Role of α2‐macroglobulin in regulating amyloid β‐protein neurotoxicity: protective or detrimental factor?

alpha2-Macroglobulin (alpha2M) has been identified as a carrier protein for beta-amyloid (Abeta) decreasing fibril formation and affecting the neurotoxicity of this peptide. The alpha2-macroglobulin receptor/low density lipoprotein receptor related protein (LRP) is involved in the internalization and degradation of the alpha2M/Abeta complexes and its impairment has been reported to occur in Alzheimer's disease. Previous studies have shown alpha2M to determine an enhancement or a reduction of Abeta toxicity in different culture systems. In order to clarify the role of alpha2M in Abeta neurotoxicity, we challenged human neuroblastoma cell lines with activated alpha2M in combination with Abeta. Our results show that in neuroblastoma cells expressing high levels of LRP, the administration of activated alpha2M protects the cells from Abeta neurotoxicity. Conversely, when this receptor is not present alpha2M determines an increase in Abeta toxicity as evaluated by MTT and TUNEL assays. In LRP-negative cells transfected with the full-length human LRP, the addition of activated alpha2M resulted to be protective against Abeta-induced neurotoxicity. By means of recombinant proteins we ascribed the neurotoxic activity of alpha2M to its FP3 fragment which has been previously shown to bind and neutralize transforming growth factor-beta. These studies provide evidence for both a neuroprotective and neurotoxic role of alpha2M regulated by the expression of its receptor LRP.     

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna, erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC₅₀s) of 2.8 · 10⁻¹¹ mol · l⁻¹, 7.0 · 10⁻⁷ mol · l⁻¹ and 3.3 · 10⁻⁷ mol · l⁻¹, respectively. KT5720 (10⁻⁷ mol · l⁻¹ and 10⁻⁶ mol · l⁻¹) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10⁻⁴ mol · l⁻¹ of adenosine and adenosine triphosphate (ATP), and up to 10⁻³ mol · l⁻¹ of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10⁻⁷ mol · l⁻¹) and forskolin (10⁻⁶ mol · l⁻¹), but were not affected by PCH (10⁻⁷ or 10⁻¹⁰ mol · l⁻¹). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated. Accepted: 11 August 1998     

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genusLoxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.     

Inhibition of nitric oxide synthase expression by a methanolic extract of Crescentia alata and its derived flavonols.

In order to validate the use of Crescentia alata (Bignoniaceae) in the traditional medicine of Guatemala as an antiinflammatory remedy, the methanolic (MeOH) extract has been evaluated in vivo for antiinflammatory activity on carrageenin paw edema in rats and in vitro on Escherichia coli lipopolysaccharide- (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774.A1 macrophage cell line. This extract exerted in vivo a significant anti-inflammatory activity at the highest dose tested. The same extract showed in vitro an inhibitory activity on inducible nitric oxide synthase expression and on NO formation in LPS-primed J774.A1 cells. Subsequent fractionation and analysis of the extract has led to the isolation and characterization as major constituents of two flavonol glycosides: quercetin 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (rutin) 1, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (kaempferol 3-O-rutinoside) 2, and flavonol aglycone, kaempferol 3. Their structures were elucidated by spectral methods. The bioassay-directed analysis of flavonols 1-3 indicated that kaempferol (3) was the most active compound contained in the MeOH extract because it reduced in vitro both NO production and iNOS expression in LPS-primed J774.A1 cells, whereas rutin (1) and kaempferol 3-O-rutinoside (2) showed no significant activity. The MeOH extract and all of flavonoids tested did not show in vitro significant cytotoxic effect in J774.A1 macrophage cell line.     

Interaction of a solitary larval endoparasitoid, Microplitis mediator, with its host, Mamestra brassicae: host acceptance and host suitability

Abstract:  Microplitis mediator (Haliday) (Hym., Braconidae) is an important parasitoid of early instar larvae of the European cabbage moth, Mamestra brassicae L. (Lep., Noctuidae). In the laboratory, we examined attack responses of female M. mediator to the first three larval instars of M. brassicae . Females were presented with M. brassicae larvae either one individual at a time in a no-choice experiment, or three individuals, one from each instar, simultaneously in a choice experiment. Whether or not there was choice, naïve female parasitoids attacked a high proportion of larvae and did not discriminate among instars. In the no-choice experiment, attacked larvae were reared, and parasitoid cocoons were produced from about 76% of larvae attacked as first and second instars, but from only 19% of larvae attacked as third instars. Dissections of attacked larvae from the choice experiment showed that about 79% of attacks on first and second instars resulted in oviposition compared with only 49% for third instars. When given choice, frequency and number of attacks on first instar larvae increased with increasing parasitoid experience. Our results suggest that first and second instar larvae of M. brassicae are suitable hosts for M. mediator , but that third instar larvae are suboptimal both because oviposition attempts were frequently unsuccessful and because immature parasitoids failed to complete development. Nevertheless, naïve attacking parasitoids exhibited minimal discrimination among instars, although experienced parasitoids most frequently attacked first instar larvae. The host selection behaviour of M. mediator is discussed in the context of optimal foraging theory and implications for biological control.     

Observations sur la significativité de la coloration aux Alcians pour les mucopolysaccharides

Resumé Les Auteurs, à la suite d'une serie des récherches sur la coloration au bleu Alcian de la cartilage, on pu démontrer que la signaléticité de ce colorant basique envers les mucopolysaccharides est due à la interférence inhibitive avec une fraction protéique présente en grande quantité dans les structures non-mucopolysaccharidiques ionisées au pH de utilisation (2,4) (DNA, collagéne etc.), fraction bien soluble dans les solutions salines et dans l'acide sulphurique 1 M, mais peu soluble dans l'acide acétique 3%.L'asportation de cette substance à l'aide de dits solvants ou de la digestion à la pepsine, augmente la colorabilité des mucopolysaccharides, mais diminue ou abolit la signaléticité.Sur cette donné, les Auteurs expliquent les differentes anomalies de la méthode aux Alcians.

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Observations about electivity of Alcian staining of mucopolysaccharides

Summary The Authors proved, in the course of a general investigation about the Alcian blue staining of cartilage, that the specificity of this dye is due to its inability of staining in the presence of a proteic fraction, which is present in a greater amount in the non-glycid structures containing ionized groups at 2,4 pH: i.e. collagen, DNA etc.This protein is easily soluble in various salt solutions and in 1 M sulphuric acid, but insoluble in 3% acetic acid. Removal of this protein by these agents or by peptic digestions result in increasing the mucopolysaccharides staining, but in decreasing or suppressing electivity.Differents abnormalities of Alcian method are explained by the Authors in this way.