On the mechanisms of glycolytic oscillations in yeast

This work concerns the cause of glycolytic oscillations in yeast. We analyse experimental data as well as models in two distinct cases: the relaxation-like oscillations seen in yeast extracts, and the sinusoidal Hopf oscillations seen in intact yeast cells. In the case of yeast extracts, we use flux-change plots and model analyses to establish that the oscillations are driven by on/off switching of phosphofructokinase. In the case of intact yeast cells, we find that the instability leading to the appearance of oscillations is caused by the stoichiometry of the ATP-ADP-AMP system and the allosteric regulation of phosphofructokinase, whereas frequency control is distributed over the reaction network. Notably, the NAD+/NADH ratio modulates the frequency of the oscillations without affecting the instability. This is important for understanding the mutual synchronization of oscillations in the individual yeast cells, as synchronization is believed to occur via acetaldehyde, which in turn affects the frequency of oscillations by changing this ratio.     

Evidence for bidirectional transverse diffusion of spin-labeled phospholipids in the plasma membrane of guinea pig blood cells

The distribution and transverse diffusion kinetics of four spin-labeled phospholipid analogues (two with choline heads: phosphatidylcholine (PC) and sphingomyelin (SM); two with amino heads: phosphatidylserine (PS) and phosphatidylethanolamine (PE) were studied in the plasma membrane of guinea pig blood cells: erythrocytes, reticulocytes, and leukemic lymphocytes. Nitroxide reduction by the internal content of the cells was used as an indicator to determine the phospholipids that penetrated the cells. The reduction rates were in the order, PS greater than PE greater than PC greater than SM in all cells. Reoxidation of phospholipids extracted by serum albumin revealed the distribution of the phospholipids at a given time. In all cells, the distribution equilibrium was reached in less than 2 h and the amounts left in the external leaflet were in the following proportional order: PS less than PE less than PC less than SM. In the erythrocytes and especially in the reticulocytes, the shape change induced by adding phospholipids relaxed partially or completely at a lower speed but kept the same proportional order as at equilibrium. All the results were analyzed quantitatively with a simple kinetic model including the rates of transverse diffusion (flip and flop), the exchange between plasma membrane and internal membranes, and the reduction rate of free radicals (determined in either the internal or external membrane leaflet). The calculated rate constants of transverse diffusion varied from 2 x 10(-3) to 1.2 x 10(-1) min-1 for the flip and from 4 x 10(-3) to 1.2 x 10(-1) for the flop, depending on the polar head and the cell type. Possible interpretations of the external phospholipid reduction mechanism and cell deformation are discussed.     

Effect of elevated [CO2] on stem wood properties of mature Norway spruce grown at different soil nutrient availability

The objective of the present study was to investigate the interactive effects of elevated [CO₂] and soil nutrient availability on secondary xylem structure and chemical composition of 41‐year‐old Norway spruce (Picea abies (L.) Karst.) trees. The nonfertilized and irrigated‐fertilized trees were, for 3 years, continuously exposed to elevated [CO₂] in whole‐tree chambers. Elevated [CO₂] decreased concentrations of soluble sugars, acid‐soluble lignin and nitrogen in stem wood, but the effects were not consistent between sampling height and/or fertilization. The effect of 2*ambient [CO₂] on wood structure depended on the exposure year and/or fertilization. Radial lumen diameter decreased and annual ring width increased in the second year of exposure (1999) in elevated [CO₂]. In the latter, the CO₂ effect was significant only in the nonfertilized trees. Stem wood chemistry and structure were significantly affected by fertilization. Fertilization increased the concentrations of nitrogen and gravimetric lignin, annual ring width, and radial lumen diameter. Fertilization decreased C/N ratio, mean ring density, earlywood density, latewood density, cell wall thickness, cell wall index, and latewood percentage. We conclude that elevated [CO₂] had only minor effects on wood properties while fertilization had more marked effects and thus may affect ecosystem processes and suitability of wood for different end‐use purposes.     

