Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Co2+ binding to α-lactalbumin

α-Lactalbumin possesses multiple Zn²⁺ binding sites, with the strongest site having an affinity constant of 5×10⁵ M^?1 [Permyakovet al. (1991),J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected in a shift of the thermal denaturation transition temperature by more than 40°. The present work examines Co²⁺ binding to bovineα-lactalbumin, where for this analog of Zn²⁺, multiple binding sites were also found from spectrofluorimetric titrations. The strong site Co²⁺ binding constant was 1.3×10⁶ M^?1. However, in contrast to Zn²⁺ binding, Co²⁺ does not cause protein aggregation nor any significant thermal destabilization of the protein. Fluroescence energy transfer measurements between Tb³⁺ in the strong calcium site to Co²⁺ in the strong Zn²⁺ site gave a distance in the range of 14–18 Å, which was in excellent agreement with recent crystallographic data for humanα-lactalbumin [Renet al. (1993), J. Biol. Chem.268, 19292–19298] However, the X-ray structure did not identify the additional zinc sites found from earlier solution studies, presumably due to restrictive crystal packing interactions. The results from the current work confirm that the strong cobalt (zinc) site in solution is the same zinc site elucidated by X-ray crystallography.     

The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

The antioxidant enzymes activity in leaves of inter-varietal substitution lines of wheat (Triticum aestivum L.) with different tolerance to soil water deficit

Understanding of the genetic basis of physiological properties, which are most relevant to water-deficit tolerance would be helpful for genomic-assisted improvement of bread wheat. A set of bread wheat inter-varietal single chromosome substitution lines (ISCSLs) of variety ‘Janetzkis Probat’ (JP) in the genetic background of ‘Saratovskaya’ 29 (S29) were used to reveal the critical chromosomes in wheat genome controlling tolerance to water deficit. The same lines were involved in the identification of chromosomes associated with the activity of antioxidant enzymes that are closely related to the detoxification of H₂O₂ [catalase (CAT), ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase (GR)]. The recipient cultivar S29 was highly drought tolerant while the donor JP was sensitive. Using non-metric multidimensional scaling of yield components and indices of drought tolerance/susceptibility chromosomes 2A and 4D, substitution in the genetic background of S29 was found to lead to a critical decrease of water-deficit tolerance. The drop of tolerance correlated with a sharp decline of cumulative activity of the catalase and the enzymes of ascorbate–glutathione cycle in wheat leaves. Clear evidence was obtained for the involvement of genes present on the homoeologous group 2 chromosomes in the control of GR and CAT activity. Substitution of the chromosome 4D had a significant reducing impact on the CAT activity level.     

Ortho-phthalic acid esters in lipophilic extract from the cell culture of <Emphasis Type="Italic">Aconitum baicalense</Emphasis> Turcz ex Rapaics 1907

Optically active bis-2R(–)ethylhexyl o-phthalate was obtained with 0.18% yield from dry cultured cells of Aconitum baicalense Turcz ex Rapaics 1907 by extraction with petroleum ether followed by silica gel column chromatography. Its structure was confirmed by the analysis of ¹³C and ¹H NMR spectra. Seasonal fluctuations of quantitative phthalate content in A. baicalense cells were identified. The tests were performed under conditions excluding the presence of phthalates in reagents, materials, and laboratory dishes. The same substance was shown to be produced by cultivated cells of other plants. Biosynthesis of esters of ortho-phthalic acid by cultivated plant cells was discovered for the first time.     

The Use of the Free Metal – Temperature ‘Phase Diagrams’ for Studies of Single Site Metal Binding Proteins

Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant K for a native protein (K _n), while the significance of the K value for the thermally denatured protein (K _u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal binding protein is defined by K _n to K _u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration – temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental and general problems coupled with the use of the phase diagrams are discussed.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin

The structure of C-terminal domain (CaD136, C-terminal residues 636-771) of chicken gizzard caldesmon has been analyzed by a variety of physico-chemical methods. We are showing here that CaD136 does not have globular structure, has low secondary structure content, is essentially noncompact, as it follows from high R(g) and R(S) values, and is characterized by the absence of distinct heat absorption peaks, i.e. it belongs to the family of natively unfolded (or intrinsically unstructured) proteins. Surprisingly, effective binding of single calmodulin molecule (K(d) = 1.4 +/- 0.2 microM) leads only to a very moderate folding of this protein and CaD136 remains substantially unfolded within its tight complex with calmodulin. The biological significance of these observations is discussed.