significantly, under the different growth conditions and tumorigenicity, as assayed by injection of cultured cells into syngeneic animals, was not decreased. Analysis of the BSA used indicated the presence of a sialoglycoprotein contaminant. This sialoglycoprotein contaminant was present in all lots examined and contains N-acetyl-and N-glycolylneuraminic acid, mannose, galactose, and glucosamine. The sialoglycoprotein can be removed by chromatography on acetate form anion-exchange resin at pH 4.3. F12 media containing the purified BSA plus selenite and the sodium salts of palmitic, oleic, and linoleic acids supported growth of the melanoma cells to the same extent as did the media containing unpurified BSA, indicating that the sialoglycoprotein has no role in sustaining the growth of the cells.

Properties of acidic saccharides produced by B16 melanoma cells treated with 1-methyl-3-isobutylxanthine.

A cyclic nucleotide phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, promoted the differentiation and maturation of B16 melanoma cells, phenomena associated with biological alterations in the surface properties of the cells. 1-Methyl-3-isobutylxanthien inhibited cell replication and increased the intracellular content of melanin and cyclic adenosine 3':5'-monophosphate. Significantly greater amounts of sialoglycoproteins were associated with 1-methyl-3-isobutylxanthine-treated cells. However, the total amount of [3H] glucosamine incorporated into anionic polysaccharide (both sialoglycopeptide and mucopolysaccharides) was not significantly changed.

Anionic polysaccharide production and tyrosinase activation in cultured human melanoma cells.

A human melanoma cell line established in our laboratory was characterized in terms of tyrosinase activity and anionic polysaccharide production. Tyrosinase levels were diluted during the growth phase and increased after the cell culture became confluent. The anionic polysaccharides produced included hyaluronic acid, heparitin sulfate, and a high-molecular-weight condroitin 4-sulfate. In contrast, a primary culture of human melanocytes derived from embryonic iris produced much greater amounts of hyaluronic acid, about 30-fold less heparitin sulfate, and a mixture of chondroitin 4-sulfate and dermatan sulfate. Saccharides secreted into the culture medium were generally identical to those remaining cell associated except for the melanoma heparitin sulfate, wherein the latter fraction appeared to be of lower molecular weight.

The production of acidic polysaccharides by 5-bromodeoxyuridine-treated B16 mouse melanoma cells.

B16 melanoma cells were treated in culture with 5-bromo-2-deoxyuridine. The cell-associated and released proteoglycans and sialoglycopeptides were compared to those of control cultures treated with thymidine. The 5-bromo-2-deoxyuridine-treated cultures showed a marked reduction in the proportion of cell-associated proteoglycans and sialoglycopeptides, an increase in the synthesis of hyaluronic acid, the absence of high-molecular-weight chondroitin sulfate, and the presence of increased amounts of heparan sulfate in the media. In addition, the 5-bromo-2-deoxyuridine-treated cells had a higher DNA content and were larger than controls.

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content.

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100 degrees C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing 35S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution is necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.

Isolation and partial characterization of a mucin-type glycoprotein from plasma membranes of human melanoma cells.

Plasma membranes were isolated from HM7 melanoma cells grown in the presence of [3H]glucosamine and Na2(35)SO4 or [3H]mannose and [14C]glucosamine. The labeled glycoconjugates were solubilized with 0.6 M lithium diiodosalicylate/0.5% Triton X-100. Fractionation of glycoconjugates by repeated chromatography on columns of Sepharose CL-6B and DEAE-Sepharose and by affinity chromatography on WGA-Sepharose yielded three radiochemically homogeneous glycoproteins. One of these having an apparent molecular weight of 100,000 was found to contain clusters of (AcNeu)1 or 2 leads to [Gal leads to GalNAc] linked O-glycosidically to the protein. One other glycoprotein contained both O-glycosidically and N-glycosidically-linked oligosaccharides, and the third contained only N-glycosidically-linked carbohydrates. Preliminary results indicate that the 100,000 molecular weight mucin-type glycoprotein is present in significantly reduced quantitites in cultured human fetal uveal melanocytes. Further, the bulk of the glycoproteins from the melanocytes were of lower molecular size compared to those from the melanoma cells.

Monoclonal antibodies to cyanogen bromide fragments of glycophorin A.