Mass Spectrometry of Human Leukocyte Antigen Class I Peptidomes Reveals Strong Effects of Protein Abundance and Turnover on Antigen Presentation

HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use.The highly polymorphic Human Leukocyte Antigen class I (HLA-I)¹ genes are encoded by three loci (HLA-A, B, and C) in a gene-rich region on chromosome 6. They produce up to six unique cell surface receptors that bind and present the so-called HLA class I peptidome, which consists of peptides derived from proteolysis of intracellular proteins. Their function is to reflect the health state of the body''s cells to CD8+ cytotoxic T cells. During thymic maturation T cells that react to self-peptides are eliminated (1), leaving T cells with the capability to recognize peptides from viruses and bacteria. This recognition is interpreted as a danger signal, leading to removal of infected cells. Transformed, preneoplastic and cancer cells also tend to display atypical self-peptides from mutated or excessively expressed self-proteins, known as tumor associated antigens (TAAs). Although HLA-I molecules are indispensable in prevention of disease, they also pose a substantial health problem by causing allergies (2), life-threatening autoimmune diseases (3), and the often fatal rejection of donor organs because of recognition of both major and minor histocompatibility antigens (4).Finding the rules for peptide generation and selection is regarded as the most important open issue in the field of HLA-I biology by leading experts (5). Although the antigen presentation pathway is well characterized, it is still unclear how basic properties such as protein abundance, turnover, and subcellular localization influence and shape the HLA-I presented peptidome (6–10). One expectation is that protein abundance should correlate with presentation (11), but previous studies have reported conflicting and contradicting results that mostly argue against a strong link (6, 7, 10, 12, 13). It is also not fully understood why only some HLA-sampled self-peptides from cancer antigens spontaneously activate T cells, whereas others do not.The majority of HLA-I peptides are derived from proteasomal degradation (5). Although the proteasome generates an excess of peptides, only some have the required sequence motifs for HLA binding, resulting in a selective sampling of available peptides (14). The presented peptides are typically nine amino acids long, but the length can range from eight to 15. The high degree of genetic variance of HLA-I receptors translates into allele-specific peptide-binding motifs defined by anchor positions, which are usually the second and the last positions in a peptide (15). Each cell has around 200,000 cell-surface-expressed HLA complexes, which bind about 10,000 unique peptide sequences (16). The affinity of a peptide toward the presenting HLA molecule does not correlate strongly with its immunogenicity, and neither does the number of presented HLA complexes (17). Instead, the most robust predictor of peptide immunogenicity appears to be the number of potential reactive T-cell clones (17–19).The longer the source protein, the higher the chances it will contain sequences that fit to a certain HLA motif, which would inflate the representation of longer proteins regardless of biological role. Furthermore, some HLA-I peptide sequences can be mapped to multiple proteins, potentially causing a problem in determining the number of observed HLA peptides per protein (13). This illustrates that careful accounting of the potentially and actually presented HLA peptides is important in properly delineating trends in propensity of peptide presentation.In cancer immunotherapy, T cells can be directed against tumors, based on the pattern of cancer associated HLA peptides. Therefore, there is great interest in determining the identity of these immunogenic peptides. Bioinformatic methods that attempt to predict HLA peptides of cancer proteins of interest are easily accessible and most commonly used. They typically score sequences with respect to proteasomal degradation, transport into the ER via the transporter associate with antigen processing (TAP) and binding to different HLA-I alleles (20). However, their precision success is modest (21, 22). The second approach is to directly capture the naturally presented peptides using mass spectrometry; however, this requires the relevant biological sample and sophisticated instruments and workflows, which have become accessible only recently for large-scale work (23–28). Although identification of cancer associated HLA peptides by MS, if performed stringently, establish the in vivo existence of the peptide, it still does not guarantee that it will elicit a potent T-cell response, which is required for further development into therapeutics (29). Therefore, like in the case of in silico predicted peptides, the immunogenicity of the peptides must in any case be tested empirically.We here present a rich and high confidence HLA-I peptidome, established by applying state-of-the-art mass-spectrometric techniques on a collection of seven cell lines. We investigate how abundance affects the propensity of proteins to be presented as measurable HLA peptides and whether or not there are specific protein classes that are overrepresented even independent of abundance. Likewise, we explore how to use in silico immunogenicity tools on the set of identified HLA peptides from cancer-associated proteins, with a view to select vaccine candidates.     

