Trace metal contamination initiates the apparent auto-aggregation,amyloidosis, and oligomerization of Alzheimer’s Aβ peptides

Nucleation-dependent protein aggregation (seeding) and amyloid fibril-free formation of soluble SDS-resistant oligomers (oligomerization) by hydrophobic interaction is an in vitro model thought to propagate -amyloid (A) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimers disease (AD), and other amyloid-associated neurodegenerative diseases. However, A is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical A deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (0.8 µM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic A1–42-mediated seeding process and A oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of A1–40 (20 µM) initiated by A1–42 (2 µM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble A oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating A amyloidosis and oligomerization.     

1H NMR studies of the mercuric ion binding protein MerP: Sequential assignment,secondary structure and global fold of oxidized MerP

Summary The oxidized form of the mercuric ion binding protein MerP has been studied by two-dimensional NMR. MerP, which is a periplasmic water-soluble protein with 72 amino acids, is involved in the detoxification of mercuric ions in bacteria with resistance against mercury. The mercuric ions in the periplasmic space are first scavenged by the MerP protein, then transported into the cytoplasm by the membrane-bound transport protein MerT, and finally reduced to elementary (nontoxic) mercury by the enzyme mercuric reductase. In this work, the ¹H NMR spectrum of oxidized MerP (closed disulfide bridge) has been assigned by using homonuclear 2D NMR techniques. The secondary structure and global fold have been inferred from the nuclear Overhauser effect (NOE) data. The secondary structure comprises four -strands and two -helices, in the order ₁₁₂₃₂₄. The protein folds into an antiparallel -sheet, ₂₃₁₄, with the two antiparallel helices on one side of the sheet. The folding topology is similar to that of acylphosphatase, the activation domain of porcine pancreatic procarboxypeptidase B, the DNA-binding domain of bovine papillomavirus-1 E2 and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins. However, there is no structural similarity between MerP and other bacterial periplasmic binding proteins.     

Improving Feedback Merging for Source-Adaptive Layered Multicast Schemes

Current source-adaptive layered multicast schemes exploit merging capabilities at special nodes in the network to combine feedback from the multicast group. The merging procedures reduce network load and avoid feedback implosion at the source. In this paper, we demonstrate how to increase the efficiency of a merger node. We show how to tune two mechanisms: feedback transmission, on the timer settings used by receivers, and temporal merging, on the timer settings used by intermediate nodes. Our approach is generic, relating purely to timer settings, and does not touch on content merging, or the decision regarding which packets to forward. We investigate the effects of temporal merging on packet suppression. We show that periodic feedback transmission coupled with a suitable timer setting at the merger node increases efficiency.     

Effects of brassinosteroids on conditioning and germination of clover broomrape (Orobanche minor) seeds

Brassinolide [2, 3, 22R,23R-tetrahydroxy-24S-methyl-B-homo-7-oxa-5--cholestan-6-one] and its related compounds, brassinosteroids, applied at the early stages of conditioning shortened the conditioning period required before clover broomrape seeds would germinate after exposure to germination stimulants, such as dl-strigol and natural stimulants from red clover (Trifolium pratense L.) root exudate. Brassinosteroids applied after conditioning increased the rate of the seed germination induced by these stimulants. The inhibitory effect of light on seed germination when it was induced by dl-strigol could be overcome by brassinosteroids. Brassinosteroids also eliminated the inhibitory effects of light and dl-strigol applied at the early stages of conditioning. GA₃ was also effective in causing seed conditioning and increased the rate of the germination induced by these stimulants. There was a relationship between brassinosteroids and GA₃ in many of the experiments conducted. These findings may have practical implications in increasing the effectiveness of applying germination stimulants in the field to soils for suicidal germination of broomrape seeds.     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis     

Do neurons have a voltage or a current threshold for action potential initiation?

The majority of neural network models consider the output of single neurons to be a continuous, positive, and saturating firing ratef(t), while a minority treat neuronal output as a series of delta pulses (t — t _i ). We here argue that the issue of the proper output representation relates to the biophysics of the cells in question and, in particular, to whether initiation of somatic action potentials occurs when a certain thresholdvoltage or a thresholdcurrent is exceeded. We approach this issue using numerical simulations of the electrical behavior of a layer 5 pyramidal cell from cat visual cortex. The dendritic tree is passive while the cell body includes eight voltage- and calcium-dependent membrane conductances.We compute both the steady-state (I ^static (V _m )) and the instantaneous (I _o (V_m)) I–V relationships and argue that the amplitude of the local maximum inI ^static (V _m ) corresponds to the current thresholdI _th for sustained inputs, while the location of the middle zero-crossing ofI _o corresponds to a fixed voltage thresholdV _th for rapid inputs. We confirm this using numerical simulations: for rapid synaptic inputs, spikes are initiated if the somatic potential exceedsV _th, while for slowly varying inputI _th must be exceeded. Due to the presence of the large dendritic tree, no charge thresholdQ _th exists for physiological input.Introducing the temporal average of the somatic membrane potential (V _m) while the cell is spiking repetitively, allows us to define a dynamic I-V relationship ^dynamic ((V _m)). We find an exponential relationship between (V _m) and the net current sunk by the somatic membrane during spiking (diode-like behavior). The slope ofI/dynamic((V _m)) allows us to define a dynamic input conductance and a time constant that characterizes how rapidly the cell changes its output firing frequency in response to a change in its input.     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Biomass protein formation by filamentous fungi on woody substrates

