Oxaloacetate decarboxylase of <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: purification,characterization, and expression of the genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The oxaloacetate decarboxylase (OAD) Na⁺ pump consists of subunits , , and , which are expressed from an oadGAB gene cluster present in various anaerobic bacteria. Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2. The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway. The gene sequences of oad-1 and oad-2 of V. cholerae strain O395-N1 were determined. The apparent frameshift in the published sequence of the oadA-2 gene from V. cholerae El Tor N16961 was not present in strain O395-N1. Upon anaerobic growth of V. cholerae on citrate, exclusively the oad-2 genes are expressed. OAD was isolated from these cells by monomeric avidin–Sepharose affinity chromatography. The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable. Decarboxylase activity was Na⁺ dependent, and the activation profile showed strong cooperativity with a Hill coefficient n_H=1.8. Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10 M and 200 M, respectively. After reconstitution into proteoliposomes, the enzyme acted as a Na⁺ pump. With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570 kDa, suggesting a tetrameric structure for OAD-2. The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cells.     

Synthesis and in vitro and in vivo evaluation of [11C]methyl-BIII277CL for imaging the PCP-binding site of the NMDA receptor by pet

A new benzomorphane derivative, [11C]methyl-BIII277CL, was evaluated as a potential radiotracer for visualizing the PCP-binding site of the N-methyl-D-aspartate (NMDA) receptor by positron emission tomography (PET). Methyl-BIII277CL was prepared by reacting the desmethyl compound (BIII277CL) with dimethylsulfate. The pharmacological profile of methyl-BIII277CL was determined by in vitro receptor-screening assays. At a concentration of 100 nM, methyl-BIII277CL showed a significant interaction with the PCP-binding site of the NMDA receptor (79% inhibition of specific binding) and the sigma-binding site (46% inhibition). In displacement assays using mice cortical membranes, methyl-BIII277CL displayed a high affinity at the PCP-binding site of the NMDA receptor (Ki = 49 +/- 14 nmol/L) and a 130-fold lower interaction with the sigma1-binding site (Ki = 6.35 +/- 0.26 micromol/L). For saturation experiments and in vivo studies, methyl-BIII277CL was radiolabeled with 11C at the O-position of the desmethyl precursor (BIII277CL) using [11C]methyliodide with a specific activity of 35-70 GBq/micromol at the end of synthesis (EOS). In saturation assays using rat whole brain membranes [11C]methyl-BIII277CL showed a Kd of 6 +/- 1 nmol/L and a Bmax of 670 +/- 154 fmol/mg protein. Biodistribution and PET studies in rats and pigs, however, indicated a lack of specific binding and unfavorable pharmacokinetics. Kinetic modeling using the 1-tissue compartment model demonstrated for [11C]methyl-BIII277CL a low distribution volume (Dv = 0.98 mL/mL(tissue)) and very high values for the kinetic parameters K1 and k2 (K1 = 0.36 mL/mL(tissue)/min and k2 = 0.37min(-1)) in pig cortex. [11C]methyl-BIII277CL, due to the lack of specificity in vivo, may not be a candidate for imaging the PCP-binding site of the NMDA receptor.     

Aggression and nest spacing in single and mixed species groups of seabirds

When heterospecific seabirds are part of a nesting colony, there may be less opportunity for conspecifics to come in direct contact with each other, resulting in lower intraspecific aggressiveness. To determine if individuals spend less time in aggressive behavior when nesting in conspecific rather than heterospecific groups, we compared the behavior of black skimmers (Rhynchops niger) nesting with gull-billed terns (Sterna nilotica) in three mixed species subcolonies to those of black skimmers in three single species subcolonies. In contrast to our predictions, black skimmers spent significantly less time in aggressive behaviors when nesting in single species subcolonies than when nesting with heterospecifics. Although skimmers in mixed species subcolonies tended to have more aggressive interactions with skimmers than terns, this may be a function of subcolony composition; the proportions of aggressive interactions with conspecifics were similar to the proportions of conspecifics in each subcolony. However, within the mixed species subcolonies, skimmers that nested nearer to terns were involved in aggressive interactions significantly less than skimmers that nested closer to conspecifics. Also, skimmers nested closer to their nearest neighbor when it was a gull-billed tern than when it was another skimmer. Regardless of which species they nested closest to, skimmers were more aggressive towards other skimmers than to terns within the mixed species subcolonies. Distance to nearest neighbor's nest did not differ significantly between the colony types, and did not seem to influence the duration of aggressive activity in the single species subcolonies. In the mixed species subcolonies, however, the time spent in aggressive behavior increased as the distance to nearest neighbor increased. It appears that of the several benefits that have been proposed of mixed species colonies, reduced time spent in conspecific aggression is not among them. However, within a mixed species colony, an individual can reduce time spent in aggressive interactions by nesting near heterospecifics. Received: 16 September 1996 / Accepted: 24 February 1997     

Platelet-activating factor production in human neutrophils by sequential stimulation with granulocyte-macrophage colony-stimulating factor and the chemotactic factors C5A or formyl-methionyl-leucyl-phenylalanine

Besides its function as a growth factor, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) "primes" polymorphonuclear leukocytes (PMN) for enhanced biologic responses to a number of secondary stimuli. We examined the effect of priming PMN with GM-CSF on the production of [3H] platelet-activating factor (PAF) from [3H]acetate upon stimulation with the chemotactic factors FMLP and C5a. In PMN stimulated with the individual peptide mediators alone [3H]PAF levels were close to controls, whereas considerable amounts of [3H]PAF are formed after stimulation of PMN which have been preexposed to GM-CSF. The priming effect was concentration and time dependent. It was optimal after a preincubation period of 2 h. A maximum of [3H]PAF accumulation is reached within 2.5 min (C5a) and 5.0 min (FMLP) after activation of GM-CSF-primed PMN. In addition, we show that PAF isolated from PMN preincubated with GM-CSF and triggered with chemotactic factors is able to enhance the respiratory burst in PMN. PAF formed by sequentially activated PMN could contribute to the enhanced oxygen radical production and cytotoxicity in effector cells and play a role in modulating and amplifying inflammatory reactions.     

