Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Die Bedeutung der RBW für die Strahlentherapie

Ohne ZusammenfassungDie vollständige Fassung des Vortrages erscheint im Kongreßband-Sonderheft der Strahlentherapie, Verlag Urban & Schwarzenberg, München-Berlin.     

β-1,3-Glucanase is highly-expressed in laticifers of Hevea brasiliensis

Clones encoding -1,3-glucanase have been isolated from a Hevea cDNA library prepared from the latex of Hevea brasiliensis using a probe Nicotiana plumbaginifolia cDNA encoding -1,3-glucanase, gnl. Nucleotide sequence analysis showed that a 1.2 kb Hevea cDNA encoding a basic -1,3-glucanase showed 68% nucleotide homology to gnl cDNA. Northern blot analysis using the Hevea cDNA as probe detected a mRNA of 1.3 kb which was expressed at higher levels in latex than in leaf. In situ hybridization analysis using petiole sections from Hevea localized the -1,3-glucanase mRNA to the laticifer cells. Genomic Southern analysis suggested the presence of a low-copy gene family encoding -1,3-glucanases in H. brasiliensis.     

Phenotypic Differences in Buoyancy and Energetics of Lean and Siscowet Lake Charr in Lake Superior

At least two phenotypes of lake charr, Salvelinus namaycush, coexist in Lake Superior. A lean morph frequents the shallow inshore waters (< 50m) and the fat morph (siscowet) occupies the deeper offshore waters (50–250 m). The objective of this study was to determine if the elevated lipid concentration of siscowets reduces the costs of swimming in deep water. First, we modelled the effects of body composition (lipids) on the costs of swimming by lake charr, and then compared these theoretical results with empirical evidence obtained from Cesium 137-based estimates of food consumption, gross energy conversion, and swimming costs (activity multiplier). The attributes of growth, energy content (kJg^-1), lipid concentrations, and Cesium 137 concentration (Bqg^-1) were obtained from multimesh gillnet catches in eastern Lake Superior (1998 and 1999). The model showed that siscowet (fat) lake charr expended less energy than lean lake charr moving through the water column. Empirical evidence derived from Cesium 137 analysis confirmed that the activity multipliers of siscowets (fat) were less than those for lean charr. These findings support the view that the restoration of the fish community of the predominately deep water of the Great Lakes might be facilitated by the introduction of the fat phenotype.     

Holes photogeneration in phthalocyanine-doped polysilane

Polysilane polymers are attractive photoconductive materials, due to the high mobility of the charge carriers (holes). The photoconductivity in the visible region is greatly enhanced by the addition of a phthalocyanine, because the dopant increases the absorption of light and the quantum yield of the holes photogeneration. The quantum yield has been measured by the technique of xerographic discharge at several values of the polarisation field. From these data the intrinsic quantum yield ₀ and the thermalisation distance r ₀ were calculated. r ₀ is similar to the value measured in the trinitrofluorenone doped polyvinylcarbazole system (27.5 ) while ₀ is much lower (2.8·10^–2 compared to 0.11). In fact, r ₀ is assumed to be mainly dependent on the polymeric environment, while ₀ depends on the nature of the coupling between the dopant and the polymer.     

Aspects of Reproductive Biology of the Scalloped Hammerhead Shark, Sphyrna Lewini, off Northeastern Brazil

Ninety four scalloped hammerhead sharks, Sphyrna lewini (53 females and 41 males) ranging in size from 121 to 321cm total length (TL), were collected from surface gillnetters operating off northeastern Brazil and throughout the southwestern equatorial Atlantic Ocean between January and December 1996. A common regression for TL and eviscerated weight (EW) was calculated as, logEW = –11.786 + 2.889 logTL. Females and males were categorised into reproductive stages (4 and 2, respectively) according to morphological changes in their gonads. Size at sexual maturity for females was estimated to be 240cm, while males appeared to mature at between 180 and 200cm. Gravid females had between 2 and 21 embryos or pups, varying in TL from 3 to 38cm. There was no relationship between maternal length and size of litter. Copulation and parturition appear to occur outside the sampled area and possibly closer to the coast. With the exception of slightly lower uterine and ovarian fecundities, the results support the few existing data on the reproductive cycle of S. lewini in other areas.     

