Effects of terrestrial dissolved organic matter on a bloom of the toxic cyanobacteria, Raphidiopsis raciborskii.

The concentration of coloured terrestrial dissolved organic matter (tDOM) from vegetation appears to be increasing in lakes in some regions of the world, leading to the term brownification. The light attenuating effect of coloured tDOM on phytoplankton growth has been a major focus of attention, but the phytotoxic effects of tDOM, particularly on cyanobacterial blooms, are less well understood. This mesocosm study tested whether coloured tDOM, leached from the leaves of a Eucalyptus tree species, inhibited a naturally occurring bloom of the toxic cyanobacterium, Raphidiopsis raciborskii, in a reservoir over a 10 day period. The study found that tDOM leachate, measured as dissolved organic carbon (DOC), inhibited photosynthesis and growth of both R. raciborskii, as well as species present at lower densities, i.e. other cyanobacteria and diatoms. However, the effect was greater at higher tDOM input loads. The photosynthetic yield (Fv/Fm) of cyanobacteria decreased rapidly in treatments with 5.9 and 25 mg L-1 DOC addition, compared to the control (reservoir water with background DOC concentration of 6.85 ± 1.09 mg L-1). tDOM had no measurable effect in the 2 and 3.3 mg L-1 DOC addition treatments. By day 5, cell densities of cyanobacteria, including R. raciborskii, and diatoms, in treatments with 5.9 and 25 mg L-1 DOC addition were significantly lower than the control with no tDOM addition, and this effect continued throughout the experiment. This is despite the leachate addition increasing phosphate concentrations which counteracted the low background concentrations of phosphate. Light attenuation and dissolved oxygen (DO) levels were also affected by the tDOM addition, but were only significantly lower in the 25 mg L-1 DOC treatment compared with the control. DOC, dissolved organic nitrogen (DON) and dissolved organic phosphorus (DOP) concentrations all decreased in the tDOM addition treatments over the first 3 days, as the microbial cell densities increased. The components of the tDOM that decreased over time were determined by 1H NMR spectroscopy in the 25 mg L-1 DOC treatment. After 5 d, the relative concentrations of fatty acids, sugars and gallic acid decreased by around 60%, while concentrations of flavonoids and myo-inositol decreased by 45 and 35% respectively. This study suggests that phytotoxic compounds in tDOM can suppress cyanobacterial blooms, despite the increased nutrient inputs. This has implications for predicting the future likelihood of cyanobacterial blooms in lakes and reservoirs with climate-change driven changes in flow events, and other changes in the amount and types of vegetation cover. Revegetation of riparian zones, resulting in increased tDOM into waterways, may also be beneficial in reducing cyanobacterial blooms.

Synthesis and secretion of a functional antibody in a vaccinia virus expression system.

A humanized rat monoclonal antibody (Campath 1H) has been expressed in HeLa cells using recombinant vaccinia viruses. Heavy and light chain recombinant viruses were constructed separately and when grown independently produced proteins of the expected molecular weights. Expressed heavy chain was entirely intracellular but light chain was mainly excreted and processed. When cells were infected at high multiplicity with both heavy and light chain recombinants a proportion of the heavy chain was then found in the extracellular medium. This secreted heavy chain was shown to be associated with light chain as judged by co-electrophoresis in non-reducing SDS polyacrylamide gels and by co-purification on protein-A sepharose. The secreted heavy and light chain complexes were functionally active as an antibody, with activity comparable to authentic Campath 1H antibody as assessed by ELISA, T-cell binding and antigen binding assays. Production of antibody in this system was achieved in the absence of serum, which is an important consideration in the production of monoclonal antibodies (MAbs). The amount of antibody produced was 0.2-0.4 micrograms/10(6) cells without optimization of expression levels. The wide host cell range of vaccinia virus together with the recently developed methods for increasing expression levels make this an attractive candidate as a flexible general vehicle for producing MAbs.

A new and improved method for 3'-end labelling DNA using [alpha-32P]ddATP.

