Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R₁ kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R₁ kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R₃ seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R₃ seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.     

Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1⁺ or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.     

Liquid serial dilution is inferior to solid media for isolation of cultures representative of the phylum-level diversity of soil bacteria

Representatives of only four well-characterized bacterial phyla were isolated from a pasture soil by using liquid serial dilution culture. In contrast, members of Acidobacteria, Verrucomicrobia, and Gemmatimonadetes and of other poorly represented bacterial lineages were isolated in earlier experiments with solidified versions of the same media. We conclude that, contrary to expectation, liquid serial dilution culture is inferior to culturing on solid media for isolating representatives of many bacterial phyla from soil.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

Sporophytic self-incompatibility in Senecio squalidus L (Asteraceae)--the search for S

Senecio squalidus (Oxford Ragwort) is being used as a model species to study the genetics and molecular genetics of self-incompatibility (SI) in the Asteraceae. S. squalidus has a strong system of sporophytic SI (SSI) and populations within the UK contain very few S alleles probably due to a population bottleneck experienced on its introduction to the UK. The genetic control of SSI in S. squalidus is complex and may involve a second locus epistatic to S. Progress towards identifying the female determinant of SSI in S. squalidus is reviewed here. Research is focused on plants carrying two defined S alleles, S(1) and S(2). S(2) is dominant to S(1) in pollen and stigma. RT-PCR was used to amplify three SRK-like cDNAs from stigmas of S(1)S(2) heterozygotes, but the expression patterns of these cDNAs suggest that they are unlikely to be directly involved in SI or pollen-stigma interactions in contrast to SSI in the Brassicaceae. Stigma-specific proteins associated with the S(1) allele and the S(2) allele have been identified using isoelectric focusing and these proteins have been designated SSP1 (Stigma S-associated Protein 1) and SSP2. SSP1 and SSP2 cDNAs have been cloned by 3' and 5' RACE and shown to be allelic forms of the same gene, SSP. The expression of SSP and its linkage to the S locus are currently being investigated. Initial results show SSP to be expressed exclusively in stigmas and developmentally regulated, with maximal expression occurring at and just before anthesis when SI is fully functional, SSP expression being undetectable in immature buds. Together these data suggest that SSP is a strong candidate for a Senecio S-gene.     

Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.     

Medfly (Diptera: Tephritidae) genetic sexing: large-scale field comparison of males-only and bisexual sterile fly releases in Guatemala

The effect of releases of bisexual (males and female) and unisexual (male only) sterilized medflies was compared in three large field evaluations over a 3-yr period (1995-1997) in southwestern Guatemala. The two strains tested were a genetic sexing strain, Vienna-4/Tol-94, carrying the temperature sensitive tsl gene to eliminate females in the egg stage, and the standard bisexual Petapa strain. Flies were mass-reared, sterilized by irradiation as pupae, shipped to a field center, and released by air as young adults over 2 km by 2 km core areas in the centers of separate 6 km by 6 km test plots. Strain performance was monitored weekly by trapping sterile and wild male adults in core and buffer areas and by collecting eggs from coffee berries to determine induced sterility. Results indicated a several-fold advantage for the males-only strain as measured by the level of induced sterility, especially at the very high release ratios of 100:1 recorded in 1997. During that final test year, sterile-fly release rates were increased to provide high sterile:wild (S:W) fly ratios in the field, and egg sterility reached levels in excess of 70% in plots were the male-only strain was used. However, in the plots where the bisexual strain was released, induced sterility only reached 12% despite S:W ratios above 1,000:1.