The de novo origin of the quiescent center regenerating root apices of Zea mays

Summary In roots from which the root cap and quiescent center have been removed new apical tissues regenerated in line with the main axis of the root. Regeneration of these tissues occurred from the region of the proximal meristem, which extends for no more than 350 m from the cut surface. Accompanying the regeneration of new apical tissues is a change in the architecture of the root apex and initiation and enlargement of a new quiescent center. A possible role for the quiescent center in the establishment of pattern at the apex is considered. Regeneration of the original apex failed to occur in those roots from which the root cap, quiescent center and proximal meristem were excised.Abbreviation QC quiescent center     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

Experiments on wavelength specific behavior ofPieris brassicae L. During drumming and egg-laying

Summary Female cabbage white butterflies, under constant laboratory conditions exhibit wavelength specific behavior in choosing sites for egg-laying and drumming.In preliminary spontaneous choice experiments with colored PVC-sheets (Table 1, results) butterflies gave somewhat different preference order than with monochromatic lights of different wavelength but equal relative quantum flux (Table 2A, B). These differences probably depended on the relative number of quanta and the spectral composition of the light reflected by the sheets.White light is ignored by the butterflies during both drumming and egg-laying, up to intensities 60 times that of the monochromatic light stimulus.The functional categories egg-laying and drumming are both associated with the spectral region from 497 nm to ca. 585 nm (Fig. 10). The functional category feeding reaction is associated with regions adjoining that for egg-laying and drumming on both the shorter-wavelength side (<497nm) and the longer-wavelength side (>578nm).Wavelength specific behavior and the significance of odor substances in the chain of stimuli that lead up to egg-laying are discussed.Respectfully dedicated to Prof. H. Autrum on the occasion of his 75th birthdaySupported by the Deutsche Forschungsgemeinschaft     

Elevated temperature and chemical modification selectively abolishes levan forming activity of levansucrase of Zymomonas mobilis

A levansucrase (SacB) of Zymomonas mobilis was purified to electrophoretic homogeneity from a recombinant Escherichia coli. The 55 kDa enzyme hydrolysed -fructosides but not -glucosides and catalysed levan formation from sucrose as well as raffinose. The optimum temperature for polymerase activity (30°C ) was lower than that for hydrolase activity (50°C ). In contrast to other levansucrases, polymerase activity of levansucrase was inhibited by para- chloromercuribenzoate (1 mM) but with little or no effect on hydrolase activity. Selective modulation of polymerase activity by this inhibitor will be useful in revealing the mechanism of levansucrase catalysis.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Computational constraints that may have favoured the lamination of sensory cortex

At the transition from early reptilian ancestors to primordial mammals, the areas of sensory cortex that process topographic modalities acquire the laminar structure of isocortex. A prominent step in lamination is granulation, whereby the formerly unique principal layer of pyramidal cells is split by the insertion of a new layer of excitatory, but intrinsic, granule cells, layer IV. I consider the hypothesis that granulation, and the differentiation between supra- and infra-granular pyramidal layers, may be advantageous to support fine topography in their sensory maps. Fine topography implies a generic distinction between where information, explicitly mapped on the cortical sheet, and what information, represented in a distributed fashion as a distinct firing pattern across neurons. These patterns can be stored on recurrent collaterals in the cortex, and such memory can help substantially in the analysis of current sensory input. The simulation of a simplified network model demonstrates that a non-laminated patch of cortex must compromise between transmitting where information or retrieving what information. The simulation of a modified model including differentiation of a granular layer shows a modest but significant quantitative advantage, expressed as a less severe trade-off between what and where. The further connectivity differentiation between infra-granular and supra-granular pyramidal layers is shown to match the mix of what and where information optimal for their respective target structures.     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Elimination of the disulphide bond alters the conformation of mature lipo-β-lactamase in yeast

Summary When expressed in Saccharomyces cerevisiae the precursor of the hybrid prokaryotic protein lipo--lactamase is accumulated in reduced form whereas the majority of the mature form contains an intra molecular disulphide bond (oxidized form). We have previously shown that mature mutant lipo--lactamases in which the cysteine residue 131 was changed to either tyrosine or threonine lack the capacity to form a disulphide bond but are nevertheless processed and secreted efficiently in yeast. Here we show that these mutant mature lipo--lactamases, in yeast cell extracts, exhibit a tenfold lower -lactamase-specific activity than the wild-type protein and that the mature mutant proteins are more susceptible to trypsin digestion. Thus, elimination of the disulphide bond alters the conformation of mature lipo--lactamase in yeast.Correspondence to: O. Pines     

Ca2+-antagonists inhibit the N-methyltransferase‐dependent synthesis of phosphatidylcholine in the heart

Evidence indicates that, in addition to the Ltype Ca²⁺ channel blockade, Ca²⁺antagonists target other functions including the Ca²⁺pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca²⁺pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca²⁺transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10^–6–10^–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10^–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10^–5 M) was associated with a depression of the V_max value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [³H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca²⁺pump and although diltiazem also inhibited the SR Ca²⁺pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca²⁺-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca²⁺ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca²⁺pumps and altered cardiac function.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

β-Mannoside mannohydrolase and the mobilization of the endosperm cell wall of lettuce seeds,cv. Grand Rapids

