A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.     

The Mechanism of Calcium-Induced Inhibition of Muscle Fructose 1,6-bisphosphatase and Destabilization of Glyconeogenic Complex

The mechanism by which calcium inhibits the activity of muscle fructose 1,6-bisphosphatase (FBPase) and destabilizes its interaction with aldolase, regulating glycogen synthesis from non-carbohydrates in skeletal muscle is poorly understood. In the current paper, we demonstrate evidence that Ca²⁺ affects conformation of the catalytic loop 52–72 of muscle FBPase and inhibits its activity by competing with activatory divalent cations, e.g. Mg²⁺ and Zn²⁺. We also propose the molecular mechanism of Ca²⁺-induced destabilization of the aldolase–FBPase interaction, showing that aldolase associates with FBPase in its active form, i.e. with loop 52–72 in the engaged conformation, while Ca²⁺ stabilizes the disengaged-like form of the loop.     

Degradation of acrylic copolymers by white-rot fungi

Various water-soluble homopolymers and copolymers of acrylamide (AAm) and acrylic acid (AA) which contained phenolic sites, such as guaiacol, lignin sulfonate (LS) and 3,4-dihydroxybenzoic acid (3,4-DHBA), were tested with regard to their degradability by white-rot fungi. Compared with Phanerochaete chrysosporium, Pleurotus ostreatus caused a significantly higher decrease in the average molecular weight (_w) of most of the copolymers and the homopolymer under the applied culture conditions. However, the _w of poly(guaiacol/AAm) increased significantly during incubation with Pl ostreatus. P. chrysosporium was able to reduce only the _w of the poly(LS/AA) to a significant degree and not that of the other polymers. The mineralization rate of AAm and AA copolymers and terpolymers of AAm, AA and phenolics (LS, 3,4-DHBA, guiacol), which were tested with P. ostreatus and Trametes versicolor, turned out to be low (0.8–3.2%). While the rates of mineralization were similar among all polymers, the decrease in radioactivity from the culture media was higher with the terpolymers bearing phenolic sites. UV spectra of the culture media revealed that the phenolic sites in the terpolymers were significantly degraded by both fungi. Obviously, the degradation of phenolics within the polymer chain caused a higher decrease in _w but did not significantly increase the mineralization rate.     

Structure- and function-based characterization of a new phosphoglycolate phosphatase from Thermoplasma acidophilum

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel beta-sheet with strand order S10, S9, S8, S1, S2 and a small beta-hairpin, strands S3 and S4. This central sheet is flanked by a set of three alpha-helices on one side and two helices on the other. The smaller domain is composed of an open faced beta-sandwich represented by three antiparallel beta-strands, S5, S6, and S7, flanked by two oppositely oriented alpha-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl- ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase.     

Non-HLA gene polymorphisms and the outcome of allogeneic hemato-poietic stem cell transplantation

Haematopoietic stem cell transplantation (HSCT) is a curative treatment of many hematological disorders. However, although significant advances have been made in donor-recipient matching or conditioning regimens, HSCT is associated with a risk of post transplant complications. Those include generation of toxic lesions, graft-versus-host-disease (GvHD) and viral reactivations. Recent studies have shown the association between polymorphic features of non-HLA encoding genes and the incidence and severity of post-transplant complications in the recipients of allogeneic HSCT, implying that the donor-recipient genotyping, extended for cytokine loci, may be of prognostic value for the transplantation outcome. Thus, the pre-transplant analysis of the patients' genetic predisposition may be considered as important factor allowing the classification of the transplant recipients as less or more susceptible for developing toxic lesions, severe and/or fatal acute GvHD or viral reactivation. This review focuses on the relationship between the polymorphic patterns of tumor necrosis factor (TNF)- alpha and TNF-beta, IFN-gamma, interleukin (IL)-6, IL-10 and heat shock protein (HSP)70-hom encoding genes and the manifestation of post-transplant complications, acute and chronic GvHD, generation of toxic lesions, viral reactivations and mortality.     

