Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Analysis of wheat resistance to the cereal aphidSitobion avenae using electrical penetration graphs and flow charts combined with correspondence analysis

The behaviour ofSitobion avenae (F.), was compared on resistant wheat lines ofTriticum monococcum (L.) and a susceptible variety ofTriticum aestivum (L.). Firstly, stylet penetration activities were monitored with the Electrical Penetration Graph (EPG) technique and subsequently analysed using flow charts combined with correspondence analysis. Plant resistance was shown to be associated with repeated penetrations without access to either the xylem or the phloem, and with numerous failures in starting a sustained sap ingestion (as represented by pattern E2). Access to sieve elements of the phloem did not seem to be much affected on resistant plants but it took the aphid three times as long to produce a sap ingestion pattern when maintained on the resistant lineT. monococcum n^o 44 (Tm44) as compared with aphids maintained on susceptible plants. As a result the total time spent in ingesting from sieve elements was reduced by 72% on Tm44. Secondly, direct observations of freely-moving apterous adults were performed. Aphids did not discriminate between resistant and susceptible wheat during the first 30 min of access to test leaves, but only 4 out of 25 aphids were still probing after eight hours on resistant Tm44. The relevance of these results to possible location of the resistance factor(s) are discussed. Although detection of plant resistance before sieve elements are reached can not be rigorously excluded, the factors involved inT. monococcum resistance toS. avenae undoubtedly occur within the phloem vessels.     

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.     

Use of sequence characterised amplified region and RAPD markers in the identification of the white truffle Tuber magnatum Pico

Isolates of white truffles were identified as Tuber magnatum Pico species using a pair of primers selected from a sequence characterised amplified region (SCAR) and a specific random amplified polymorphic DNA (RAPD) marker. The present study reveals that PCR-fragment-pattern polymorphisms, the construction of probes and couples of primers from one or more of these polymorphic fragments may provide a useful and rapid tool for identifying species of ectomycorrhizal fungi in addition to conventional methods (morphological parameters).     

Amphiphysin II (SH3P9; BIN1), a Member of the Amphiphysin/Rvs Family, Is Concentrated in the Cortical Cytomatrix of Axon Initial Segments and Nodes of Ranvier in Brain and around T Tubules in Skeletal Muscle

Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrin_G), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.     

Genetic differentiation and detection of cryptic species in the genus Lepismachilis (Insecta: Microcoryphia) from the Western Mediterranean region

Different species of the bristletail genus Lepismachilis were collected in 14 localities in Italy and Spain and an allozyme electrophoretic survey was carried out to estimate the degree of genetic variability and differentiation at intra- and interspecific levels. Four morphological species were initially identified (L osellai, L. y-signata, L. affinis, L. targionii), but the electrophoretic analysis demonstrated the presence of two additional species among the individuals of L. targionii (Lepismachilis spl and sp2). The validity of these species and their differentiation from L targionii were demonstrated by the fixation of alternative allelic patterns at several loci (7 in Lepismachilis spl and 8 in Lepismachilis sp2), coupled with fixed, previously undetected, morphological differences. In addition, Lepismachilis sp2 was sympatric with L. targionii in three collecting sites, where the fixation of alternative allelic patterns unequivocally demonstrated reproductive isolation. Genetic variability did not seem to be correlated with local ecological factors, and differences between species should rather be explained by different historical factors. Low levels of gene flow, estimated with two different indirect methods, were observed in L. targionii and L. y-signata, and were due to high levels of structuring among populations. Genetic differentiation among conspecific populations was not correlated to their geographical arrangement and the presence of loci fixed for different alleles among them suggested that stochastic factors (such as genetic drift) may have played a role in determining genetic differentiation of geographically isolated populations. Genetic divergence values indicated that the six species are well differentiated and allozyme profiles were diagnostic for all of them. On the other hand, allozyme data did not provide adequate information to resolve evolutionary relationships among the species, nor did they confirm the validity of the two subgenera (Lepismachilis and Berlesilis) in which the genus Lepismachilis is traditionally divided.     

Fourier harmonic analysis of the sagittal profile of the Plesianthropus transvaalensis (STS5) skull

An analytical morphometrical investigation of fronto facial profile of Plesianthropus transvaalensis was carried out by means of Fourier spectral series. Amplitude of contributors was evaluated in comparison with harmonic sets of a modern skull and two "mosaics" obtained by a convenient merging of segments of modern and hominid skull profiles. Patterns are distinctly different in Plesianthropus and modern profile because amplitude of second harmonic is greater than first in the former and viceversa in the latter; moreover while in Plesianthropus first and second harmonic give the same phase, it does not occur for modern skull. Fourier series of "mosaic" obtained by frontal profile of Plesianthropus and facial profile of modern resembles the set of modern skull while "mosaic" of modern frontal profile and hominid facial one resembles closely pattern of Plesianthropus.     

Changes in the surface morphology of the rabbit endometrium related to the estrous and progestational stages of the reproductive cycle

Summary Changes occurring on the surface of the uterine luminal epithelium of the rabbit during the estrous and progestational stages of the reproductive cycle were examined by scanning and transmission electron microscopy. The findings demonstrate that the uterine epithelium, or endometrium, contains two cell types: ciliated cells and nonciliated, microvillous cells. In estrous animals, ciliated cells, although not very numerous, were usually observed in small groups. However, at increasing intervals of time following mating, ciliated cells progressively disappeared from the endometrium until approximately eight to ten days post coitum, when they became scare. From several hours to four to five days following mating, extensive changes occurred on the surfaces of microvillous cells. When observed by TEM, these elements contained organelles typical of cells involved in the synthesis and secretion of glycoproteins. Furthermore, microvillous cells during this period displayed numerous apical protrusions of different sizes and shapes and containing material of varying electron density. Parallel SEM examinations of the same material confirmed the presence of these protrusions. Some of the protrusions appeared as spheroidal masses attached to the cytoplasm by means of a cytoplasmic strand. Other surface masses were clearly unattached to the cell surface and were distributed (1) on the surface of microvillous cells, (2) on the cilia of adjacent ciliated cells, and (3) on the surface of spermatozoa.Changes occurring on the luminal surface during the early postcoital period are interpreted as an expression of morphodynamic processes likely involving coupled secretion (exocytosis) and resorption (endocytosis) of luminal material. The observations presented here also demonstrate that between six and ten days post coitum, the rabbit endometrium contained increasing numbers of enlarged, nonciliated cells that probably arose by the fusion of smaller, microvillous elements.The work reported here was supported by C.N.R. contracts No. CT 760128809 and CT 77014239 (to P.M.) and NIH. Grant HD-04274 (to J.V.B.)