Unraveling protein interaction networks with near-optimal efficiency

The functional characterization of genes and their gene products is the main challenge of the genomic era. Examining interaction information for every gene product is a direct way to assemble the jigsaw puzzle of proteins into a functional map. Here we demonstrate a method in which the information gained from pull-down experiments, in which single proteins act as baits to detect interactions with other proteins, is maximized by using a network-based strategy to select the baits. Because of the scale-free distribution of protein interaction networks, we were able to obtain fast coverage by focusing on highly connected nodes (hubs) first. Unfortunately, locating hubs requires prior global information about the network one is trying to unravel. Here, we present an optimized 'pay-as-you-go' strategy that identifies highly connected nodes using only local information that is collected as successive pull-down experiments are performed. Using this strategy, we estimate that 90% of the human interactome can be covered by 10,000 pull-down experiments, with 50% of the interactions confirmed by reciprocal pull-down experiments.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

Dynamics of memory T cell proliferation under conditions of heterologous immunity and bystander stimulation

By examining adoptively transferred CSFE-labeled lymphocytic choriomeningitis virus (LCMV)-immune donor T cells in Thy-1 congenic hosts inoculated with viruses or with the cytokine inducer poly(I:C), strikingly different responses of bona fide memory T cells were found in response to different stimuli. Poly(I:C) (cytokine) stimulation caused a limited synchronized division of memory CD8 T cells specific to each of five LCMV epitopes, with no increase and sometimes a loss in number, and no change in their epitope hierarchy. Homologous LCMV infection caused more than seven divisions of T cells specific for each epitope, with dramatic increases in number and minor changes in hierarchy. Infections with the heterologous viruses Pichinde and vaccinia (VV) caused more than seven divisions and increases in number of T cells specific to some putatively cross-reactive but not other epitopes and resulted in substantial changes in the hierarchy of the LCMV-specific T cells. Hence, there can be memory T cell division without proliferation (i.e., increase in cell number) in the absence of Ag and division with proliferation in the presence of Ag from homologous or heterologous viruses. Heterologous protective immunity between viruses is not necessarily reciprocal, given that LCMV protects against VV but VV does not protect against LCMV. VV elicited proliferation of LCMV-induced CD8 and CD4 T cells, whereas LCMV did not elicit proliferation of VV-induced T cells. Thus, depending on the pathogen and the sequence of infection, a heterologous agent may selectively stimulate the memory pool in patterns consistent with heterologous immunity.     

Automated detection of remote homology

The classification of a newly identified protein as a member of a superfamily is important for focusing experiments on its most likely functions. Such classification, often performed by hand, has now been fully automated. This sophisticated new approach takes into account not only alignment scores but also a number of other computable attributes, such as functional sites deduced from sequence conservation patterns.     

Translocation of phosphatidylthreonine and -serine to mitochondria diminishes exponentially with increasing molecular hydrophobicity

Some cultured cells contain significant amounts of a rarely recognized phospholipid, phosphatidylthreonine. Since phosphatidylthreonine is a structural analog of phosphatidylserine, the question rises whether it is transported to mitochondria and decarboxylated to phosphatidylisopropanolamine therein. We studied this issue with hamster kidney cell-line using a novel approach, i.e. electrospray mass-spectrometry and stable isotope-labeled precursors. Scanning for a neutral loss of 155, which is characteristic for phosphatidylisopropanolamine, indicated that this lipid is indeed present. The identity of phosphatidylisopropanolamine was supported by the following: (i) it co-chromatographed with phosphatidylethanolamine; (ii) its molecular species profile was similar to that of phosphatidylethanolamine; (iii) its head group was labeled from ¹³C-threonine; and (iv) its concentration increased in parallel with phosphatidylthreonine. Tests with solubilized decarboxylase and subcellular fractionation studies indicated that the low cellular content of phosphatidylisopropanolamine is due to inefficient decarboxylation, rather than poor translocation of phosphatidylthreonine to mitochondria. Importantly, the average hydrophobicity of phosphatidylisopropanolamine molecular species was significantly less than that of phosphatidylthreonine species, indicating that hydrophilic phosphatidylthreonine species translocate to mitochondria far more rapidly than hydrophobic ones. Parallel results were obtained for phosphatidylserine. These findings imply that efflux from the ER membrane could be the rate-limiting step in the phosphatidylthreonine and -serine translocation to mitochondria.     

Detection of bacterial contamination in starch and resin-based papermaking chemicals using fluorescence techniques

Rapid fluorescence techniques were evaluated for the detection of bacterial contaminants in papermaking chemicals including starch and the resin-based sizes and starch slurries used in the paper industry. Viable and non-viable bacterial cells were visualised by fluorescent probes and detected by epifluorescence microscopy and flow cytometry. The best discrimination ability was obtained with the fluorescent probes LIVE/DEAD and SYBR Green, based on the staining of cellular nucleic acid, and ChemChrome V3, which demonstrated cellular enzymatic activity. The process samples had to be diluted and filtered before fluorescence staining and analysis because they were viscous and contained solid particles. Fluorescence microscopic counts of bacteria in highly contaminated process samples were similar to plate counts, but flow cytometric enumeration of bacterial cells in process samples yielded 2- to 10-fold lower counts compared with plate counts, depending on the consistency of the sample. The detection limits in flow cytometric analysis and in epifluorescence microscopy were 10³–10⁶ cells ml⁻¹ and 10⁵–10⁶ cells ml⁻¹, respectively. Intrinsic bacterial contamination was detectable with fluorescence techniques and highly contaminated process samples could be analysed with fluorescence methods. Electronic Publication     

Two Endophytic Fungi in Different Tissues of Scots Pine Buds (Pinus sylvestris L.)

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.     

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively. The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10(3). An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C. In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety. However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed.     

False-positive apoptosis signal in mouse kidney and liver detected with TUNEL assay

Apoptosis is a physiological, programmed process for the elimination of cells from living organisms. Currently, one of the most frequently used methods to detect apoptosis is TUNEL assay. It has provided valuable information about apoptosis in various tissues. However, the sensitivity and the specificity of TUNEL technique have also been criticized. We detected an intense false-positive apoptotic signal in nude and Balb/c mice kidney and liver. In kidney the signal was confined to the proximal, distal and collecting tubular cells, and in liver to hepatocytes. Both tissues appeared normal in light microscopy, and no DNA ladder formation or increase in caspase-3 enzyme activity was detected. BrdU labelling and Ki-67 immunostaining did not reveal increased cell proliferation in these tissues. On the other hand, false-positive signal was not detected in testis, spleen, pancreas or renal cell carcinoma from the same animals. Also, no false-positive signal was seen in human liver or kidney samples. Although factors known to produce false-positive staining related to sample harvesting, preparation and staining protocols were eliminated, the cause of the false- positive apoptotic signal remains unknown. We conclude that caution must be exercised when examining apoptosis in mouse tissues with TUNEL assay.