A plea for the ‘de-migranticization' of research on migration and integration1

ABSTRACT

Migration and integration research has been institutionalized over the last few decades. However, an increasing number of voices has been calling for more reflexivity, criticizing the nation-state- and ethnicity-centred epistemology that often informs this discipline. Consistently with this line of reasoning, I argue that migration and integration research originates in a historically institutionalized nation-state migration apparatus and is thus entangled with a particular normalization discourse. Therefore, this field of study contributes to reproducing the categories of this particular migration apparatus. This entanglement poses some serious dilemmas for this research tradition, dilemmas that ask for further consideration and possible solutions. My main proposition is to ‘de-migranticize' migration and integration research. I outline possible ways of doing so and discuss the consequences of such a strategy for the future of migration and integration studies.     

The Protector Species Hypothesis: Do Black Skimmers Find Refuge from Predators in Gull-billed Tern Colonies?

The protector-species hypothesis explains mixed-species coloniality on the basis of benefits individuals of a species may receive by nesting with another species, the ‘protector’ species, that responds aggressively to potential threats. The reactions of nesting individuals to both natural and model predators were observed to determine whether black skimmers (Rhynchops niger) gain an antipredator advantage by nesting with gull-billed terns (Sterna nilotica). Observations of natural predators were gathered from three mixed-species and three single-species (black skimmers) subcolonies. Natural predators most commonly encountered by the colonies were herring gulls (Larus argentatus), laughing gulls (Larus atricilla), and ruddy turnstones (Arenaria interpres). Gull-billed terns responded to the gulls, but not to the turnstones, in higher proportions than did black skimmers. Two decoys, a mink and a gull, were used to simulate predatory encounters, and a duck decoy was used as a control at two mixed-species and one single-species subcolonies. Gull-billed terns responded in significantly higher proportions than did skimmers to all decoy treatments in the mixed-species subcolonies. Mobbing of both natural and model predators by the terns suggests that skimmers may gain a reproductive advantage by nesting with these terns. However, the response of black skimmers to both natural and simulated predators was independent of the presence of gull-billed terns in the colony, indicating that black skimmers may not perceive these objects as threats, or may react differently to predators than do gull-billed terns.     

Functional reconstitution of a receptor-activated signal transduction pathway in Xenopus laevis oocytes using the cloned human C5a receptor.

We have used the polymerase chain reaction to isolate and clone the cDNA encoding the human C5a receptor, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes for functional characterization of the receptor protein. Receptor activity was determined either electrophysiologically by measuring the agonist-dependent opening of [Ca2+]i-dependent Cl- channels, or by analysing the agonist-dependent efflux of 45Ca2+ from the oocytes. Using both methodologies, injection of pure C5a receptor cRNA failed to confer C5a sensitivity on the oocytes. In contrast, marked responses to C5a were observed when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells, which is devoid of C5a receptor mRNA. Binding studies using radioiodinated C5a revealed that the C5a receptor polypeptide was in fact synthesized and targeted to the oocyte plasma membrane in oocytes injected with receptor cRNA alone, and that the level of receptor expression was not influenced by coinjection of poly(A)+ RNA from undifferentiated HL-60 cells. These results strongly suggest that the human C5a receptor requires a specific cofactor(s) lacking in Xenopus oocytes but present in undifferentiated HL-60 cells, to generate intracellular signals in oocytes. Identification and characterization of this factor will provide important information about the molecular mechanisms by which G-protein-coupled receptors activate phospholipase C.     

Novel anti-inflammatory and analgesic agents: synthesis,molecular docking and in vivo studies

Twelve new derivatives of benzothiazole bearing benzenesulphonamide and carboxamide were synthesised and investigated for their in vivo anti-inflammatory, analgesic and ulcerogenic activities. Molecular docking showed an excellent binding interaction of the synthesised compounds with the receptors, with 17c showing the highest binding energy (–12.50?kcal/mol). Compounds 17c and 17i inhibited carrageenan-induced rat paw oedema at 72, 76, and 80% and 64, 73, and 78% at 1?h, 2?h, and 3?h, respectively. In the analgesic activity experiment, compounds 17c, 17?g, and 17i had ED₅₀ (µM/kg) of 96, 127, and 84 after 0.5?h; 102, 134, and 72 after 1?h and 89, 156, and 69 µM/kg after 2?h, respectively, which were comparable with 156, 72, and 70 µM/kg for celecoxib. The ulcerogenic index of the most active derivatives 17c and 17i were 0.82 and 0.89, respectively, comparable to 0.92 for celecoxib. The physicochemical studies of the new derivatives showed that they will not have oral bioavailability problems.