Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans

The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.     

Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins

Membrane proteins destined for the vacuolar or lysosomal lumen are typically ubiquitinated, the ubiquitin serving as a targeting signal for the multivesicular body pathway. The RING-domain ubiquitin ligase Tul1 is an integral membrane protein that modifies the yeast vacuolar enzyme carboxypeptidase S (Cps1), the polyphosphatase Ppn1/Phm5 and other proteins containing exposed hydrophilic residues within their transmembrane domains (TMDs). Here we show that Bsd2 provides an alternative ubiquitination mechanism for Cps1, Phm5 and other proteins. Bsd2 is a three-TMD protein with a PPXY motif that binds the HECT domain ubiquitin ligase Rsp5. It can thus act as a specific adaptor linking Rsp5 to its substrates. Like Tul1, the Bsd2 system recognises polar TMDs. Bsd2 also controls the vacuolar targeting of a manganese transporter and a mutant plasma membrane ATPase, and together with the ER retrieval receptor Rer1, it protects cells from stress. We suggest that Bsd2 has a wide role in the quality control of membrane proteins. Bsd2 is the yeast homologue of human NEDD4 binding protein N4WBP5, which may therefore have similar functions.     

Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Oral immunogenicity of human papillomavirus-like particles expressed in potato

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.     

Genetic population structure of the Dakota skipper (Lepidoptera: Hesperia dacotae): A North American native prairie obligate

A range-wide survey of Dakotaskipper (Hesperia dacotae) populationsassessed levels of genetic variability andgeographic scale of population structure inthis species of conservation concern. Thisspecies exists on isolated patches of nativetall- and mixed-grass prairie within a highlymodified landscape dominated by agriculture. It has been extirpated from the southernportion of its range and has sufferedrange-wide declines. Nine populations weresampled from western Minnesota, eastern SouthDakota, and southern Manitoba. Starch gelelectrophoresis was used to resolve 21 isozymeloci in 278 Dakota skippers. Dakota skipperpopulations were approximately as variable asother lepidopterans found in isolated habitats. Genetic distances indicated that Manitobapopulations were somewhat distinct from ones inMinnesota and South Dakota. Isolation-by-distance was detected range-wideand among the seven southern-most populations. Genetically effective immigration rates weresmall at both range-wide and regional scalesand effective populations sizes were lowsuggesting that Dakota skipper populations aregenetically isolated from one another, althoughthey were likely more connected in the recentpast. Genotype assignment tests revealed twoclusters of populations in Minnesota and SouthDakota that were not apparent from theisolation-by-distance results. Significantheterozygote deficiencies relative toHardy-Weinberg expectations and high inbreedingcoefficients suggest structure within samplelocations. Management recommendations includethe maximization of effective population sizein each Dakota skipper population to offset theeffects of drift and habitat corridors in somecases. Habitat management should consider thewithin-site population structure and possibletemporal population structure detected in thisstudy.     

Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 micro M) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation. We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.     

Pilot trophic model for subantarctic water over the Southern Plateau, New Zealand: a low biomass, high transfer efficiency system

The Southern Plateau subantarctic region, southeast of New Zealand, is an important feeding area for birds, seals and fish, and a fishing ground for commercially significant species. The Southern Plateau is a major morphometric feature, covering approximately 433,620 km² with average depth of 615 m. The region is noted for its relatively low levels of phytoplankton biomass and primary production that is iron-limited. In order to evaluate the implications of these attributes for the functioning of this ecosystem a steady-state, 19-compartment model was constructed using Ecopath with Ecosim software of Christensen et al. [www.ecopath.org]. The system is driven by primary production that is primarily governed by the supply of iron and light. The total system biomass of 6.28 g C m⁻² is very low compared with systems so far modelled with a total system throughput of 1136 g C m⁻² year⁻¹. In the model, the Southern Plateau retains 69% of the biomass in the pelagic system and 99% of total production. Although fish are caught demersally, most of their food is part of production in the pelagic system. Top predators represent about 0.3% of total biomass and account for about 0.24 g C m⁻² year⁻¹ of food consumed made up of birds 0.058 g C m⁻² year⁻¹, seals 0.041 g C m⁻² year⁻¹, and toothed 0.094 g C m⁻² year⁻¹ and baleen whales 0.051 g C m⁻² year⁻¹. This amounts to 105,803 tonnes carbon over the whole of the Southern Plateau and is about 17% of the total amount of food eaten by non-mesopelagic fish. Mean transfer efficiencies between trophic levels II and IV of 23% are at the high end of the range reported in the literature. In the model, adult fish production is almost completely accounted for by the fisheries take (32%), consumption by seals (7%), toothed whales (21%), other adult fish (13%), and squid (20%). Fish and squid catches are at the trophic levels of 4.8 and 5.0, respectively. The gross efficiency of the fishery is 0.018% (catch/primary production). Although not all data come from direct knowledge of this system, the model reflects its general characteristics, namely a low primary production system dominated by the microbial loop, low sedimentation to the seafloor, high transfer efficiencies, a long food web and supporting high-level predators.     

Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase

The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.     

The inhibitory effect of ethanol on ethanol production by Zymomonas mobilis

Summary In an effort to establish the reasons for the limitations in the final ethanol concentration of Zymomonas mobilis fermentation, the effects of CO₂ and ethanol on the fermentation were investigated using continuous and fed-batch cultivation systems. The nucleation and stripping out of CO₂ from the fermenter using diatomaceous earth or nitrogen gas or both exhibited a profound effect on the glucose uptake rate during the early stages of fed-batch fermentation, but did not improve final ethanol yields. The addition of ethanol together with above mentioned experiments confirmed conclusively that ethanol inhibition is responsible for the final ethanol concentration obtainable during Zymomonas mobilis fermentation. The final concentration lies between 90 and 110 gl⁻¹ or approximately 12–15% (v/v) ethanol.