Improving soil carbon estimates of Philippine mangroves using localized organic matter to organic carbon equations.

BACKGROUND: Southeast Asian (SEA) mangroves are globally recognized as blue carbon hotspots. Methodologies that measure mangrove soil carbon stock (SCS) are either accurate but costly (i.e., elemental analyzers), or economical but less accurate (i.e., loss-on-ignition [LOI]). Most SEA countries estimate SCS by measuring soil organic matter (OM) through the LOI method then converting it into organic carbon (OC) using a conventional conversion equation (%Corg = 0.415 * % LOI + 2.89, R2 = 0.59, n = 78) developed from Palau mangroves. The local site conditions in Palau does not reflect the wide range of environmental settings and disturbances in the Philippines. Consequently, the conventional conversion equation possibly compounds the inaccuracies of converting OM to OC causing over- or under-estimated SCS. Here, we generated a localized OM-OC conversion equation and tested its accuracy in computing SCS against the conventional equation. The localized equation was generated by plotting % OC (from elemental analyzer) against the % OM (from LOI). The study was conducted in different mangrove stands (natural, restored, and mangrove-recolonized fishponds) in Oriental Mindoro and Sorsogon, Philippines from the West and North Philippine Sea biogeographic regions, respectively. The OM:OC ratios were also statistically tested based on (a) stand types, (b) among natural stands, and (c) across different ages of the restored and recolonized stands. Increasing the accuracy of OM-OC conversion equations will improve SCS estimates that will yield reasonable C emission reduction targets for the country's commitments on Nationally Determined Contributions (NDC) under the Paris Agreement. RESULTS: The localized conversion equation is %OC = 0.36 * % LOI + 2.40 (R2 = 0.67; n = 458). The SOM:OC ratios showed significant differences based on stand types (x2 = 19.24; P = 6.63 × 10-05), among natural stands (F = 23.22; p = 1.17 × 10-08), and among ages of restored (F = 5.14; P = 0.03) and recolonized stands (F = 3.4; P = 0.02). SCS estimates using the localized (5%) and stand-specific equations (7%) were similar with the values derived from an elemental analyzer. In contrast, the conventional equation overestimates SCS by 20%. CONCLUSIONS: The calculated SCS improves as the conversion equation becomes more reflective of localized site conditions. Both localized and stand-specific conversion equations yielded more accurate SCS compared to the conventional equation. While our study explored only two out of the six marine biogeographic regions in the Philippines, we proved that having a localized conversion equation leads to improved SCS measurements. Using our proposed equations will make more realistic SCS targets (and therefore GHG reductions) in designing mangrove restoration programs to achieve the country's NDC commitments.

Activation of the beta interferon promoter by paramyxoviruses in the absence of virus protein synthesis.

Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-ß promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.

Catalytic turnover of STAT1 allows PIV5 to dismantle the interferon-induced anti-viral state of cells.

A dynamic model of STAT1 degradation by the V protein of parainfluenza virus 5 (PIV5; formerly SV5) has been proposed. In it, the V protein functions as a bipartite adaptor linking DDB1, a component of a cellular SCF-like ubiquitin E3 ligase complex, to STAT2, which in turn binds STAT1 and presents STAT1 to the E3 ligase complex for ubiquitination and subsequent degradation. Furthermore, it appears that loss of STAT1 from the complex results in decreased affinity of V for STAT2 such that STAT2 either dissociates from V or is displaced by STAT1/STAT2 complexes, facilitating the cycling of the DDB1/PIV5 V containing E3 complex for further rounds of STAT1 ubiquitination and degradation. By determining the approximate number of molecules of V, DDB1, STAT1 and STAT2 present in IFN-treated 2fTGH cells, we provide additional evidence for this dynamic model of STAT1 degradation. These results show that (i) in IFN-treated cells there is approximately 4-fold less STAT2 and 15-fold less DDB1 than STAT1 per cell and thus DDB1 and STAT2 must repeatedly acquire more STAT1 for degradation to go to completion, and (ii) approximately 600 molecules of V protein per cell can target as many as 120,000 molecules of STAT1 for degradation in the absence of either viral or cellular protein synthesis. The importance of this mechanism in terms of the ability of the virus to dismantle the IFN-induced anti-viral state of cells is discussed.

Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs.

Mapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.

Simian virus 5 V protein acts as an adaptor, linking DDB1 to STAT2, to facilitate the ubiquitination of STAT1.

The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.

In vitro and in vivo specificity of ubiquitination and degradation of STAT1 and STAT2 by the V proteins of the paramyxoviruses simian virus 5 and human parainfluenza virus type 2.

Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous.

Novel modifications to the C-terminus of LTB that facilitate site-directed chemical coupling of antigens and the development of LTB as a carrier for mucosal vaccines.

To facilitate site-directed chemical coupling of antigens to the heat labile enterotoxin B subunit of Escherichia coli (LTB), a series of genetically modified fusion proteins of LTB were constructed. The LTB fusion proteins had modified versions of a short (14 amino acid) spacer epitope called the Pk tag attached at their C termini. The LTB-Pk.cys mutants differed from each other in the position of a single cysteine residue within the Pk tag. The presence of a cysteine residue at any position within the Pk spacer tag did not prevent the LTB-Pk.cys proteins from forming pentamers or binding to GM1 gangliosides, but the position of the cysteine had variable impact on the yield of the fusion proteins. Following site-directed chemical coupling of antigens to the cysteine residue within the Pk tag, the LTB antigen conjugates retained their ability to bind GM1 on the surface of eukaryotic cells. Intranasal immunisation of mice with an experimental antigen (HRP) chemically linked to LTB-Pk.cys induced high levels of anti-HRP antibodies that could be detected in the serum, saliva and nasal and lung washes. No antibody responses to HRP were detected when HRP was co-administered with, but not linked to, LTB-Pk.cys.