We have synthesised dideoxyadenosine-5'-[alpha-32P]triphosphate [( alpha-32P] ddATP ) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5'-[alpha-32P]triphosphate [( alpha-32P] KTP ) as a means to 3'-end label DNA. The [alpha-32P] ddATP was found to be three to five times more efficient than [alpha-32P] KTP . Blunt and 3'-protruding ends were labelled more efficiently with [alpha-32P] ddATP using terminal transferase than were the 5'-ends with [gamma-32P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3'-end labelling of DNA to become a realistic alternative to 5'-end labelling. We have also compared [alpha-32P] ddATP - and [alpha-32P] KTP -labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.

The sequence of foot-and-mouth disease virus RNA to the 5' side of the poly(C) tract.

The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O1 compared with 362 nt for A10, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure.

Synthetic peptides mimic subtype specificity of foot-and-mouth disease virus.

The major immunogen of foot-and-mouth disease virus (FMDV) is located between amino acids 141-160 of the capsid protein VP1. Synthetic peptides corresponding to the major immunogenic region give good neutralising antibody responses and protection in guinea pigs. To define more precisely the immunogenic site of the virus, we have examined serological differences between subtypes of the A serotype using synthetic peptides covering the 141-160 region. We show that these synthetic peptides carry determinants which mimic the subtype specificity of the virus. The correlation between these results and predictive structural models, based on the amino acid sequence, is discussed.

Nonnative intermediate state of acid-stable beta-sheet protein.

Cell adhesion molecule, CD2, from the immunoglobulin superfamily, is comprised of antibodies and Ig-like domains and plays a fundamental role, not only in the immune system, but also in the interactions between cells, specifically in cell-cell adhesion. This study examines the N-terminal domain 1 of CD2 (CD2-1) at different pHs, and in 2,2,2-trifluoroethanol (TFE), using nears- and far-UV circular dichroism (CD), fluorescence, and 1H nuclear magnetic resonance to elucidate factors contributing to the Ig beta-structure. Contrary to the complete unfolding induced by guanidinehydrochloride, CD2-1 retains its native tertiary structure at pHs from 1.0 to 10.0. Like the effects of high temperatures that have previously been observed, TFE reduces the integrity of the tertiary structure, while reorganizing the secondary structure from a native all-beta-sheet to a significantly alpha-helical conformation. The induced helicity of CD2-1 correlates with the helicity inherent in its primary sequence. Our results suggest that electrostatic interactions are less important for the formation of the native secondary and tertiary structure of CD2-1, although they are crucial for CD2's adhesion function. Interference with the protein's hydrophobic interactions and hydrogen-bonding networks, however, causes significant changes in its conformation. Residues of CD2-1, with high conformational flexibility, may contribute for the formation of a metastable dimer by domain-swapping.

Effect of alpha-amyrin palmitate on adjuvant arthritis.

alpha-amyrin palmitate was synthesized and tested on adult male Wistar rats made arthritic by subplantar injection of complete Freund's adjuvant. When administered orally at 66 mg/kg BW every 48 h for 5 days from days 32 to 40 post-adjuvant and assessed on day 50, alpha-amyrin palmitate returned the increases in serum hyaluronate and blood granulocytes toward non-arthritic levels and corrected the moderate anaemia of adjuvant arthritis. Histological examinations of the second and third proximal foot interphalangeal joints showed reduced synovial proliferation and invasion of joints and reduced leucocyte infiltration of bone marrow in alpha-amyrin palmitate-treated rats. In addition, the drug prevented or reduced bone (subchondral or cortical) and cartilage (articular) destruction in arthritic rats. The results suggest that alpha-amyrin palmitate is antiarthritic in the adjuvant model of arthritis in rats.

Antiinflammatory activity of a Ghanaian antiarthritic herbal preparation: III.

alpha-Amyrin palmitate, present in a Ghanaian antiarthritic herbal preparation of Alstonia boonei, Elaies guineensis and Rauvolfia vomitoria, was synthesised and tested on complete Freund's adjuvant-induced arthritic rats. Administered orally at 56 mg/kg body weight (BW) daily for 8 days from days 11 to 18 post adjuvant (acute) or at 66 mg/kg BW every 48 h for 5 days from days 32 to 40 (chronic), the drug returned the increases in serum hyaluronate and blood granulocytes towards non-arthritic levels and corrected the moderate anaemia of adjuvant arthritis. Histological examinations of the proximal interphalangeal foot joints showed reduced synovial proliferation and invasion of joints and reduced leucocyte infiltration of bone marrow and periarticular tissue in treated rats. The results suggest that alpha-amyrin palmitate contributes to the previously shown antiarthritic effect of the herbal preparation.