Summary Growth and biomass protein formation by filamentous fungi grown on pretreated tropical woods of Mesta (Hibiscus cannabinus Linn.) and Subabul [Leucaena leucocephala (Lam.) de Witt] as well as their isolated hemicellulose and cellulose fractions have been studied. Penicillium janthinellum and Penicillium funiculosum produced a biomass having 20 to 30% crude protein when grown on either hemicellulose, while growth on pretreated (autoclaved in 1% NaOH) wood or isolated cellulose fractions was comparatively poor and crude protein content only 5 to 8% in the biomass.NCL Communication no.3550     

A morphometric analysis of the subcellular distribution of LHβ and FSHβ in secretory granules in the pituitary gonadotrophs of the frog (Rana japonica)

Immunohistochemical localization of lutropin (LH) and follitropin (FSH) in the pituitary gland of the frog Rana japonica was studied by the peroxidase-anti-peroxidase method and the two-face, double-labeling method with different-sized gold particles at the light-and electron-microscopic levels, respectively, using monoclonal antibodies against bullfrog LH and FSH. Light-microscopic immunohistochemistry indicated that approximately 66.0% of all the gonadotrophs in the pituitary contained both LH and FSH, whereas 33.4% of gonadotrophs contained only LH, and 0.6% contained only FSH. The staining intensity of LH and FSH varied from cell to cell. The gonadotrophs were classified into four types (Types I–IV) in terms of their ultrastructural and immunolabeling characteristics. Moreover, several secretory granule types were recognized according to differences in their shape and electron density. In all the cell types, both LH and FSH were often seen in the same secretory granules, but the proportion of granules bearing both hormones ranged from 5.5% in Type I to 32.7% in Type IV. Most secretory granules in Types I and II were immunolabeled with LH alone, whereas a small number of granules were immunolabeled with FSH alone. More immunolabeled FSH granules were present in Types III and IV than in Types I and II.     

Medicarpin and maackiain 3-O-glucoside-6′-O-malonate conjugates are constitutive compounds in chickpea (Cicer arietinum L.) cell cultures

Summary The pterocarpan phytoalexin conjugates medicarpin 3-O-glucoside-6-O-malonate and maackiain 3-O-glucoside-6-O-malonate were isolated from cell suspension cultures of chickpea (Cicer arietinum L.) cultivar ILC 3279 and structurally elucidated. Both pterocarpan conjugates are constitutive metabolites of the chickpea cell cultures. Upon application of an elicitor from yeast to the cell cultures a substantial increase in the level of the phytoalexin aglycones medicarpin and maackiain was observed although a delayed but significantly higher rise of the conjugates also occurred. The significance of the pterocarpan conjugates for phytoalexin production is discussed.Abbreviations MeGM medicarpin 3-O-glucoside-6-O-malonate - MaGM maackiain 3-O-glucoside-6-O-malonate - MeG medicarpin 3-O-glucoside - MaG maackiain 3-O-glucoside - FGM formononetin 7-O-glucoside-6-O-malonate - BGM biochanin A 7-O-glucoside-6-O-malonate - IFR NADPH: 2-hydroxyisoflavone oxidoreductase - PTS pterocarpan synthase - IGT UDP-glucose: isoflavone 7-O-glucosyltransferase - IMT malonyl-coA: isoflavone 7-O-glucoside-6 -O-malonyltransferase - RT retention time - sh shoulder - d duplette - m multiplette - s singulette     

Studies on aneuploids of Petunia

Summary The seven possible primary trisomics of Petunia (2 n= 14) located in the progenies of triploid, hypertriploid and hypotriploid plants were distinguished from one another and from diploid on the basis of cytological and morphological criteria. They were provisionally named as Oval, Semi, Slender, Pseudonormal, Arrow, Narrow and Giant. In three of the trisomics, the extra chromosome was identified for the first time at pachytene stage. Postpachytene studies revealed no precise relationship between the length of extra chromosome and the frequency of multiple association.     

