Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I     

Genome size and ploidy level among wild and cultivated <Emphasis Type="Italic">Prunus</Emphasis> taxa in Slovakia

2C DNA content and ploidy level variation of Prunus spinosa and closely related taxa together with Prunus domestica L. and Prunus insititia L. was studied in Slovakia. The aim of the study was to define genome sizes and find differences between closely related taxa within Prunus spinosa sensu lato mentioned in previous works. According to our results, investigated taxa can be divided into three groups according to ploidy level: Prunus spinosa, Prunus dasyphylla, Prunus ×fruticans, Prunus ×dominii and Prunus ×schurii are tetraploids, Prunus ×fechtneri is pentaploid, and P. domestica and P. insititia are hexaploids. Genome size differences within tetraploid taxa were relatively small (Prunus spinosa: 1.40?±?0.02, P. ×domini: 1.44?±?0.01, P. ×fruticans: 1.48?±?0.02, P. ×schurii: 1.44?±?0.02), but statistically significant. Although further research is needed, it seems that the concept of several taxa as product of hybridization between P. spinosa and cultivated plum species has been supported by our study.     

In vitro identification of mitochondrial oxidative stress production by time-resolved fluorescence imaging of glioma cells

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker? Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.     

Thermal behavior of round and wagtail dancing honeybees

The thermal behavior of round and wagtail dancing honeybees (Apis mellifera carnica) gathering sucrose solutions of concentrations between 0.5 and 2 mol·l^-1 was investigated under field conditions by infrared thermography (30–506 m flight distance). During the stay inside the hive thoracic surface temperature ranged from 31.4 to 43.9 °C. In both round and wagtail dancing honeybees the concentration of sucrose in the food influenced dancing temperature in a non-linear way. Average dancing temperature was 37.9 °C in foragers gathering a 0.5 mol·l^-1 sucrose solution, 40.1°C with a 1 mol·l^-1, 40.6°C with a 1.5 mol·l^-1 and 40.7°C with a 2 mol·l^-1 solution. The variability of thoracic temperature was highest with the 0.5 mol·l^-1 and lowest with the 1.5 and 2 mol·l^-1 concentrations. Thoracic temperatures during trophallactic contact with hive bees were similar to dancing temperature at 1.5 mol·l^-1 but lower at the other concentrations. During periods of distribution of food to hive bees (trophallactic contact >2.5s) the dancers' thorax cooled down by more than 0.5°C considerably more frequently with the 0.5 mol·l^-1 solution (65% of cases) than with the 1.5 mol·l^-1 solution (26%). By contrast, heating the thorax up by more than 0.5°C was infrequent with the 0.5 mol·l^-1 solution (2%) but occurred at a maximum rate of 26% with the 1.5 mol·l^-1 solution. Bees gathering the 1 or 2 mol·l^-1 solutions showed intermediate behavior. Linear model analysis showed that at higher concentrations the dancers compensated better for variations of hive air temperature: per 1 °C increase of hive temperature dancing temperature increased by 0.34, 0.22, 0.12, and 0.13 °C with 0.5, 1, 1.5, and 2 mol·l^-1 sucrose solutions, respectively. The results furnish evidence that dancing honeybees follow a strategy of selective heterothermy by tuning their thermal behavior to the needs of the behavior performed at the moment. Thoracic temperature is regulated to a high level and more accurately when fast exploitation of profitable food sources is recommended. Thoracic temperature is lowered when the ratio of gain to costs of foraging becomes more unfavorable.Abbreviations SD standard deviation - SD _reg SD around regression line - H _rel relative humidity at feeding station - T _a air temperature at feeding station - T _i air temperature near the dancers - T _d Thoracic surface temperatures - T _d dancing - T _tr trophallactic contact (distribution of food) - T _w walking - T _stay mean temperature of total stay in the hive     

Evolutionary relationships in the genus <Emphasis Type="Italic">Secale</Emphasis> revealed by DArTseq DNA polymorphism

Till this day, there is not much known about the phylogeny of the Secale genus; therefore, in our research, we tried to shed some lights on the issue of rye (Secale genus) taxonomy. The genetic diversity and phylogenetic relationships were evaluated using 13,842 DArTseq? polymorphic markers. The model-based clustering (STRUCTURE software) separated our 84 samples into three main clusters: perennial cluster, annual cluster, and S. sylvestre cluster. The same result was obtained using Neighbour Joining tree and self-organizing maps. Secale sylvestre, S. strictum, and S. cereale are the three main species of the Secale genus. Three samples of rye are in basal positions in phylogenetic trees. These accessions share ancient morphological characters and are probably the ancestors of different lineages within Secale. Annual Secale taxa, with the exception of S. sylvestre, create one mutual taxon. We have found out that the semi-perennial taxa of S. cereale var. multicaule and S. strictum subsp. ciliatoglume are genetically closest to the annual species of S. cereale. Phylogenetic signals for semi-perennial and annual taxa are also present in S. strictum subsp. africanum. SNP-based analysis revealed that evolution of annual S. cereale has already begun in S. strictum subsp. africanum. The results showed that S. vavilovii cannot be considered as a separate species but a subspecies of S. cereale Secale cereale subsp. dighoricum is a hybrid. It is still not clear whether we can consider S. strictum subsp. strictum and S. strictum subsp. kuprijanovii as two separate species.     