Adenosine-5'-O-(3-thiotriphosphate) as an affinity probe for studying leader RNA's transcribed by vesicular stomatitis virus.

The ATP required for in vitro transcription by vesicular stomatitis virus can be replaced by the analogue adenosine-5'-O-(3-thiotriphosphate) without affecting the rate of transcription or the nature of the mRNA products. RNA chains (less than 5 S) initiated with adenosine-5'-O-(3-thiotriphosphate) can be isolated by affinity chromatography on mercury-agarose columns used in these experiments. At least 80% of the leader RNA synthesized in vitro by infectious B virions and 70% of the leader RNA synthesized by defective-interfering particles of vesicular stomatitis virus contained a 5'-terminal triphosphate as judged by binding to mercury-agarose of leader RNA synthesized in the presence of the 5'-thiotriphosphate adenosine analogue.

Role of the membrane (M) protein in endogenous inhibition of in vitro transcription by vesicular stomatitis virus.

An endogenous transcriptase inhibitor active at high concentrations of vesicular stomatitis (VS) virus was present in trypsinized whole virions but was absent from ribonucleoprotein cores containing only the L, N, and NS proteins. Poly(L-glutamic acid) effectively reversed the transcriptase inhibition. Transcription under noninhibited, inhibited, and poly(L-glutamic acid)-reversed conditions did not appear to greatly affect the nature of the RNA transcription product. The VS virion matrix (M) protein was purified to greater than 98% homogeneity and was found to have an isoelectric point of approximately 9.0. Purified M protein inhibited transcription by ribonucleoprotein cores, an effect that was partially reversed by poly(L-glutamic acid). Two group III temperature-sensitive (ts) mutants of VS virus (tsO23 and ts G31) with lesions in the M protein exhibited little or no endogenous inhibitor activity compared with two wild-type strains and a group V mutant (tsO45) with a lesion in the G protein. The data presented strongly suggest that the virion M protein is responsible for the endogenous inhibition of in vitro RNA synthesis seen at high concentrations of VS virus.

Inhibition of transcription by immunoglobulins directed against the ribonucleoprotein of homotypic and heterotypic vesicular stomatitis viruses.

Specific antisera were raised by immunization of rabbits with purified nucleocapsids containing only RNA and N protein (ribonucleoprotein, RNP) obtained from vesicular stomatitis (VS) virions of the Indiana (VSInd) and the New Jersey (VSNJ) serotypes. The specificity of anti-RNPInd serum was demonstrated by selective precipitation of homotypic RNPInd devoid of L and NS proteins; anti-RNPInd serum also selectively precipitated soluble N protein present in cytoplasm of infected cells, but co-precipitated a limited amount of contaminating soluble NS protein. Immunoglobulins prepared from each homotypic antiserum markedly inhibited in vitro transcription of VSInd and VSNJ virions. Anti-RNPInd and anti-RNPNJ immunoglobulins also exhibited cross-reactivity by inhibiting transcription of heterotypic virions, but only to a much lesser degree than in the homotypic reaction. Anti-RNPInd immunoglobulin did not inhibit transcription of the antigenically unrelated Chandipura rhabdovirus, but anti-RNPNJ immunoglobulin did to a very limited extent. The transcription inhibitory activity of anti-RNPInd immunoglobulin was not dependent on RNP immunoprecipitation activity, which could be diluted out well before loss of antitranscriptase activity. Anti-RNPind immunoglobulin appeared to exert its effect on transcription by blocking elongation rather than initiation or reinitiation of RNA transcripts.

Reversal by certain polyanions of an endogenous inhibitor of the vesicular stomatitis virus-associated transcriptase.