Eleven mouse monoclonal antibodies directed against epitopes on CNBr peptides of the major sialoglycoconjugate of the human red blood cell, glycophorin A, have been produced by hybridomas derived from P3-X63-Ag8.653 myeloma cells and spleen cells from BALB/c mice immunized with purified glycophorin. The monoclonal antibodies could be divided into four groups according to their reactivities with CNBr peptides in a direct ELISA assay: one antibody (6B5) that binds solely to the aminoterminal octapeptide (CNBr3); two antibodies (8F10 and 9C3) that bind to CNBrl (residues 9-81); two antibodies (3D2 and 4C6) that are reactive with CNBr2, The C-terminal portion of the molecule (residues 82-131); six antibodies (1B4, 4C3, 4E7, 7B10, 7C11 and 9D6) which are cross-reactive with an epitope on both CNBr1 and CNBR3 glycopeptides. This cross-reactive epitope(s) appears to involve both carbohydrate and protein residues.

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes.

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.

Transcriptional regulation of tracheo-bronchial mucin (TBM) gene by ethanol.

The epithelia of the respiratory tract are protected by a mucin glycoprotein. The expression of mucin changes when epithelia come in contact with toxic agents such as ethanol. Previously, we have identified and characterized the expression of a tracheo-bronchial mucin (TBM) gene. In the present study, we observed that ethanol regulates TBM expression at the transcription level. Ethanol enhanced the expression of TBM mRNA in a dose- and time-dependent manner in HBE1 cells. At 100 mM concentration (a concentration reported to be present in alcoholics), ethanol induced an eight-fold increase in TBM transcription as determined by reporter gene expression analysis.

Primary structure of the canine U1 snRNA.

The nucleotide (nt) sequence of the canine U1 snRNA was determined. It exhibited significant homology (90-98%) with known U1 sequences. The RNA can be folded according to the secondary structure previously proposed for the U1 snRNA. It contained the conserved sequence UUACCUG in loop A (nt 6-12), required for the recognition of the 5' splice site, and the sequence UGCACU in loop B (nt 68-73), required for recognition of the U1-70K protein. The U1 snRNA was localized in the nucleus and its transcription was sensitive to alpha-amanitin, suggesting that it is transcribed by RNA polymerase II. Southern analysis revealed that the canine genome possesses 5-10 copies of U1 snRNA-encoding genes.

Glycobiology of Plasmodium falciparum.

The human malaria parasite, Plasmodium falciparum, has as its only glycoconjugate GPI anchors. These structures, present in essentially all parasite surface proteins, are associated with disease pathology. In contrast, the parasite depends for essential recognition events on saccharides associated with host cell glycoproteins and proteoglycans.

Non-sequence-specific antimalarial activity of oligodeoxynucleotides.

The effects of exogenously applied oligodeoxynucleotides on Plasmodium falciparum proliferation was investigated. A fluorescence-activated cell sorter assay was employed to measure parasitemia after administration of either phosphodiester or phosphorothioate oligodeoxynucleotides. We report sequence-independent antimalarial activity preferentially with phosphorothioate congeners with IC50 values in the 1-2 microM range. Phosphorothioate oligodeoxynucleotides which were antisense, sense or nonsense to Plasmodium mRNA, as well as homopolymers (30-mers containing all A or T bases) were equally effective inhibitors of parasitemia. The antimalarial activity was dependent upon oligomer length, concentration, and time of addition to the cultures but was independent of the parasite strain tested. Four P. falciparum strains, including a multi-drug-resistant strain (MDR-K), a drug-sensitive strain (FCR-3), a erythrocyte membrane sialic acid-independent strain (7G8) and a strain isolated from a cerebral malaria patient (CM-87) were equally susceptible to treatment with a phosphorothioate oligomer. Inhibition of red cell invasion is primarily responsible for the observed decrease in proliferation as determined by a study of parasite maturation in the presence of a 30-mer nonsense phosphorothioate oligodeoxynucleotide.

Molecular cloning and antigenic mapping of heat-shock protein 70 from the malaria species Plasmodium bergheI.

We have isolated a 70-kD heat-shock protein (hsp-70) cDNA from Plasmodium berghei. A cDNA clone encoding the P. berghei hsp-70 was isolated and sequenced, demonstrating that it is highly homologous with other Plasmodium hsp-70s. One of the common features is a series of GGMP amino acid repeats at the carboxy terminus; there is also a long, AT-rich 5' untranslated region, a hallmark of other malarial RNAs. Hydropathy and antigenicity analyses suggest the presence of two hydrophilic domains. Recombinant peptides comprising different fragments of hsp-70 were expressed in Escherichia coli and assessed for antigenicity with antiserum from mice immunized with sonicated extracts of P. berghei. Antigenic sites map to regions that include the two hydrophilic domains.

Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei.

Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.