Amyloid Beta Peptide Membrane Perturbation Is the Basis for its Biological Effects

Experimental studies have indicated that the mechanisms offered for explaining the neurotoxicity of amyloid beta peptide (AP) are diverse, and include altered enzyme activities, disrupted calcium homeostasis, and increased free radical formation. AP appears to interact at the cell membrane with a multitude of receptor sites and also inserts physically into the membrane matrix. This membrane insertion affects the membrane fluidity and potentially influences the function of resident membrane proteins. We propose a unifying hypothesis to explain the experimental observations of the diverse cellular responses to AP. The indiscriminate physical insertion of AP into the cell membrane unspecifically activates a host of membrane processes by perturbation of the membrane proteins. This recurrent activation of membrane processes eventually culminates in neuronal cell death. We recommend that successful therapeutic interventions should be directed at reducing or preventing the interaction of AP with neuronal cell membranes.     

Alzheimer’s Disease: Soluble Oligomeric Aβ(1–40) Peptide in Membrane Mimic Environment from Solution NMR and Circular Dichroism Studies

Amyloid beta peptide (A) is a small peptide present in normal cells and aggregated A is the main constituent of the extracellular amyloid plaques found in Alzheimers disease (AD) brain. Recent studies suggest that soluble A oligomers are neurotoxic rather than amyloid fibrils found in amyloid plaques. This study using multidimensional NMR spectroscopy and circular dichroism (CD) provides the first direct evidence that alterations in membrane structure can trigger the conversion of soluble -helical monomeric A into oligomeric A in a -sheet conformation.     

Field experiments on the optical orientation of pelagic rotifers

Avoidance of shore by pelagic rotifers is considered to be the result of an optical orientation. Field experiments show that the spatial light distribution in the shore region determines the preferred direction of migration. The behaviour of Eudiaptomus gracilis was tested in comparison to that of rotifers.This publication is dedicated to Pater Dr. Josef Donner on the occasion of his 70th birthday     

Isolation of a raw starch-binding fragment from barley alpha-amylase

Barley -amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with -cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel -sheets in domain C and the ₇-and ₈-helices of the (/)₈ domain.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

Unexpected Transformations of Natural Chlorins under Treatment with Dichlorodicyanobenzoquinone

The treatment of natural chlorins with 2,3-dichloro-5,6-dicyanobenzoquinone resulted not only in the intramolecular cyclization of the propionic acid residue in position 17 with the formation of an additional -lactone cycle at the pyrrole ring D, but also in the oxygen-assisted oxidation of 8-ethyl group in ring B to an -methoxyethyl substituent.     

Allomonal defence secretions of the American dog tick Dermacentor variabilis (Acari: Ixodidae) promote clustering

The release of the defence secretion from the large wax glands (sensilla sagittiformia) of Dermacentor variabilis ticks modifies the behaviour of other ticks by inducing clustering. A coating of natural tick secretion onto test objects (delipidized ticks, glass beads or filter paper discs) also elicits a clustering response, but a topically applied squalene, its major component, does not have this effect. The clustering response appears to be species specific: D. variabilis cluster on conspecific ticks that secreted but they fail to cluster on Amblyomma americanum or Ixodes scapularis ticks unless coated with secretions from D. variabilis. Volatile components in the defence secretion are involved in recruiting conspecific ticks to those that secreted. When attacked by predatory fire ants, Solenopsis invicta, D. variabilis clustered on individuals that had released the defence secretion. This suggests that the secretion protects ticks from predation by functioning as an alarm pheromone. If confirmed, this is the first report of an alarm pheromone and its glandular source in ticks. The terminology applicable to the integumental glands of ticks is discussed.     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

Amelanchier alnifolia vegetation in eastern Idaho,USA and its environmental relationships

Fifteen shrub stands of Amelanchier alnifolia vegetation were studied using Braun-Blanquet procedures, tabulated, and ordered by Twinspan, Decorana, and field experience into 5 groups of stands. The stands occurred on the lower slopes of the southwestern Teton and southeastern Big Hole mountains of eastern Idaho and in-and outside a U.S. Forest Service exclosure above the Hoback River in adjacent Wyoming. The ecology of this Amelanchier vegetation as a whole and of the 5 groups of stands is discussed using Jenny's factors of soil formation (1941, 1958, 1980) as a framework. Plant indicator values (Ellenberg 1979, 1988) which rank species on their occurrences in relation to light, temperature, continentality of climate, water availability, soil reaction (pH), and soil nitrogen supply, were independently assigned to the 121 species of vascular plants encountered in the described stands.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

Chromosome and isoenzyme studies on cells derived from protoplast fusion of Nicotiana glauca with glycine max-Nicotiana glauca cell hybrids

Summary The somatic hybrids of Glycine max (L)Merr.-Nicotiana glauca Grah. exhibited a preferential loss of N. glauca chromosomes. When protoplasts from such hybrid cells were back fused twice to N. glauca protoplasts, a considerable increase in stability of the N. glauca chromosomes was observed. Gel electrophoresis studies of aspartate aminotransferase showed that the chromosome(s) responsible for this enzyme was stabilized in the back fused hybrid cell lines. The data suggest that the back fusion technique described in this study might aid in stabilizing somatic hybrids.NRCC No. 18040     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