The RNA-dependent RNA polymerase associated with vesicular stomatitis virus has been found to be markedly inhibited at high concentrations of virus. This endogenous inhibitor of the virion transcriptase was completely reversed by the action of two negatively charged polyamino acids: poly(L-glutamic acid) and pepsin (EC 3.4.23.1). Two other polyanions, heparin and polyethylene sulfonate, strongly inhibited the activity of the virion transcriptase even at low virus concentrations. Poly (L-glutamic acid) rapidly released the block in transcription of concentrated vesicular stomatitis virus, possibly owing to competition for binding sites of the inhibitor on the virion nucleocapsid transcription complex.

Variability of terpene content in the soft coralSinularia flexibilis (Coelenterata: Octocorallia), and its ecological implications.

Colonies of the soft coralSinularia flexibilis (Quoy & Gaimard) (Coelenterata, Octocorallia) were collected at Lizard Island (14°40'S and 145°28'E) Research Station. Extraction of the corals and quantitative chemical analysis for the three major diterpene components, flexibilide, dihydroflexibilide, and sinulariolide, afforded average ratios of 4∶3∶1 respectively. Colonies, sized on the basis of the sterile stalk circumference, were analyzed for possible correlations between size and chemical composition. The major metabolite, flexibilide, was inversely correlated with colony size, while sinulariolide concentration showed a direct correlation. The concentration of dihydroflexibilide was independent of colony size. Samples were further analyzed with respect to site of collection. Colonies were collected at three distinct reefal sites. One was characterized by large monospecific stands ofParites cylindrica, a second was a sandy bottom site with a mixed community of soft corals and occasional scleractinians, while the third site was a very diverse reef community with many species of scleractinian corals.Sinularia flexibilis was well represented at each site, and the concentration of flexibilide and sinulariolide varied significantly among sites. The concentration of flexibilide was significantly higher at the third, highly competitive site, while the concentration of sinulariolide was highest at thePorites-dominated site. Dihydroflexibilide levels were independent of site. It seems likely that concentrations of flexibilide, a highly cytotoxic molecule involved in interference competition, and sinulariolide, a known algicide probably responsible for colony maintenance, may be influenced by their environments.

The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus.

The complete nucleotide sequence of the coding region of foot and mouth disease virus RNA (strain A1061) is presented. The sequence extends from the primary initiation site, approximately 1200 nucleotide from the 5' end of the genome, in an open translational reading frame of 6,999 nucleotides to a termination codon 93 nucleotides from the 3' terminal poly (A). Available amino acid sequence data correlates with that predicted from the nucleotide sequence. The amino acid sequence around cleavage sites in the polyprotein shows no consistency, although a number of the virus-coded protease cleavage sites are between glutamate and glycine residues.

Eudistomin V, a new beta-carboline from the Australian ascidian Pseudodistoma aureum.

Chemical investigation of the Australian ascidian Pseudodistoma aureum has resulted in the isolation of a new beta-carboline, eudistomin V (1). The known compounds eudistomin H (2) and I (3) were also isolated, and all compounds had their structures determined by spectroscopic means.

Use of UV irradiation to identify the genetic information of vesicular stomatitis virus responsible for shutting off cellular RNA synthesis.

UV irradiation of infectious vesicular stomatitis virus was employed to study the relationship between the expression of certain viral gene functions and viral inhibition of RNA synthesis in mouse myeloma (MPC-11) cells. Viral infectivity, protein synthesis, and viral mRNA synthesis were all highly susceptible to inactivation by UV radiation; however, low levels of viral transcriptase activity were detected in vitro in virus preparations subjected to large doses of UV radiation. In sharp contrast, the capacity of vesicular stomatitis virus to shut off cellular transcription was quite resistant to UV radiation. The data presented here indicate that viral transcription is essential to inhibit host RNA metabolism, even though synthesis of viral polypeptides in the inhibited cells could not be detected. At those levels of UV radiation that inactivated all viral gene functions, except viral inhibition of cellular RNA synthesis, the only viral product detected was non-adenylated, low-molecular-weight RNA species.