Effect of climate change on sporulation of the teleomorphs of <Emphasis Type="Italic">Leptosphaeria</Emphasis> species causing stem canker of brassicas

Leptosphaeria maculans and L. biglobosa are closely related sibling fungal pathogens that cause phoma leaf spotting, stem canker (blackleg) and stem necrosis of oilseed rape (Brassica napus). The disease is distributed worldwide, and it is one of the main causes of considerable decrease in seed yield and quality. Information about the time of ascospore release at a particular location provides important data for decision making in plant protection, thereby enabling fungicides to be used only when necessary and at optimal times and doses. Although the pathogens have been studied very extensively, the effect of climate change on the frequencies and distributions of their aerially dispersed primary inoculum has not been reported to date. We have collected a large dataset of spore counts from Poznan, located in central-west part of Poland, and studied the relationships between climate and the daily concentrations of airborne propagules over a period of 17 years (1998–2014). The average air temperature and precipitation for the time of development of pseudothecia and ascospore release (July–November), increased during the years under study at the rates of 0.1 °C and 6.3 mm per year. The day of the year (DOY) for the first detection of spores, as well as the date with maximum of spores, shifted from 270 to 248 DOY, and from 315 to 265 DOY, respectively. The acceleration of the former parameter by 22 days and the latter by 50 days has great influence on the severity of stem canker of oilseed rape.     

PCR-RFLPs within the lactate dehydrogenase (LDH-A) gene of the domestic pigeon (Columba livia var. domestica)

A total of 45 racing pigeons were genotyped using PCR-RFLP method. PCR product of the LDH-A gene was amplified according to the Long-PCR procedure. The amplification products were digested with restriction enzymes. PCR-RFLPs for two restriction enzymes, HaeIII and NlaIV, were observed. Two pairs of alleles LDH-A(A) and LDH-A(B) for LDH-A-NlaIV polymorphism and LDH-A(C) and LDH-A(D) for LDH-A-HaeIII polymorphism were detected in the homozygous and heterozygous states. Frequencies of alleles were as follows: A - 0.622, B - 0.378 and C - 0.256, D - 0.744.     

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5'-monophosphates from such natural metabolites as adenosine 5'-phosphosulfate and adenosine 5'-phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5'-O-phosphorofluoridate and adenosine 5'-O-(gamma-fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P-F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.     

Comparative studies on metabolic activity of planktonic,benthic and epiphytic bacteria

Summary Differences have been noted in metabolic activity of bacteria isolated from water, mud and from Canadian pondweed (Elodea canadensis).The epiphytic bacteria showed the greatest metabolic activity in every instance, benthic bacteria were least active and the water isolates were intermediate.Non-chromogenic bacteria were metabolically more active than the chromogens.Casamino acids proved to be the most readily oxidized substrate among the compounds studied. Glucose, succinate, fumarate and gluconate were readily used by most epiphytic and planktonic bacteria. Acetate was intermediate and fructose was the least suitable substrate for these organisms. Casamino acids proved to be the best substrate for all three groups, although the benthic bacteria were less active with this substrate than the former two groups of bacteria. Gluconate, succinate, pyruvate and acetate were intermediate, and glucose and fructose were least suitable for most of the benthic bacteria.     

Delignification of Wood Chips and Pulps by Using Natural and Synthetic Porphyrins: Models of Fungal Decay

Kraft pulps, prepared from softwoods, and small chips of birch wood were treated with heme and tert-butyl hydroperoxide in aqueous solutions at reflux temperature. Analyses of treated pulps showed decreases in kappa number (a measure of lignin content) from about 36 to less than 2, with concomitant increases in brightness (80% increase in the better samples). Analyses of treated wood chips revealed selective delignification and removal of hemicelluloses. After 48 h of treatment, lignin losses from the wood chips approached 40%, and xylose/mannose (hemicellulose) losses approached 70%, while glucose (cellulose) losses were less than 10%. Examination of delignified chips by transmission electron microscopy showed that the removal of lignin occurred in a manner virtually indistinguishable from that seen after decay by white rot fungi. Various metalloporphyrins, which act as biomimetic catalysts, were compared to horseradish peroxidase and fungal manganese peroxidase in their abilities to oxidize syringaldazine in an organic solvent, dioxane. The metalloporphyrins and peroxidases behaved similarly, and it appeared that the activities of the peroxidases resulted from the extraction of heme into the organic phase, rather than from the activities of the enzymes themselves. We concluded that heme-tert-butyl hydroperoxide systems in the absence of a protein carrier mimic the decay of lignified tissues by white rot fungi.