Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Análisis farmacogenético retrospectivo de una paciente pediátrica en tratamiento anticoagulante: caso clínico

We present the clinical case of a 10-year-old patient diagnosed with dilated cardiomyopathy who registered INR values above 10 upon receiving standard doses of acenocoumarol, as well as other values reported as uncoagulable, forcing the discontinuation and restart of treatment more than once. Expected and stable INR levels were achieved after more than 30 days of treatment, surprisingly with half the recommended dose for a patient of her age and weight. We decided to conduct a retrospective pharmacogenomic analysis including nucleotide genetic polymorphisms (SNPs) with different degrees of association with the dose/response to antivitamin K (AVK) drugs: rs2108622 (gene CYP4F2), rs9923231, rs7294 (gene VKORC1), rs1799853, and rs1057910 (CYP2C9 gene) using TaqMan^® RT-PCR. The patient was homozygous for rs9923231 (VKORC1) and heterozygous for rs2108622 (CYP4F2), a genetic profile strongly associated with a requirement of lower AVK doses as shown by national and international evidence.In conclusion, the pharmacogenetic analysis confirmed that this patient''s genetic conditions, involving low expression of the VKA therapeutic target, required a lower dose than that established in clinical protocols as recommended by the Food and Drug Administration (FDA) and the PharmGKB^® for coumarin drugs. A previous genotypic analysis of the patient would have allowed reaching the therapeutic range sooner, thus avoiding potential bleeding risks. This shows the importance of pharmacogenetic analyses for highly variable treatments with a narrow therapeutic range.     

Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation

Treatment of lung epithelial cells with different kinds of nano-sized particles leads to cell proliferation. Because bigger particles fail to induce this reaction, it is suggested that the special surface properties, due to the extremely small size of these kinds of materials, is the common principle responsible for this specific cell reaction. Here the activation of the protein kinase B (Akt) signaling cascade by carbon nanoparticles was investigated with regard to its relevance for proliferation. Kinetics and dose-response experiments demonstrated that Akt is specifically activated by nanoparticulate carbon particles in rat alveolar type II epithelial cells as well as in human bronchial epithelial cells. This pathway appeared to be dependent on epidermal growth factor receptor and beta(1)-integrins. The activation of Akt by these receptors is known to be a feature of adhesion-dependent signaling. However, intracellular proteins described in this context (focal adhesion kinase pp125(FAK) and integrin-linked kinase) were not activated, indicating a specific signaling mechanism. Inhibitor studies demonstrate that nanoparticle-induced proliferation is mediated by phosphoinositide 3-kinases and Akt. Moreover, overexpression of mutant Akt, as well as pretreatment with an Akt inhibitor, reduced nanoparticle-specific ERK1/2 phosphorylation, which is decisive for nanoparticle-induced proliferation. With this report, we describe the activation of a pathway by carbon nanoparticles that was so far known to be triggered by ligand receptor binding or on cell adhesion to extracellular matrix proteins.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

Galectin-9 recognizes and exhibits antimicrobial activity toward microbes expressing blood group–like antigens

While adaptive immunity recognizes a nearly infinite range of antigenic determinants, immune tolerance renders adaptive immunity vulnerable to microbes decorated in self-like antigens. Recent studies suggest that sugar-binding proteins galectin-4 and galectin-8 bind microbes expressing blood group antigens. However, the binding profile and potential antimicrobial activity of other galectins, particularly galectin-9 (Gal-9), has remained incompletely defined. Here, we demonstrate that while Gal-9 possesses strong binding preference for ABO(H) blood group antigens, each domain exhibits distinct binding patterns, with the C-terminal domain (Gal-9C) exhibiting higher binding to blood group B than the N-terminal domain (Gal-9N). Despite this binding preference, Gal-9 readily killed blood group B–positive Escherichia coli, whereas Gal-9N displayed higher killing activity against this microbe than Gal-9C. Utilization of microarrays populated with blood group O antigens from a diverse array of microbes revealed that Gal-9 can bind various microbial glycans, whereas Gal-9N and Gal-9C displayed distinct and overlapping binding preferences. Flow cytometric examination of intact microbes corroborated the microbial glycan microarray findings, demonstrating that Gal-9, Gal-9N, and Gal-9C also possess the capacity to recognize distinct strains of Providencia alcalifaciens and Klebsiella pneumoniae that express mammalian blood group–like antigens while failing to bind related strains that do not express mammalian-like glycans. In each case of microbial binding, Gal-9, Gal-9N, and Gal-9C induced microbial death. In contrast, while Gal-9, Gal-9N, and Gal-9C engaged red blood cells, each failed to induce hemolysis. These data suggest that Gal-9 recognition of distinct microbial strains may provide antimicrobial activity against molecular mimicry.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

