Five genetic loci involved in the synthesis of acidic exopolysaccharides are closely linked in the genome of Rhizobium sp strain NGR234

Summary R-prime plasmids were constructed from a derivative of Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn5-induced EPS-deficient (Exo^–) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo^–. Exo⁺ revertants occurred at a low rate (1×10^-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo^– mutants of strain ANU280. Complementation of these Exo^– mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the exo genes. The 30 group 2 Exo^– mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was cis-dominant to two other exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.     

The genetic control of alkaline phosphatase activity in the duodenum of the mouse

The inheritance of duodenal alkaline phosphatase activity has been studied in two inbred strains of Swiss mice. These two strains consistently maintained a three- to fourfold difference in duodenal phosphatase activity for six generations before the start of the genetic studies. Males of both the high-activity strain (HAS) and the low-activity strain (LAS) were mated to females of the other strain to produce an F₁ generation, members of which were sib-mated to produce an F₂ generation. The frequency distributions of the parental, F₁, and F₂ generations reveal that the activity of the enzyme is under polygenic control. Distribution of activities in the F₂'s derived from HAS grandmothers differs from that of the F₂'s from LAS grandmothers, indicating that a maternally inherited factor, probably contained in the milk of one strain, influences the activity. Heritability estimates based on half-sib correlation coefficients show that additive genetic variance makes up about 50–70% of the total variance of duodenal phosphatase activity in LAS, and approximately 30–45% in HAS. The latter strain is the more variable, both within generations and within litters; its activity is also more strongly enhanced by injection of substrate into the stomach, and is more severely reduced by starvation. Chromatographic analysis of butanol extracts of phosphatase from 11-day-old mice of the two strains reveals that both contain the same two isozymes. HAS does, however, appear to have 6–8 times as much as LAS of an isozymic form of phosphatase having a relatively high phenylphosphate/-glycerophosphate ratio, and only about twice as much of a low PhP/bGP ratio form.Supported by Research Grant GM 03937 from the National Institutes of Health, U.S. Public Health Service.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Catalytic facilitation by diffusion of adsorbed substrate on membrane surface

Membrane-alkaline phosphatase shows greater velocity of reaction than solubilized enzyme at low substrate concentration, whereas at saturation-concentration the opposite is true. The catalytic rate enhancement with the membrane-enzyme, when substrate availability is limiting, is attributed to non-specific adsorption of substrate to the membrane followed by its surface-diffusion to the active site resulting in an enhanced collision rate for the substrate with the enzyme. Experimental evidence for the adsorption-diffusion is provided by the dynamic quenching of 1-anilino-naphthalene-8-sulphonate, a membrane-bound probe's fluorescence by the substrate, 4-nitrophenyl-phosphate.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.

     

Luteinizing hormone has a stage-limited effect on preantral follicle development in vitro

Although it is known that LH receptors are present from the time of thecal differentiation, the role of LH during early follicle development is not yet clear. The effect of LH on preantral follicle development has therefore been investigated in vitro using a culture system that supports the development of intact follicles. We have previously shown that although preantral follicles 150 micrometer in diameter (2-3 granulosa cell layers) do not require LH to proceed through antral development, smaller follicles (1-2 granulosa cell layers, 85-110 micrometer in diameter) do not develop beyond the large preantral stage in the presence of only FSH and 5% mouse serum. Follicles of this size were therefore used to determine the effects of LH and serum on their development in vitro. The results showed that although FSH must be continuously present, a low concentration of LH together with a slight increase in serum concentration was necessary, specifically during the primary stage of follicle development (from 85 micrometer in diameter until the follicles had reached 150 micrometer in diameter) to induce the capacity for subsequent LH-independent rapid growth and antral development. The in vitro development of maturable oocytes with normal spindle and chromatin morphology was also supported. These results indicate that LH probably induces changes in the early differentiating thecal cells, which are critical for the completion of subsequent follicular and oocyte development.     

Desialylated alkaline phosphatase: activation by 4-nitrophenol

Mouse ileal alkaline phosphatase is a sialyl enzyme (12-14 moles per mole of enzyme). When partially desialylated by treatment with neuraminidase, the enzyme loses most of its activity, associated with reduced apparent Vmax and Km. Part of that loss, however, is recovered as the product 4-nitrophenol's concentration builds up in the cuvette. Experimental results are presented to demonstrate that the activation is due to the binding of 4-nitrophenol as a ligand by the partially desialylated enzyme and that both the loss of activity by sialic acid removal and activation by ligand-binding are correlated with changes in protein conformation.