Water and Sulphate Uptake at Root Reduction in Ricinus communis

The aim of the investigation was to study the influence of the rate of water uptake on the uptake of sulphate at supernormal rates of water flow. This was achieved by reducing the size of the root system of 42 days old Ricinus plants. The rate of water flow through the root increased 3 times by reducing the root system to 20 percent. This did not change the retention of sulphate in the roots. The uptake of sulphate was proportional to the size of the root system and thus independent of the rate of water flow while the water uptake (transpiration) was a function of the size of the shoot and the resistance of the root. This was contrary to the conditions at a moderate rate of water flow, when water and sulphate uptake followed each other. The results are discussed in terms of the salt uptake as a series of active and passive processes.     

Fibrin Degradation Products and Ovarian Tumours

Fibrin degradation products (F.D.P.) were determined in the serum of 163 women in whom ovarian tumours had been suspected on palpation at gynaecological examination and who were afterwards examined by laparoscopy or subjected to laparotomy. F.D.P. were found in the serum (0·5-30 mg/100 ml) of 23 (72%) out of 32 patients with malignant tumours. Of 131 patients with benign findings F.D.P. (traces to 2 mg/100 ml) were found in six (4·5%), and in most of these the occurrence of F.D.P. could be explained on other clinical grounds. The findings suggest that the examination of F.D.P. in suspected malignant ovarian tumour may be of diagnostic value.Determination of F.D.P. in malignant ascitic fluid showed very high values, ranging between 40 and 350 mg/ 100 ml. This argues for the occurrence of F.D.P. in the blood being due to an extravascular breakdown of fibrin caused by tumour cells, but they may also be due to thromboplastic and fibrinolytic agents from the tumour entering the blood stream.     

The external promoter in the guinea pig 5S rRNA gene is different from the rodent promoter

The guinea pig has about 100 copies of the 5S rRNA gene per haploid genome and they are present in 2.1 kb tandem repeats. Three bona fide 5S rRNA genes and four pseudo genes were sequenced. The conserved external promoter (D box) found in rodents and primates is only partially present in the guinea pig. The "D box like" sequence in guinea pig only has eight of the 12 nucleotides in the conserved D box. The results are in accordance with investigations showing that the guinea pig is not a rodent. Conserved sequences in the non-transcribed spacer can therefore be useful in phylogenetic studies.     

Acoustic rhinometry in dog and cat compared with a fluid-displacement method and magnetic resonance imaging.

An increasing number of studies have used acoustic rhinometry (AR) for study of pharmacological interventions on nasal cavity dimensions in dogs and cats, but there have been no attempts to validate AR in these species. This is done in the present study. We compared area-distance relationships of nasal cavities from five decapitated dogs (3.5-41 kg) and cats (3.8-6 kg). AR was compared with magnetic resonance (MR) imaging and a fluid-displacement method (FDM) using perfluorocarbon. AR measured 88% (98-79%) (mean and 95% confidence interval) of nasal cavity volume in dogs determined by FDM and 71% (83-59%) in cats. AR markedly underestimated nasal cavity dimensions when minimum areas were below 0.1 cm2 in dogs and 0.05 cm2 in cats. AR underestimation increased with the severity of the constriction and with distance. Cross-sectional areas in the deeper parts of the cavity measured 76% (99-54%) of FDM in dogs and 52% (66-39%) in cats. AR agreed well with MR, especially in the deeper part of the cavity. MR images showed that the nasal cavities had a very complex structure not expected to be reproduced by AR. MR could not be considered a "gold standard" because definition of the cross-sectional area of the lumen depended critically on subjective choices. FDM produced repeatable measurements and possibly offers the most adequate reference in future evaluation of AR. AR underestimated what we believed were the most correct cross-sectional areas determined by FDM, especially in the deeper part of the dog and cat nasal cavities. Despite these difficulties, AR has been shown to be useful to describe qualitative changes in cross-sectional area.     

Structure determination of T cell protein-tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130-132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.