Effects of Cadmium on Aquatic Hyphomycetes

Two kinds of experiments, sporulation and growth experiments, were carried out to demonstrate the effect of cadmium on aquatic hyphomycetes. Oak (Quercus petraea L.) leaves were exposed in a hard-water stream (Lüssel, Swiss Jura) and a soft-water stream (Ibach, Black Forest) for 2 months. In the laboratory, fungal sporulation on the leaves in stream water enriched with cadmium (as CdCl₂) was studied. A measurable effect was found when the cadmium concentration exceeded 0.1 ppm (0.1 mg/liter). Concentrations higher than 100 ppm inhibited conidium production completely. This toxic effect of cadmium was species dependent and much higher in soft water (water with low concentrations of calcium and magnesium) than in hard water. Growth experiments with Alatospora acuminata Ingold, Clavariopsis aquatica De Wildeman, Flagellospora curvula Ingold, Heliscus lugdunensis Saccardo and Therry, and Tetracladium marchalianum De Wildeman showed the same pattern of cadmium sensitivity as that seen in the sporulation experiments. Mycelial growth was less sensitive to cadmium than was fungal sporulation. High concentrations of competing cations (e.g., calcium and zinc) or potential ligands could reduce cadmium toxicity. Calcium content seems to be the most important factor responsible for the different sensitivity of aquatic hyphomycetes in hard and soft water.     

Spherical vs. granular immobilization support selection and performance on an optical flow cell immunosensor based on the fluorescence of Cyanine-5

A spherical porous glass support Trisoperl (TRISO) with four pore diameters (? 47.8; 55.9; 102.6, and 108.8 nm) was characterized and selected for application in an optical flow cell immunosensor, in comparison with controlled pore glass (CPG). The TRISO support was functionalized with aldehyde and isothiocyanate (-NCS) groups to attach bovine serum albumin and alkaline phosphatase (AP). The TRISO isothiocyanate pore diameter 47.8 nm (TRISO(-NCS) 47.8 nm) showed the better potential to be used in the immunosensor. It immobilized more protein (19.3 mg AP per g support) while presenting an optical performance comparable to the CPG. CPG(-NCS) and TRISO(-NCS) 47.8 nm were tested in the immunosensor model where the saturation of the Goat IgG immobilized in the supports with Monoclonal Anti-Goat IgG conjugated with Cyanine-5 was reached, followed by regeneration with the elution buffer modified PBS pH 2.0. The TRISO(-NCS) 47.8 nm presented lower fluorescence intensity at saturation (around 39 AU) than CPG(-NCS) (150 to 104 AU), but revealed a major advantage related to the uniform arrangement of the spherical particles in the flow cell, generating no significant fluorescence differences between gravity and flow package.     

Changes in medium radioactivity and composition accompany high-affinity uptake of glutamate and aspartate by mouse brain slices

In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an infinitely large pool. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well.     

Zur Ethologie von Putzsymbiosen einheimischer Süßwasserfische im natürlichen Biotop

