Biomass and litter dynamics in a Melaleuca forest on a seasonally inundated floodplain in tropical,northern Australia

Litterfall from a Melaleuca forest was investigated as part of chemical cycling studies on the Magela Creek floodplain in tropical, northern Australia. The forest contained two species of tree, Melaleuca cajaputi and Melaleuca viridiflora, with a combined average density of 294 trees ha^–1. The M. viridiflora trees had diameter breast height measurements ranging from 11.8 to 62.0 cm, median class 25.1–30.0cm and a mean value of 29.2±1.0 cm, compared to 13.0 to 66.3 cm, 30.1–35.0cm and 33.5±1.0cm for M. cajaputi trees. A regression model between tree height, diameter breast height and fresh weight was determined and used to calculate average tree weights of 775±1.6kg for M. viridiflora and 1009±1.6kg for M. cajaputi, and a total above-ground fresh weight of 263±0.3t ha^–1. The weight of litter recorded each month on the ground beneath the tree canopy ranged from 582±103 to 2176±376 g m^–2 with a monthly mean value of 1105±51 g m^–2. The coefficient of variation of 52% on this mean indicates the large spatial and temporal variability in litter distribution over the study site. This variability was greatly affected by the pattern of water flow and litter transport during the Wet season. Litterfall from the trees was evaluated using two techniques - nets and trays. The results from these techniques were not significantly different with annual litterfall collected in the nets being 705 ± 25 g m^–2 and in the trays 716±49 g m^–2. The maximum monthly amount of litterfall, 108 ±55g m^–2, occurred during the Dry season months of June–July. Leaf material comprised 70% of the total annual weight of litter, 480±29 g m^–2 in the nets and 495 ± 21 g m^–2 in the trays. The tree density and weight of litter suggest that the Melaleuca forests are highly productive and contribute a large amount of material to the detrital/debris turnover cycle on the floodplain.     

Coincidence painting: a rapid method for cloning region specific DNA sequences.

We have developed a novel coincidence cloning strategy, termed Coincidence Painting, which enables the rapid generation of large numbers of region specific sequences. Coincidence Painting utilises Degenerate Oligonucleotide Primed PCR (DOP-PCR) amplification of flow sorted derivative translocation chromosomes. The PCR products are hybridised in situ onto specific flow sorted chromosomes for coincident sequence selection. Eluted and reamplified material is then cloned using a novel insert end revelation and ligation technique. Cloned inserts range in size from 150-1300 bps of which approximately 54% appear to be single copy sequences. The cloning method permits the excision of vector free probe for library hybridisation screening and the small insert size facilitates analysis for the generation of sequence tagged sites (STSs). We have used such clones successfully for YAC screening by PCR and for cosmid screening by filter hybridisation. This new methodology should allow the rapid saturation with probes of regions defined by specific translocation breakpoints.     

Homogeneous amperometric immunoassay for theophylline in whole blood

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 μl whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of + 150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: Y = 0·90x − 1·01, (r = 0·98, N = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.     

Studies in C-terminal sequencing: New reagents for the synthesis of peptidylthiohydantoins

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Baileyet al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50°C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.     

A simple most probable number technique for the sensitive recovery of a genetically-modified Pseudomonas aureofaciens from soil

A strain of Pseudomonas aureofaciens (SBW25EeZY-6KX) that was chromosomally marked with a lacZY and a km^r-xylE cassette could be recovered from non-sterile soil by a selective Pseudomonas enrichment broth amended with 100 ppm kanamycin and 50 ppm X-gal in an MPN (most probable number) assay. The assay was sensitive and reliable, allowing detection of as few as one recombinant cell in a 1% (w v) soil suspension. The soil used contained a large ( ca 5% of total culturable bacteria) background of indigenous bacteria that were either able to utilize lactose, were resistant to kanamycin, or both.     

Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugar beet; gene transfer to a recombinant pseudomonad

Abstract The presence of transfer proficient plasmids in bacteria isolated from the leaves of sugar beet ( Beta vulgaris L.) was studied. Of 435 bacteria sampled 79 (18%) contained plasmids. Pseudomonads (30%), Erwinia (12%) and Klebsiella (9%) were the largest populations sampled of which 22%, 33% and 29%, respectively, contained plasmids. The ability of these plasmids to self-transfer or mediate the mobilization of the tra⁻ mob⁺ broad host range IncQ plasmid R300B was determined. R300B was maintained in 61/79 natural plasmid containing isolates, the Gram positive isolates could not support R300B. Pseudomonas aureofaciens SBW25, isolated from sugar beet leaves, was chromosomally marked with a tetracycline resistance gene and used as a recipient (SBW25ETc). Five isolates of Erwinia herbicola and one of Erwinia salicis containing natural plasmids were able to mobilize R300B into the recombinant, SBW25ETc. These mobolizing ( tra⁺ ) plasmids were not maintained in transconjugant SBW25 cells. Analysis of the fragment patterns of Pst I digested plasmid DNA demonstrated that four (pSB139, pSB140, pSB142, pSB146; 110 kb) were identical, one (pSB153; 65 kb) was common to a subset of fragments in these four and another (pSB169; 100 kb) was unique. Other natural isolates were able to transfer copper resistance ( Erwinia rhapontici , 2 strains) or mercury resistance ( Pseudomonas fluorescens SBW340) to a rifampicin resistant recipient Pseudomonas putida UWC1 but not to SBW25ETc. These self-transferable plasmids were not able to mobilize R300B. These data demonstrate that the phyllosphere supports indigenous microbial populations which have the capacity to transfer genetic material between bacteria of different genera.     

Chemical composition and oxygen consumption rates of the ctenophore Bolinopsis infundibulum from the Gulf of Maine

Quantitative determinations of chemical composition and oxygenconsumption rates were made for a deep-living population ofthe lobate ctenophore Bolinopsis infundibulum. Animals werecollected in the Gulf of Maine with the submersible ‘Johnson-Sea-Link’during September 1989 at depths ranging from 120 to 240 m. Carbonand nitrogen contents were similar to values reported for epipelagicctenophores. Lipid and protein levels were lower than valuestypical of epipelagic ctenophores, but higher than those ofmesopelagic species. Carbohydrate was nearly an order of magnitudehigher than previously recorded for B.infundibulum. Oxygen consumptionrates ranged from 0.004 to 0.235 µl O₂ mg^–1 dryweight h^– at temperatures ranging from 5 to 7°C. Carbon-specificmetabolic rates ranged from 0.21 to 12.73 µl O₂ mg^–1C h^–1. Energy expenditures estimated from respirationdata (     

Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).     

A structural comparison of 21 inhibitor complexes of the aspartic proteinase from Endothia parasitica.

The aspartic proteinases are an important family of enzymes associated with several pathological conditions such as hypertension (renin), gastric ulcers (pepsin), neoplastic disease (cathepsins D and E), and AIDS (HIV proteinase). Studies of inhibitor binding are therefore of great importance for design of novel inhibitors for potential therapeutic applications. Numerous X-ray analyses have shown that transition-state isostere inhibitors of aspartic proteinases bind in similar extended conformations in the active-site cleft of the target enzyme. Upon comparison of 21 endothiapepsin inhibitor complexes, the hydrogen bond lengths were found to be shortest where the isostere (P1-P'1) interacts with the enzyme's catalytic aspartate pair. Hydrogen bonds with good geometry also occur at P'2, and more so at P3, where a conserved water molecule is involved in the interactions. Weaker interactions also occur at P2, where the side-chain conformations of the inhibitors appear to be more variable than at the more tightly held positions. At P2 and, to a lesser extent, P3, the side-chain conformations depend intriguingly on interactions with spatially adjacent side chains, namely P'1 and P1, respectively. The tight binding at P1-P'1, P3, and P'2 is also reflected in the larger number of van der Waals contacts and the large decreases in solvent-accessible area at these positions, as well as their low temperature factors. Our analysis substantiates earlier proposals for the locations of protons in the transition-state complex. Aspartate 32 is probably ionized in the complexes, its charge being stabilized by 1, or sometimes 2, hydrogen bonds from the transition-state analogues at P1. The detailed comparison also indicates that the P1 and P2 residues of substrate in the ES complex may be strained by the extensive binding interactions at P3, P'1, and P'2 in a manner that would facilitate hydrolysis of the scissile peptide bond.