Ranking genes with respect to differential expression

Background  

In the pharmaceutical industry and in academia substantial efforts are made to make the best use of the promising microarray technology. The data generated by microarrays are more complex than most other biological data attracting much attention at this point. A method for finding an optimal test statistic with which to rank genes with respect to differential expression is outlined and tested. At the heart of the method lies an estimate of the false negative and false positive rates. Both investing in false positives and missing true positives lead to a waste of resources. The procedure sets out to minimise these errors. For calculation of the false positive and negative rates a simulation procedure is invoked.     

A sensitive quantification of HHV-6B by real-time PCR

Human herpesvirus (HHV)-6B is a pathogen causing latent infection in virtually all humans. Nevertheless, the interaction of HHV-6B with its host cells is poorly understood. Although HHV-6B is approximately 90% homologous to HHV-6A, it expresses certain B-specific genes. In order to quantify the amount of expressed viral mRNA we have developed a method using real-time PCR on a LightCycler instrument. Here we describe an assay for the detection of the HHV-6B B6 mRNA, but our approach can easily be extended to involve other mRNAs. This method is useful during the study of HHV-6B biology and offers reliable and reproducible, quantitative detection of viral mRNA below the attomol range. Published: December 9, 2002     

Heterobinary Adhesins Based on the Escherichia coli FimH Fimbrial Protein

The FimH adhesin of Escherichia coli type 1 fimbriae confers the ability to bind to d-mannosides by virtue of a receptor-binding domain located in its N-terminal region. This protein was engineered into a heterobifunctional adhesin by introducing a secondary binding site in the C-terminal region. The insertion of histidine clusters into this site resulted in coordination of various metal ions by recombinant cells expressing chimeric FimH proteins. In addition, libraries consisting of random peptide sequences inserted into the FimH display system and screened by a “panning” technique were used to identify specific sequences conferring the ability to adhere to Ni²⁺ and Cu²⁺. Recombinant cells expressing heterobifunctional FimH adhesins could adhere simultaneously to both metals and saccharides. Finally, combining the metal-binding modifications with alterations in the natural receptor-binding region demonstrated the ability to independently modulate the binding of FimH to two ligands simultaneously.

Expression systems for the display of heterologous protein segments facilitate the presentation of both defined and random peptide sequences at exposed regions of surface proteins of filamentous bacteriophage virions, bacteria, and yeasts (2, 4). We are particularly interested in the display of heterologous peptides in type 1 fimbriae. Such surface organelles are found on the majority of Escherichia coli strains and confer the ability to bind to specific surfaces. A single type 1 fimbria is a heteropolymer that is 7 nm wide and approximately 1 μm long. It consists of approximately 1,000 subunits of the major element, FimA, that are polymerized in a right-handed helical structure that also contains low levels of the minor components FimF, FimG, and FimH (9). The FimH protein has been shown to be the actual receptor-binding molecule which recognizes α-d-mannose-containing structures (10). Because of this, type 1 fimbriated bacteria readily agglutinate yeast cells (a rich source of mannan).The FimH adhesin is located at the tip of each fimbria and also is interspersed along the fimbrial shaft (6, 10). The results of linker insertion mutagenesis (16) and analyses of naturally occurring variants (17–19) and hybrid proteins constructed by fusing FimH to FocH (8) and MalE (21) suggest that the FimH protein consists of two major domains, each constituting roughly one-half of the molecule; the N-terminal domain seems to contain the receptor-binding site, while the C-terminal domain seems to contain the recognition sequences for export and bioassembly.In previous studies we investigated the ability of FimH to display heterologous peptides in connection with the development of vaccine systems. Various heterologous sequences, representing immune-relevant sectors of foreign proteins, were authentically displayed on the bacterial surface in FimH (12). These observations led us to believe that the FimH protein is an ideal candidate for display of random peptide sequences and for construction of designer adhesins (i.e., proteins manipulated to bind to targets of choice). Here we describe simultaneous heterobifunctional binding of recombinant cells expressing chimeric FimH proteins to metal and d-mannose targets.     

Serum Leptin Concentration and Insulin Sensitivity in Men with Abdominal Obesity

JOHANNSSON, GUDMUNDUR, CECILIA KARLSSON, LARS LÖNN, PER MÅRIN, PER BJÖRNTORP, LARS SJÖSTRÖM, BJÖRN CARLSSON, LENA M.S. CARLSSON, BENGT-ÅKE BENGTSSON. Serum leptin concentration and insulin sensitivity in men with abdominal obesity. Obes Res. 1998;6:416–421. Objective : We have examined the association between generalized adiposity, abdominal adiposity, insulin sensitivity, and serum levels of leptin in a cross-sectional study of abdominally obese men. Research Methods and Procedures : Thirty men, 48 to 66 years of age with a body mass index (BMI) of between 25 kg/m² and 35 kg/m² and a waist hip ratio of <0.95, were included in the study. Serum leptin concentration was measured using radioimmunoassay. Total body fat percentage was determined from total body potassium, abdominal adiposity was measured by computed tomography, and the glucose disposal rate (GDR) was measured during an euglycemic, hyperinsulinemic glucose clamp. Results : Significant correlations were found between serum leptin concentration and BMI, percentage body fat, abdominal subcutaneous adipose tissue, serum insulin, GDR, and 24-hour urinary-free Cortisol. In a multiple regression analysis, it was shown that abdominal subcutaneous adipose tissue, GDR, and BMI explained 72% of the variability of serum leptin concentration. GDR demonstrated an independent inverse correlation with serum leptin concentration. Discussion : In abdominally obese men with insulin resistance, it was demonstrated that most of the individual variability in serum leptin concentration was explained by the amount of subcutaneous abdominal adipose tissue, insulin sensitivity, and BMI.     

