Affinity labeling of dihydrofolate reductase with an antifolate glyoxal

Dihydrofolate reductases from different species contain several highly conserved arginines, some of which have been shown by x-ray crystallography to have their guanido groups near the p-aminobenzoyl glutamate moiety of enzyme-bound methotrexate. The orientation of one of these (Arg-52) appears to be completely reversed in comparing the crystal structures of Escherichia coli with Lactobacillus casei enzyme (Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J. (1982). J. Biol. Chem. 257, 13650-13662). We synthesized a novel antifolate containing a glyoxal group designed to react specifically with active-site guanido groups which are able to approach the p-aminobenzoyl carbonyl of methotrexate. The binding of this compound to the enzyme was competitive with dihydrofolate (DHF) in ordinary buffers. In borate buffer at pH 8.0 it inactivated dihydrofolate reductases from both E. coli and L. casei at similar maximum rates, while the chicken liver enzyme was more slowly inactivated. The inactivation was stoichiometric, paralleled the loss of the glyoxal chromophore, and showed saturation kinetics. Inhibitor binding and thus inactivation was enhanced by NADPH, while DHF protected the enzyme. This allowed calculation of the Kd for DHF which was found to be identical with its Km. The stoichiometrically inactivated enzyme displayed the 340-nm chromophore characteristic of 4-aminopteridines bound to dihydrofolate reductase confirming active-site labeling with normal orientation of the ligand. The ligand remained covalently bound to inactivated enzyme upon denaturation at low pH but dissociated at neutral pH. Computer graphic modeling of the crystal structures predicted reaction of Arg-31 but not Arg-52 in L. casei dihydrofolate reductase and of only Arg-52 in the E. coli enzyme. Purification of the CNBr fragments from the inactivated enzymes gave a single labeled peptide for each species. The particular peptide tagged in each case was unaffected by the presence of NADPH and was in excellent agreement with the crystallographic predictions.     

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.     

Influence of prey species and stages on predatory efficiency and development of two phytoseiid mites

The effect of prey species and the different stages of prey on the predatory efficiency and biology of the phytoseiid mites,Amblyseius gossipi Elbadry andTyphlodromus mangiferus sp. n. was studied. It was found that feeding either predator onTetranychus cucurbitacearum (Sayed) promoted faster development and a higher rate of oviposition than rearing on the twospotted spider mite,T. urticae (Koch). Different stages of both prey species also produced different responses in the biological activities of these predaceous mites.

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Résumé On a étudié l'effet de l'espèce de la proie et de ses différents stades sur l'efficacité et la biologie des acariens phytoseiides,Amblyseius gossipi Elbadry etTyphlodromus mangiferus sp. n. Il a été constaté que l'alimentation des 2 prédateurs avecTetranychus cucurbitacearus (Sayed) assure un dévelopment plus rapide et une fécondité plus élevée que leur élevage surT. urticae (Koch). Les différents stades de ces 2 proies produisent des réponses différentes dans les activités biologiques des 2 acariens prédateurs.

     

The distribution and abundance of species of Danthonia DC on the New England Tablelands (Australia)

The distribution and percent frequency of the 13 species and one variety of Danthonia recorded on the New England Tablelands were examined in a survey of 110 predominantly pastoral sites. The relationships between site factors and the presence and percent frequency of the different Danthonia species were subjected to canonical correlation analysis. Individual species were found to have particular sets of environmental factors associated with their distribution and percent frequency; a fact not well recognized in the past. The most common factor associated with the abundance of Danthonia was either a long time since the last cultivation or no cultivation at all. Other factors such as altitude, drainage, soil parent material, soil texture and total soil phosphorus status were also of some importance. The six species most commonly recorded throughout the New England Tablelands were D. racemosa RBr, D. pilosa RBr, D. laevis J. Vickery, D. linkii Kunth., D. richardsonii Cashmore and D. tenuior (Steud.) Conert. Of these the first three are ecologically wide species. The first two can coexist with other naturalized species under grazing while D. laevis tends to disappear with disturbance. D. linkii and D. richardsonii require fairly narrow habitat conditions but these conditions and consequently these species are common. Both species respond positively to disturbance. Danthonia linkii increases with cultivation and D. richardsonii colonizes eroded soil surfaces. Danthonia tenuior has more restricted requirements and is not so frequent The other seven species and one variety did not occur often enough for detailed study and the occurrence of two of these species, D. carphoides and D. caespitosa must be considered as rare.     

Excitation of a magnetospheric MHD cavity by Kelvin-Helmholtz instability

The Kelvin-Helmholtz instability is investigated analytically by using a one-dimensional nonuniform model of the Earth’s magnetosphere and the adjacent solar wind region. Its properties are shown to be essentially governed by the presence of an MHD cavity that arises in the magnetosphere because of the non-uniformity of the latter and also because of the jump in the parameters of the medium at the magnetopause (the outer boundary of the magnetosphere). System oscillations constitute a discrete spectrum of eigenmodes, which are determined by the wave vector k _t along the tangential discontinuity and also by the mode number n = 0, 1, 2, …, playing the role of the wavenumber along a coordinate normal to the magnetopause. Analytic expressions are obtained for the frequency and instability growth rate of each eigenmode and for the functions describing its spatial structure. All these quantities depend parametrically on the solar wind velocity V _W , or more precisely, on the Doppler frequency shift ω _W = k _t · V _W . For each eigenmode, there is a lower instability threshold depending on the parameter ω _W and a sharp maximum in the growth rate at the eigenfrequency of the magnetospheric cavity. For ω _W values below the threshold, the properties of an eigenmode are highly sensitive to the type of solar wind nonuniformity. Three cases are considered: a uniform solar wind and solar winds in which the speed of sound increases or decreases away from the magnetopause.     

Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.     

Human dynein and sperm pathology

Human spermatozoa with normal structure and with different axonemal deficiencies (absence of axoneme, of arms, or of central structures) were studied by electron microscopy, SDS-polyacrylamide gel electrophoresis, and ATPase activity measurements. Normal human sperm possess a complement of high molecular weight polypeptides with an electrophoretic migration similar to that of sea urchin and other mammalian sperm dyneins. Human high molecular weight bands are numbered one to four in order of increasing of electrophoretic mobility; all of them are absent in spermatozoa that lack axoneme. The absence of doublet arms, coincides with the absence of bands 2, 3, and 4; the absence of central structures coincides with a reduction in intensity of band 2. In the latter two abnormal conditions, band 1 has an increased intensity. The data are tentatively interpreted by attributing the polypeptides forming bands 3 and 4 to the arm structure, whereas band 2 is supposed to contain a mixture of polypeptides localized in the arms and in the central structures; these abnormal sperm contain modified polypeptides which gather in band 1. Histochemical ATPase stainings indicate that this enzyme is localized mainly in the doublet arms and, to a minor extent, in the central structures.     

Effect of adrenaline, glucagon and insulin on glucose metabolism in carp: influence of temperature]

In carps living since 4 months at 6, 20 or 30 degrees C, epinephrine or glucagon injections produce increase in plasma glucose but affect only slightly liver glycogen: lower is the temperature, slower and longer are the effect. Insulin injection induces more or less delayed hypoglycaemia according to temperature acclimatization; decrease in blood glucose is accompanied by a slight increase of glycogen in all tissues at 6 degrees C and on the contrary by a very strong depletion of this polysaccharide in liver and even heart at 20 and 30 degrees C.