A side-reaction in the methylation of galacturonate derivatives with diazomethane-boron trifluoride etherate

Partial p-nitrobenzoylation of methyl (methyl 2-O-methyl-α-d-galactopyranosid)uronate (1) gave the 3-p-nitrobenzoate 2 in good yield. Treatment of 2 or methyl (methyl 2,3-di-O-benzoyl-α-d-galactopyranosid)uronate (11) with diazomethane-BF₃-etherate gave, in addition to the expected 4-methyl ethers, by-products resulting from lengthening of the carbon chain. The by-products were formulated as derivatives of methyl 4,7-anhydro-α-d-galacto-heptopyranosid-6-ulose dimethy acetal on the basis of p.m.r. and i.r. spectral data, by analysis of their mass-spectral fragmentation pattern, and by chemical transformations.     

Polymorphic and autoreactive H-2-specific monoclonal antibody isolated after injections of syngeneic sendai virus-coated lymphocytes

An H-2-specific monoclonal antibody (mAb Q-1) was obtained from B10.Q (H-2 ^q) mice injected with syngeneic Sendai virus-coated cells. The IgM monoclonal antibody recognizes the public determinant H-2.25 shared by H-2 ^k (K ^k) and H-2 ^r haplotypes and cross-reacts with H-2^d, H-2^s, H-2^p, and H-2^q cells, the latter being the haplotype of the challenged B-cell donor. The binding of mAb Q-1 to H-2^d, H-2^s, H-2^q, and H-2^p cells was lower than to H-2^k and H-2^r and of decreasing affinity but could be clearly distinguished from the negative reactions with H-2^b and H-2^f cells. MAb Q-1 distinguishes between Sendai virus-coated and uncoated lymphocytes only cells with low-affinity binding. On virus-coated or infected (H-2^p, H-2^q, H-2^d, H-2^s) cells lysis was stronger than on normal lymphocytes. We interpret the enhanced lysis of Sendai virus-positive cells by mAb Q-1 to be due to recognition of a modified exposure of public H-2 determinants induced by Sendai virus.On leave from The Institute of Immunology and Experimental Therapy, Wroclaw, Poland     

Identification of 3-hydroxy-6-methyl-2H-pyran-2-one from pyrolysis of phosphoric acid-treated cellulosic materials

The hydrogen isotope-effect that occurs in vitro during myo-inositol 1-phosphate synthase-catalyzed conversion of d-[5-³H]glucose 6-phosphate into myo-[2-³H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the ³H/¹⁴C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70 % ethanol-insoluble fraction of d-[5-³H, 1-¹⁴-C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of d-glucose that occurred during its conversion into d-galactosyluronic residues of pectic substance is not explained by loss of ³H when UDP-d-[5-³H, 1-¹⁴C]glucose is oxidized by UDP-d-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.     

PERIODIC THERMAL STRESS: OPTIMAL SPACING PREDICTED BY A SIMPLE SET-BACK MODEL OF CELL DIVISION SYNCHRONIZATION

Digital computer simulations have been used to make quantitative predictions based on a simple set-back model of cell division synchronization. According to the model appropriate thermal stress reverses progress within a segment of the division cycle called the set-back interval. In the simulations normally distributed cell-to-cell variations in division cycling rate between periodic thermal shocks were produced with the Monte Carlo method.
The simulations have shown that reasonably good synchronization with the single shock per division strategy requires a relatively long set-back interval and small cell-to-cell variations in rate of progress through the division cycle. The simulations have shown that the degree of synchrony produced by such periodic shocks is highly dependent on the time interval between shocks—with a series of as many as seven shocks inappropriately spaced producing less synchrony than a single shock! The optimal time interval between successive thermal shocks was found approximately equal to the mode division cycle time at synchrony equilibrium multiplied by 1 plus half the fraction of the division cycle occupied by the set-back interval. Position of the set-back interval within the division cycle had little effect on synchrony at the end of the final shock.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: A small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca²⁺ and Mg²⁺ cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA = 1:1 mol/base and in the range of concentration of the cation²⁺ 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L^x phase with repeat distance d_Lx ∼ 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L_DOPC phase with repeat distance d_DOPC ∼ 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L_DOPC phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC + DNA + Ca²⁺ aggregates was investigated in the range 20-80 °C. The transversal thermal expansivities of both phases were evaluated.