13C–13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin

Molecular size has limited solution NMR analyses of proteins. We report ¹³C–¹³C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting ¹³C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C connectivities between nuclei separated by 1–4 bonds. These results show the potential of ¹³C–¹³C NOESY for solution studies of molecular assemblies >100 kDa.     

Suitability of Solanum lycopersicum L. ‘Microtom’ for growth in Bioregenerative Life Support Systems: exploring the effect of high‐LET ionising radiation on photosynthesis,leaf structure and fruit traits

• The realisation of manned space exploration requires the development of Bioregenerative Life Support Systems (BLSS). In such self‐sufficient closed habitats, higher plants have a fundamental role in air regeneration, water recovery, food production and waste recycling. In the space environment, ionising radiation represents one of the main constraints to plant growth.
• In this study, we explore whether low doses of heavy ions, namely Ca 25 Gy, delivered at the seed stage, may induce positive outcomes on growth and functional traits in plants of Solanum lycopersicum L. ‘Microtom’. After irradiation of seed, plant growth was monitored during the whole plant life cycle, from germination to fruit ripening. Morphological parameters, photosynthetic efficiency, leaf anatomical functional traits and antioxidant production in leaves and fruits were analysed.
• Our data demonstrate that irradiation of seeds with 25 Gy Ca ions does not prevent achievement of the seed‐to‐seed cycle in ‘Microtom’, and induces a more compact plant size compared to the control. Plants germinated from irradiated seeds show better photochemical efficiency than controls, likely due to the higher amount of D1 protein and photosynthetic pigment content. Leaves of these plants also had smaller cells with a lower number of chloroplasts. The dose of 25 Gy Ca ions is also responsible for positive outcomes in fruits: although developing a lower number of berries, plants germinated from irradiated seeds produce larger berries, richer in carotenoids, ascorbic acid and anthocyanins than controls.
• These specific traits may be useful for ‘Microtom’ cultivation in BLSS in space, in so far as the crew members could benefit from fresh food richer in functional compounds that can be directly produced on board.

     

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

Background  

Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear.     

Molecular population genetics of the alcohol dehydrogenase locus in the Hawaiian drosophilid D. mimica

Sequence variation among 10 alleles of the alcohol dehydrogenase (Adh) gene of the Hawaiian drosophilid D. mimica was analyzed with reference to the evolutionary history of the Hawaiian subgroup as well as to levels and patterns of polymorphism of the Adh gene in continental drosophilid species. The Adh gene of D. mimica is less polymorphic than that of other drosophilid species, and no replacement substitutions were found. Statistical analyses of the Adh alleles suggested the action of balancing selection and revealed significant linkage disequilibrium among three of the variable sites. The effective population size was estimated to be only slightly smaller than that of continental species and, surprisingly, on the same order of magnitude as the actual size.      

Differential scanning calorimetry of chicken erythrocyte nuclei.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.     

Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures

Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.     

Glycosylated forms of nuclear lamins

Chromatin and pore complex-lamina preparations were obtained from pig and chicken tissues, and their proteins were analysed by mono- and bidimensional electrophoresis. A glycosylated form of lamin A, recognized by concanavalin A, was shown to be present in at least 3 of the tissues examined. Glycosylation is suggested to be a further postsynthetic modification, besides phosphorylation and methylation, which can modify the properties of lamins.     

Development of horse polyclonal antiserum inhibiting all in vitro biological functions of human IFN-gamma

Following a standard immunization protocol with recombinant human interferon-gamma (IFN-gamma), a horse polyclonal antiserum was obtained and evaluated for its ability to interfere with multiple IFN-gamma activities in vitro. Data obtained show that polyclonal horse antiserum neutralizes the antiproliferative activity of IFN-gamma, inhibits the binding of IFN-gamma to cellular receptors, and can up-regulate HLA-DR antigen expression and interfere with its antiviral activity. The broad neutralizing capacity of horse polyclonal antiserum has been assessed on cell lines which differ as to origin and sensitivity to IFN-gamma. Moreover, we observed that this antiserum could inhibit the binding of radiolabeled IFN-gamma to its cellular receptor, its subsequent internalization into the target cell, and its antiviral activity. As it is able to inhibit all the biological activities of IFN-gamma, this antiserum might provide new therapeutic approaches to diseases with evidence of activated cell-mediated immunity.     

Gamma Aminobutyric Acid (GABA) and Plant Responses to Stress

4-aminobutyrate (GABA) is a non-protein amino acid that is widely distributed throughout the biological world. In animals, GABA functions as the predominant inhibitory neurotransmitter in the central nervous system by acting through the GABA receptors. The neuromuscular system enables animals to escape from environmental stresses. Being nonmotile, plants have evolved chemical responses to mitigate stress. Mechanisms by which GABA may facilitate these responses are discussed in this review. Environmental stresses increase GABA accumulation through two different mechanisms. Stresses causing metabolic and/or mechanical disruptions, resulting in cytosolic acidification, induce an acidic pH-dependent activation of glutamate decarboxylase and GABA synthesis. Extremely marked declines in cytosolic pH occur under oxygen deprivation, which is the primary stress factor in flooded soils, and this stress induces the greatest accumulation of GABA. Other stresses, including cold, heat, salt, and mild or transient environmental factors, such as touch, wind, rain, etc. rapidly increase cellular levels of Ca²⁺. Increased cytosolic Ca²⁺ stimulates calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. A review of the kinetics of GABA accumulation in plants reveals a stress-specific pattern of accumulation that is consistent with a physiological role for GABA in stress mitigation. Recent physiological and genetic evidence indicates that plants may possess GAB A-like receptors that have features in common with the animal receptors. The mechanism of action of animal GABA receptors suggests a model for rapid amplification of ion-mediated signals and GABA accumulation in response to stress. Metabolic pathways that link GABA to stress-related metabolism and plant hormones are identified. The survival value of stress-related metabolism is dependent on metabolic changes occurring before stress causes irreversible damage to plant tissue. Rapid accumulation of GABA in stressed tissue may provide a critical link in the chain of events leading from perception of environmental stresses to timely physiological responses.