Pod development of groundnut (Arachis hypogaea L.) in solution culture

Normal pods (containing seed) of groundnut (Arachis hypogaea L.) (cv. TMV-2) were successfully raised in darkened, aerated, nutrient solution, but not in the light. The onset of podding was evident 7 to 8 d after gynophores were submerged in the darkened nutrient solution. An examination of pods and submerged portions of gynophore surfaces by scanning electron microscopy showed the presence of two distinctly different protuberances: unicellular root-hair-like structures that first developed from epidermal cells of the gynophores and developing pods; and branched septate hairs that developed later from cells below the epidermal layer. The septate hairs became visible only after the epidermal and associated unicellular structures had been shed by the expanding gynophore and pods. Omission of Mn and Mg from the podding environment increased pod and seed weight, whilst omission of Zn reduced pod and seed weight.     

The origin of crop weed communities composed of summer annuals

Summary Evidence for the origin and dating of crop weed communities composed of summer annuals is to be found in palaeobotanical and archaeological data. There is archaeological evidence that around the middle of the first millennium BC fields were taken into permanent cultivation, even on the poorer soils.Fertilization with lime and manure and Plaggenwirtschaft were evidently involved in this early agriculture. In this paper the idea is put forward that Plaggenwirtschaft resulted in a sudden increase in the number of weed species brought together in cornfields and that this practise led to the very first beginning of our modern weed communities.Translated into English by Drs. C. van Driel-Murray.     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

The study, addressed to understand whether or not human platelets possess a unique thiol-oxidase whose activity could be modulated by signalling pathway initiated upon the activation of Receptor-C_k revealed the existence of disulphide-dependent oxidation within these cells and this phenomenon was regulated by Receptor-C_k-dependent generation of second messengers especially phosphatidic acid (PA); cAMP and cGMP. Purification of this activity revealed the existence of 47 kDa protein having thiol-oxidase activity. Keeping in view these results we propose that the existence of this novel 47 kDa Thiol-oxidase within human platelets may provide a crucial switch for the regulation of Receptor-C_k-dependent mevalonate pathway in human platelets.     

Mictic patterns of the rotifer Brachionus plicatilis Müller in small ponds

Populations of the rotifer Brachionus plicatilis were monitored in three small ponds in a marsh on the Mediterranean coast. Samples were taken approximately every three weeks from July 1992 to November 1993. Salinity, temperature, conductivity, pH and oxygen concentration were measured in the field. Population density was determined from preserved quantitative samples. Individuals were classified as mictic females, amictic females, non-ovigerous females, and males, differentiating between two morphotypes (S and L). From these counts, a level of mixis was calculated. We also determined the proportion of mictic females in natural populations by culturing females isolated from fresh samples. From these data, mictic patterns over time and correlation between levels of mixis and environmental and population parameters were analyzed. From a previous study S and L morphotypes were known to correspond to genetically different clonal groups. Our data showed that reproduction was predominantly parthenogenetic in these clonal groups, but mictic females were found in most samples, the proportion of mictic females ranging from 0 to 29%. The clonal groups showed different patterns of mixis. L clonal group presented a continuous sexual reproductive pattern. In contrast, S clones showed a rather punctuated mictic pattern. A positive correlation between levels of sexual reproduction and population density was found for S and L groups. However, they differed in their density threshold for mictic reproduction. The adaptive meaning of these patterns and their implications in maintaining genetic diversity within and between populations are discussed.     

Sun-compass orientation in the characidCheirodon pulcher

Synopsis Small microcharacidsCheirodon pulcher were netted during the dry season in pools of the savanna llanos at a considerable distance from the main river channel. Fish could have moved from the river to these pools using sun-compass orientation. Orientation responses of these fish were tested in a four-armed experimental device, exposed to direct sunlight or to azimuth-inverted sunlight reflected by a mirror. Groups of 20 fish were placed in a central area, released and then counted in the arm traps. When exposed to direct sunlight, fish oriented south, both in the morning and afternoon. Nevertheless, when the sun azimuth was inverted they oriented north, indicating that they use a sun-compass and that they compensate for sun-azimuth inversion at noon in a way that geographic headings remain constant throughout the day. These results reinforce previous laboratory interpretations which suggested that these flood plain species migrate inland during the high-water period and return to the main river channels before the dry season begins.     

Induction of Somatic Embryogenesis and Plant Regeneration of ‘Ataulfo’ Mango (Mangifera indica)

Nucellar explants from the immature fruit of Ataulfo mango, 3–4 cm long, were used. The process had a very low efficiency (<10%); however, Ataulfo was more embryogenic than other cultivars such as Tommy Atkins or Haden in our hands. Overall, the process from nucellus to plantlet stage took 5 months. Twelve regenerated plants were obtained and are currently growing in the greenhouse. They appear to be normal and free of abnormalities. Some biochemical and molecular tests are currently being carried out.