Zusammenfassung Erstmals konnten Putzsymbiosen zwischen Süßwasserfischen im natürlichen Biotop beobachtet werden. Die sowohl intra-als auch interspezifischen fakultativen Symbiosen wurden bei Bitterling (Rhodeus sericeus amarus), Laube (Alburnus alburnus), Schleie (Tica tinca), sowie bei juvenilen Rotaugen (Rutilus rutilus), Rotfedern (Scardinius erythrophthalmus) und Flußbarschen (Perca fluviatilis) festgestellt. Bei Übertragung der Putzstimmung im Schwarmverband kann ein Putzen im Kollektiv auftreten, wo jeder jeden putzt bzw. dazu auffordert. Das Ausgangsverhalten zum Geputztwerden ist die Einstellung der Lokomotion (Putzstarre), wobei die beobachteten Süßwasserfische im Gegensatz zu marinen Fischen selbst in der Aufforderungsstellung kompensatorische Flossenbewegungen durchführen. Diese einleitende Reglosigkeit kann bereits Putzen durch den Partner auslösen, dessen Bereitschaft mit zunehmender Schrägstellung des Putzkunden gesteigert wird. Das Kopfabwärtsstehen erfolgt durch passives Absinken des Körpervorderteiles, woraus sich stammesgeschichtlich das aktive Schräg-bis Kopfstehen als Aufforderungssignal entwickelt haben dürfte (Laube, Rotfeder, Rotauge). Wahrscheinlich kann die steile Aufforderungsstellung durch Selbstdressur und durch Nachahmen individuell gelernt werden. Fische, die auffordernde Partner anschwimmen, nehmen bei diesen häufig statt zu putzen ebenfalls die Aufforderungsstellung ein; entweder durch Stimmungsübertragung, oder das Anschwimmen erfolgte bereits in der Erwartung, dort ebenfalls geputzt zu werden. Selten treten Kopfzucken (Alburnus) oder Körperrütteln (Scardinius) als wahrscheinlich zusätzliche Aufforderungssignale auf. Attrappenversuche zeigen, daß alle Details des auffordernden Fisches realisiert sein müssen, um als Aufforderung zu wirken, was auf einen gelerten Auslösemechanismus schließen läßt. Die juvenilen Flußbarsche zeigen als Aufforderung neben leichter Schrägstellung ein Zeitlupen-Scheuern vor dem Partner im freien Wasser, was aus dem Verhalten des Normalscheuerns der selbständigen Körperreinigung abzuleiten ist, wobei der Bitterling Zwischenstufen aufweist (Schein-Scheuern).Ein weit ausholender Schwansschlag, möglicherweise ein ÜbersprungverhaIten, löst die Spannung der Putzstarre. Diese überbetonte Intentionsbewegung zum Schwimmstart läßt noch keinen Ansatz zur Signalbildung erkennen. Zu denken wäre an das Körperschütteln mancher Putzkunden tropischer Meere, um die spezialisierten Putzerfische fortzuschicken. Diese fehlen jedoch im einheimischen Süßwasser, wodurch auch das Überwiegen der Putzaufforderungen gegenüber der Putztätigkeit verständlich wird.

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Summary Cleaning symbioses are described for the first time between fresh water fishes in their natural habitat. The symbioses are only facultative and were observed intra-and intorspecifically between members of the species Rhodeus amarus, Alburnus alburnus, Tinca, tinca and juvenile Rutilus rutilus, Scardinius erythrophthalmus and Perca fluviatilis.When the motivation to become cleaned is transmitted within a school of fish, mutual cleaning and inviting each other is common. The first stage in cleaning behaviour is cessation of movement and maintenance of a rigid posture which in these species of fish—contrary to marine species—is accompanied by compensatory fin movements.The more vertical the motionless posture of a fish the more active is the cleaning response of the partner. The vertical position is attained by passive sliding down of the head part. This form of display may be learned individually by imitation and (or) by trial and error.Fish approaching an inviting partner very often assume the invitation posture themselves instead of starting with the cleaning activity. This may be explained by transmission of the mood of invitation or because the invitation display raises the expectation in the approaching fish to become cleaned. Occasionally the invitation display is enhanced by head jerks (in Alburnus) or by body wriggling (in Scardinius).Tests with dummies prove that all details of the invitation pattern have to be fulfilled in order to make it acceptable as such by the partner. This points towards a learned releasing mechanism. Juveniles of Perca fluviatilis, besides assuming a slightly oblique posture, also show an additional feature of the invitation display by slow rubbing motions in front of swimming partners. This sslow motion rubbing may have developed from the normal cleaning behaviour which consists in rubbing the body against the ground. Intermediate stages between these two functions of rubbing motions are shown by Rhodeus amarus (apparent rubbing).The motionless posture of invitation is broken by a large beating of the tail, which may be a displacement activity. This exaggerated intention movement has, however, not yet evolved into a social signal, comparable, for example, to the body shaking of some marine fish by which these induce their cleaners to leave. There are no specialized cleaning fishes in fresh water which explains the dominance of invitation dispays over cleaning activities.