Selective staining of Y chromosomal loops in Drosophila hydei,D. neohydei,and D. eohydei

Modified Giemsa procedures have been developed which elicit differential and highly selective staining of individual Y chromosomal lamp-brush loops in spermatocyte nuclei of Drosophila hydei, D. neohydei, and D. eohydei. In all three species the Y loop pair known as the clubs stains a brilliant dark red with Giemsa at pH 10. With the same treatment other loop pairs either remain unstained, e.g. the threads, or show a differentiation between light blue and pink staining matrical material, e.g. pseudonucleolus and cones in D. hydei and D. eohydei. With eosin at pH 2.8 the threads in D. hydei can be stained intensely, as well as one matrical component of the pseudonucleolus. Pretreatment with RNase or TCA removes all stainability from the Y loops with Giemsa at pH 10. TCA treatment enhances eosin staining at pH 2.8. These and other variations of Giemsa may be utilized to establish homologies between Y loops in different species. The molecular basis of the staining reactions remains to be elucidated.     

Über die Teilungsfolge in den Fäden von zwei Grünalgen

Zusammenfassung In den Fäden der GrünalgenMicrospora amoena undSpirogyra crassa wechseln geschlossene Abschnitte von eigentlich ruhenden Zellen mit Abschnitten von präprophasischen, das sind Mitose-bereiten, mitotisch aktiven und posttelophasischen, also unmittelbar nach der Mitose stehenden Zellen ab. Nur ausnahmsweise schieben sich Zellen des einen Typus in Abschnitte von Zellen des anderen Typus ein.Dieses Verhalten wird hypothetisch mit dem Auftreten und der Weiterleitung teilungsinduzierender Hormone erkärt. Daß die Teilungsstadien einschließlich der vor- und nachmitotischen Stadien nicht entsprechend einem Gefälle streng geordnet aufeinanderfolgen, beruht wahrscheinlich auf der Verschiedenartigkeit der äußeren und inneren Faktoren, die auf die einzelnen Zellen einwirken. Genealogische Faktoren spielen offenbar eine untergeordnete Rolle.     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

Interpretation of metameric architecture in dominant shrubs of the Chilean matorral

Summary The seasonal progression of phenophases in 21 shrub species of the Chilean matorral was analyzed. Five modules or basic units that are responsible for the aboveground architecture of the plants were characterized. These modules appear to be organized in seven different spatial arrangements. In drought-deciduous species a module type with an absolute short shoot with limited apical growth, leafy or spiny, predominated. In evergreen species long shoot and temporal leafy short shoot module types were more frequent. The spatial arrangement of morphologically different modules and the temporal sequence of their formation allow a dynamic interpretation of the modular architecture of the plants.     

Contextualizing Images in Dreams and Daydreams

A contextualizing image (CI) is a powerful central image of a dream which appears to contextualize (provide a picture-context for) the dreamer's emotion. For instance, dreamers who have experienced any serious traumatic event sometimes dream, I was overwhelmed by a tidal wave. This appears to picture their feeling of terror and/or vulnerability.A scoring system for CIs is examined here and is applied to dreams and daydreams supplied by 40 students. Two raters scoring dreams on a blind basis showed good inter-rater reliability. Recent dreams were shown to have more as well as more intense CIs than recent daydreams; likewise, dreams that stand out had more intense CIs than daydreams that stand out. Students with thin boundaries had more and more intense CIs than students with thick boundaries in their recent dreams and nightmare, but not so clearly in dreams and nightmares that stand out. The emotions judged as contextualized by the powerful images tended towards fear/terror and helplessness/vulnerability in dreams (especially in dreams that stand out) whereas emotions contextualized by images in daydreams showed a wide range with no clusters.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Analysis of primary food sources and trophic relationships of aquatic animals in a mangrove-fringed estuary,Khung Krabaen Bay (Thailand) using dual stable isotope techniques

Stable carbon (¹³C) and nitrogen (¹⁵N) isotopes were used to elucidate primary food sources and trophic relationships of organisms in Khung Krabaen Bay and adjacent offshore waters. The three separate sampling sites were mangroves, inner bay and offshore. The ¹³C values of mangrove leaves were –28.2 to –29.4, seagrass –10.5, macroalgae –14.9 to –18.2, plankton –20.0 to –21.8, benthic detritus –15.1 to –26.3, invertebrates –16.5 to –26.0, and fishes –13.4 to –26.3. The ¹⁵N values of mangrove leaves were 4.3 to 5.7, seagrass 4.3, macroalgae 2.2 to 4.4, plankton 5.7 to 6.4 , benthic detritus 5.1 to 5.3, invertebrates 7.2 to 12.2 , and fishes 6.3 to 15.9. The primary producers had distinct ¹³C values. The ¹³C values of animals collected from mangroves were more negative than those of animals collected far from shore. The primary carbon sources that support food webs clearly depended on location. The contribution of mangroves to food webs was confined only to mangroves, but a mixture of macroalgae and plankton was a major carbon source for organisms in the inner bay area. Offshore organisms clearly derived their carbon through the planktonic food web. The ¹⁵N values of consumers were enriched by 3–4 relative to their diets. The ¹⁵N data suggests that some of aquatic animals had capacity to change their feeding habits according to places and availability of foods and as a result, individuals of the same species could be assigned to different trophic levels at different places.