Consumer–resource matching in a food chain when both predators and prey are free to move

The classical theory of the ideal free distribution (IFD) predicts that the spatial distribution of consumers should follow the distribution of the resources they depend on. Here, we study consumer–resource matching in a community context. Our model for the community is a food chain with three levels. We study whether the primary consumers are able to match resources both under predation risk and in its absence. Both prey and predators have varying degrees of knowledge of the global and local resource distribution. We present two versions of the model. In the "resource maximising" model, the consumers consider the availability of their resource only. In the "balancing" model, individual consumers minimise predation risk per unit of resource that they can gain access to. We show that both models can lead to perfect matching of consumers on resources and predators on consumers, assuming that individuals have full knowledge of the whole environment. However, when the consumers' information and freedom of movement are greater than those of the predators, then the predators generally undermatch the consumers. In the opposite case, we observe overmatching and high consumer movement rates. Furthermore, undermatching of predators on consumers tends to induce overmatching of consumers on resources.     

Ontogeny and Structure of a New, Miniaturised and Spiny Olenid Trilobite from Southern Sweden

Several hundred specimens of a tiny olenid trilobite, Ctenopyge ceciliae sp. nov., have been found in stinkstone nodules in the upper Cambrian Peltura scarabaeoides Zone in southern Sweden. This exceptionally spinose form is known only from disarticulated specimens, but is quite well preserved, and all growth stages are represented. The early ontogenetic stages are exceptionally small, the protaspis being only half the size of that of the associated Peltura species. There may have been no more than three thoracic segments. Thus the whole ontogeny was compressed, and this together with the very small size of the adult indicates a true miniaturisation. Whereas the likely control of the origin of the tiny C. ceciliae was basically progenesis, the extreme spinosity had a different origin; allometric growth or possibly peramorphosis. C. ceciliae is small enough for the spines to have appreciably retarded sinking through frictional effects, and this small trilobite is interpreted as a free-swimming or floating form.     

Isolation and polypeptide composition of l,3-ß-glucan synthase from plasma membranes of Brassica oleracea

The l,3-ß-glucan synthase (callose synthase, EC 2.4.1.34) was solubilized from cauliflower ( Brassica oleracea L.) plasma membranes with digitonin, and partially purified by ion exchange chromatography and gel filtration [fast protein liquid chromatography (FPLC)] using 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) in the elution buffers. These initial steps were necessary to obtain specific precipitation of the enzyme during product entrapment, the final purification step. Five polypeptides of 32, 35, 57, 65 and 66 kDa were highly enriched in the final preparation and are thus likely components of the callose synthase complex. The purified enzyme was activated by Ca²⁺, spermine and cellobiose in the same way as the enzyme in situ, indicating that no essential subunits were missing. The polyglucan produced by the purified enzyme contained mainly 1,3-linked glucose.     

Detection and identification of gibberellins in Sitka spruce (Picea sitchensis) of different ages and coning ability by bioassay, radioimmunoassay and gas chromatography – mass spectrometry

Gibberellins Al (GA₁), GA₃, GA₄, GA₉, and after enzymatic hydrolysis of GA-conjugate-like fractions, GA₉ and GA₁₅, were identified in shoots of Sitka spruce [ Picea sitchensis (Bong.) Carr.] of different ages by combined gas chromatography-mass spectrometry (GC-MS). The purification and separation of the GAs involved the use of reverse phase and normal phase high performance liquid chromatography (HPLC). The Tan-ginbozu dwarf rice bioassay and binding to antibodies raised against GA₁, GA₄ and GA₉ were used for detection of GA-like substances. The qualitative differences between the three ages of plant material were the presence of GA₃ and GA₁ in the 48-year-old material and the absence of detectable amounts of GA₄ in the same material. This indicates a difference in GA metabolism which may reflect the difference in ability to form reproductive buds.     

Maintaining and restoring biodiversity in European boreal forests by developing natural disturbance regimes

Abstract. A conceptual model is presented as a guide to the maintenance and restoration of ecologically sustainable boreal forest. The model is based on the hypothesis that self-sustained forest ecosystems can be (re-)created, and their biodiversity developed, if forest management can simulate the composition and structure of boreal forest landscapes by introducing and maintaining disturbances leading to naturally dynamic spatial and temporal patterns of forest regeneration. The major explanatory variable in the model is the effect of wildfire on sites with different fuel characteristics and climates found in the European boreal forest. Four levels of fire intensity are distinguished, based on mean fire frequencies. These range from extremely low in some wet tall-herb sites or sites at high altitudes or latitudes with a humid climate, where fire is absent or rare, to dry lichen-rich sites where fire occurs often. The model is called ASIO, after the words Absent, Seldom, Infrequent and Often, indicating the four levels. Three main disturbance regimes are distinguished in the European boreal forest, based on the complex interactions between probabilistic (e.g. mean fire intervals at different site types) and random events (e.g. where and when a fire occurs): (1) gap-phase Picea abies dynamics; (2) succession from young to old-growth mixed deciduous/coniferous forest; and (3) multi-cohort Pinus sylvestris dynamics. The model stems mainly from studies in Fennoscandia, but some studies from outside this region are reviewed to provide support for a more general application of the model. The model has been implemented in planning systems on the landscape level of several large Swedish forest enterprises, and is also used as an educational tool to help private land owners with the location and realization of forest management regimes. Finally, the model can be used to develop an administrative system for the monitoring of biodiversity in boreal forest.