Mobilization of the endosperm cell-wall reserves of Lactuca sativa L. cv. Grand Rapids requires endo--mannanase and -galactosidase activity. A third enzyme, -mannoside mannohydrolase (EC 3.2.1.25) is also involved. We have determined the optimum extraction and assay conditions for this enzyme, which is soluble only in high-salt (1 M NaCl) buffer. It is located exclusively within the cotyledons, in association with a cellulosic cell-wall fraction. Its substrates are the products of endosperm cell-wall mobilization, mannobiose and mannotriose, which diffuse to the cotyledons and are hydrolyzed extracellularly by the -mannoside mannohydrolase.Abbreviation PNPM p-nitrophenyl--mannopyranoside We dedicate this paper to the memory of our friend and colleague Dr. Jacob D. Duerksen     

The RF-cinematogram

We present a spike-triggered averaging method capable of mapping the visual receptive fields of several neurons simultaneously. The stimulation is general and the mapping proceeds automatically without the need to match the stimulation to the cells' preference for position, orientation, direction, etc. The maps are spatiotemporal; receptive field (RF) structures are quantitatively determined in three dimensions: the two dimensions of visuotopic space, and time. The method presented is one of a family of reverse correlation or spike-triggered averaging techniques (DeBoer and Kuyper 1968) capable of revealing linear aspects of stimulus-response coupling. The formal relationship of these methods to stimulus-response crosscorrelation is shown. The analysis is extended to provide some second-order axis-of-motion information (direction marks). The stimulus is a constantly illuminated, randomly jumping bright or dark spot, not an elongated bar. Spot diameters between one-third to 1 × RF width are effective. The method ascertains for each recorded action potential or spike the prior visual field position of the spot. The average or most probable spot positions define the receptive field spatially. Repeating the process for a succession of times prior to observed spikes defines the field temporally, presented here as a succession of spatial maps. We term this portrayal a receptive field cinematogram, RFc or ciné. The RFc reveals and economically portrays the spread of excitability and suppression across the receptive field, culminating in the generation of a spike. RFcs for LGN neurons and for simple cells recorded in cat cortical areas 17 and 18 are presented and interpreted in terms of classic ON/OFF regions. The availability of temporal information permits the separation of an excitatory exit response, generated when a moving bright spot leaves an OFF region, from an excitatory entrance response occurring when a bright spot enters an ON region, because these responses occur at different times (exit responses earlier). Spike emission remains coupled to (cross-correlated with) stimulus events over time periods as long as 96 ms, implying that some stimulus drive or afferent visual input is delayed by as much as 96 ms more than other input. This is a striking instance of temporal dispersion in the visual system. In some cells, said to be spatiotemporally inseparable, the delay (latency) varies systematically across the visual field; i.e., the place for optimal stimulation varies with the time prior to spike emission. In these cells, the RFc shows receptive field structures which move across the visual field over trajectories equal to approximately twice the total conventional RF width. Exit and entrance responses, on the other hand, arise in a simple way from separated ON and OFF RF subregions. ON/ OFF mechanisms thus appear unrelated to spatiotemporal inseparability. The RFc method is easily automated, efficient, and characterizes multiple RFs simultaneously, as required in work with multiple electrode arrays.     

Sequence analysis of a small early chorion gene subfamily interspersed within the late gene locus in Bombyx mori

A comprehensive sequence analysis of three early chorion genes (6F6.1, 6F6.2, 6F6.3) which form a small subfamily is presented. Two main features characterize this subfamily: (1) the 6F6 gene copies are -branch genes and, unlike typical chorion genes which are organized in divergent gene pairs, they are unpaired, and (2) they are not clustered in genetic locus Ch3 but are dispersed in Ch1-2, which is about 3 to 4 centiMorgans away and contains middle and late chorion genes. Sequence comparisons show that members of this subfamily exhibit high identity values in their major coding region (94–96%) and that similarities also extend, but to a lesser degree, into their noncoding regions. The putative 6F6 promoter regions have no significant similarities with the corresponding regions of other early -genes but quite surprisingly share common elements with middle and late genes. The main difference among the 6F6 gene introns is the presence of inserted sequences: the insert into 6F6.2 (IR; 248 bp) is flanked by a 102–103-bp inverted repeat, while those into 6F6.1 (FIB; 184 bp) and 6F6.3 (HOPE; 951 bp) are carried by a partial Bm1 element. HOPE has features of a non-LTR retrotransposable element. Preliminary experiments indicate that the copy number of IR and HOPE in the Bombyx mori genome is about 5,000 and 20,000, respectively. The great similarity of 6F6 genes cannot be accounted for by selective pressure but rather appears to be the result of gene-conversion-like events, which are supposed to operate frequently in middle and late chorion genes but not in other known early -genes. Using the relative position and orientation of the 6F6 gene copies, it is possible to propose an evolutionary scheme for the formation of chorion locus Ch1-2. Correspondence to: G.C. Rodakis