Inducible expression of the P, V, and NP genes of the paramyxovirus simian virus 5 in cell lines and an examination of NP-P and NP-V interactions.

The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.

Two nontemplated nucleotide additions are required to generate the P mRNA of parainfluenza virus type 2 since the RNA genome encodes protein V.

The nucleotide sequence of the "P/V gene" of parainfluenza virus type 2 is presented. To determine the nature of any nontemplated additions of nucleotides that may arise during the synthesis of mRNA from this gene the polymerase chain reaction was used to amplify specific sequences of both genomic RNA and mRNA. These results demonstrated that the V protein is encoded by the genome while insertion of two nontemplated G residues are required to synthesize P mRNA. The predicted P protein has 44% identical amino acid homology with that of simian virus 5 and 37% with mumps virus; 25% of the amino acids is conserved between all three viruses.

Sequence analysis of the HN gene of parainfluenza virus type 2.

A cDNA library was constructed in lambda gt10 using mRNA purified from cells infected with parainfluenza virus type 2 (PIV2). Virus-specific clones were identified by screening the library with 32P-labelled cDNA probes made from randomly primed vRNA. Clones containing the haemagglutinin-neuraminidase (HN) gene were identified by sequence comparisons with known parainfluenza virus HN gene sequences. The largest HN clone isolated had a nucleic acid sequence of 2065 bp with a single long open reading frame encoding a protein of 571 amino acids. The HN protein has nine predicted glycosylation sites and an amino-terminal membrane-spanning region. The PIV2 HN protein shares 43% amino acid identity with the HN protein of simian virus 5 and 40% with mumps virus, 30% of the amino acids being common to all three viruses.

The urease of Ureaplasma urealyticum.

The urease from Ureaplasma urealyticum (serotype 8) has been purified by immuno-affinity column chromatography. Two active nickel-containing forms of the enzyme were demonstrated by non-denaturing electrophoretic analysis and a single active peak of apparent molecular mass 190 kDa was shown by FPLC. Total inactivation and denaturation of the enzyme to give three subunit polypeptides (one of 72 kDa containing nickel, one of 14 kDa and one of 11 kDa) was achieved by treatment with SDS and boiling. Densitometry suggested that the active enzyme contains equimolar ratios of the three subunits and hence is a hexamer. The enzyme displayed a pH optimum of 6.9 and pI values were determined. Storage of the purified enzyme at -70 degrees C followed by thawing to 20 degrees C caused a partial breakdown to inactive subunits. Anti-urease monoclonal antibodies bound both to the active enzyme and to the inactive 72 kDa subunit, and the antibodies cross-reacted with ureases from all of the other human serotypes. Competition assays with the antibodies revealed four distinct epitopes of the enzyme, all distinct from its active site.

Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum.

Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.

Differential promotion and suppression of Z leads to B transitions in poly[d(G-C)] by histone subclasses, polyamino acids and polyamines.

The right-handed (B) conformation of poly[d(G-C)] in 7.5 mM sodium cacodylate and 25% ethylene glycol can be readily converted to the left-handed (Z) conformation by the addition of 250 microM MnCl2 and this transition can be reversed by chelation of the Mn ions with EDTA or by addition of NaCl. This ability to obtain such reversible transitions in solvent and solute conditions which allow DNA-protein interactions and their assessment by c.d. permitted an analysis of the effect of purified histones, polyamino acids, protamine and polyamines on these transitions. Individual core histones H3, H4, H2a and H2b or protamine stabilised the Mn-induced Z form and prevented the transition to B DNA normally observed after chelation with EDTA or on dialysis to physiological salt concentrations. A similar suppression of Z leads to B transition was also achieved with poly-L-arginine (but not with poly-L-lysine). In contrast, histones H1 and H5 promoted the Z leads to B transition. Polyamines (spermine and spermidine) converted the B form to another right-handed (A) form which transformed to the Z form after the addition of EDTA and this Z form was restored to the B conformation on the addition of NaCl. These results suggest that sequence-dependent variations in the conformation of natural DNA may be modulated by interaction with histones and other basic cellular components and may provide a conformational basis for nucleosome formation and possibly for the control of gene expression.

Nucleic acid-binding properties of adenovirus structural polypeptides.

The ability of adenovirus structural polypeptides to bind nucleic acids was assessed by separating the polypeptides on SDS-polyacrylamide gels, transferring them electrophoretically to nitrocellulose and probing with 32P-labelled nucleic acids. Polypeptides IVa2, V, VI and VII, as well as trace amounts of pVII and a polypeptide of apparent mol. wt. 40 X 10(3) were able to bind label under these conditions. Labelling was also detected with a smaller polypeptide, possibly related to the cleavage products of pVII and/or pVI. The binding of DNA to polypeptide VI appeared to be more sensitive to detergents than the others. No sequence specificity could be detected in the DNA binding.

Monoclonal antibodies against adenovirus type 5: preparation and preliminary characterization.

Eight different hybridoma cells lines producing monoclonal antibodies against the major antigens of human adenovirus type 5 have been obtained. They were selected by screening initial hybridomas by the fluorescent antibody technique followed by radioimmune precipitation and they reacted with hexon, penton, fibre and 100K polypeptides. Five apparently different epitopes against the hexon antigen were detected showing a spectrum of activity against the hexons of other serotypes, suggesting that the hexon contained a variety of subgroup specificities as well as the previously described group and type specificities.