An evaluation of the putative human mammary tumour retrovirus associated with peripheral blood monocytes.

The primary aims of this study were purification and molecular cloning of a putative retrovirus designated human mammary tumour virus (HMTV). However, our preliminary unpublished data of negative reverse transcriptase (RT) activity in ostensibly 'infected' cells led us to re-examine the evidence for this virus; namely multinucleate giant cell (MNGC) formation and RT activity in cultured blood monocytes from breast cancer patients versus benign breast tumour and normal control subjects. MNGCs from by fusion of monocytes and we estimated the total number of cell fusions which had occurred after 10 days of culture in vitro by counting cells with two, three, four and five or more nuclei (n) and by measuring the density of adherent mononuclear cells for each subject studied. We found no clear-cut difference in MNGC formation between the three subject groups. Moreover, a substantial number of cultures, encompassing the three groups, showed far more MNGCs per 10(5) monocytes than previously reported. Various parametric and nonparametric statistical analyses were performed on the multinucleate cell data and only one parametric test, which utilised the density of monolayers as a co-variate, showed a statistically significant difference at the 5% level between the breast cancer and the normal subject groups. We observed marked subject-to-subject variation in multinucleate cell formation and we suggest that the evidence for a difference between the breast cancer and the normal groups is marginal. Further, MNGC formation by breast cancer monocytes may not be attributed to the presence of a retrovirus since 5'-Azacytidine (AZA), an agent known to stimulate replication of latent retroviruses showed no effect on the MNGC formation. In addition, culture supernatants from the three groups were assayed for RT activity and no test sample gave a significant signal above background. Preliminary transmission electron microscopy analysis failed to identify viral particles in MNGCs.

Block to influenza virus replication in cells preirradiated with ultraviolet light.

Ultraviolet (uv) irradiation of CEF cells immediately before infection with influenza A (fowl plague) virus inhibited virus growth; no inhibition of the growth of a parainfluenza virus (Newcastle disease virus) could be detected in irradiated cells. The kinetics of inhibition after various doses of uv irradiation were multihit, with an extrapolation number of two. When irradiated cells were allowed to photoreactivate by exposure to visible light for 16 hr their capacity to support influenza virus replication was largely restored; this process was sensitive to caffeine, suggesting that it required DNA repair. In CEF cells exposed to 360 ergs/mm(2) of uv radiation the rate of synthesis of host cellular RNA was reduced by more than 90%, and that of host cellular protein by 40-50%, as judged by incorporation of precursor molecules into an acid-insoluble form. When such irradiated cells were infected with influenza virus all the genome RNA segments were transcribed, but the overall concentration of virus-specific poly (A)-containing cRNA was reduced about 50-fold. Within this population of cRNA molecules, the RNAs coding for late proteins (HA, NA, and M) were reduced in amount relative to the other segments. The rates of synthesis of the M and HA proteins were specifically reduced in uv-irradiated cells, but the rates of synthesis of the P, NP, and NS proteins were only slightly reduced compared to normal cells. Immunofluorescent studies showed that, in uv-irradiated cells, NP migrated into the nucleus early after infection and later migrated out into the cytoplasm, as in normal cells. In contrast to normal cells, no specific immunofluorescence associated with M protein could be observed in uv-irradiated cells. It is concluded that uv-induced damage to host cellular DNA alters the pattern of RNA transcription in CEF cells infected with influenza virus, and that this results in a block to late protein synthesis which stops virus production.

Potential secondary and tertiary structure in the genomic RNA of foot and mouth disease virus.

The nucleotide sequence of the 5' untranslated region of foot and mouth disease virus (FMDV), serotype A10 has been determined. This completes the first total genomic sequence for any one serotype of FMDV. Analysis of the sequence to the 3' side of the poly (C) tract reveals the presence of a 24 nucleotide repeated motif which has homologies with a sequence located upstream of the transcriptional initiation site from several mammalian fibrinogen genes. The function of this element in FMDV is unclear. However, computer analysis of this region predicts the presence of a high degree of secondary and tertiary structure in which these repeats form an important part. The implications of these predictions are discussed.