Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are impo... more Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing
Abstract: STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calci... more Abstract: STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin-like peptides or “insectatachykinins,” such as Locusta tachykinin (Lom-TK) III, produced dose-dependent calcium responses in stably transfected S2-STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10-5M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom-TK III-induced calcium response. Further analysis showed that the agonist-induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom-TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5-trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin-dependent signaling processes.
The bioluminescent Ca2+-sensitive reporter protein, aequorin, was employed to develop an insect c... more The bioluminescent Ca2+-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca2+-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC50 value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca2+-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.
Abstract: Tachykinin-like peptides have been identified in many vertebrate and invertebrate speci... more Abstract: Tachykinin-like peptides have been identified in many vertebrate and invertebrate species. On the basis of the data reviewed in this paper, these peptides can be classified into two distinct subfamilies, which are recognized by their respective sequence characteristics. All known vertebrate tachykinins and a few invertebrate ones share a common C-terminal sequence motif, -FXGLMa. The insect tachykinins, which have a common -GFX1GX2Ra C-terminus, display about 30% of sequence homology with the first group.Tachykinins are multifunctional brain/gut peptides. In mammals and insects, various isoforms play an important neuromodulatory role in the central nervous system. They are involved in the processing of sensory information and in the control of motor activities. In addition, members of both subfamilies elicit stimulatory responses on a variety of visceral muscles. The receptors for mammalian and insect tachykinins show a high degree of sequence conservation and their functional characteristics are very similar. In both mammals and insects, angiotensin-converting enzyme (ACE) plays a prominent role in tachykinin peptide metabolism.
Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are impo... more Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing
Abstract: STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calci... more Abstract: STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin-like peptides or “insectatachykinins,” such as Locusta tachykinin (Lom-TK) III, produced dose-dependent calcium responses in stably transfected S2-STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10-5M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom-TK III-induced calcium response. Further analysis showed that the agonist-induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom-TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5-trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin-dependent signaling processes.
The bioluminescent Ca2+-sensitive reporter protein, aequorin, was employed to develop an insect c... more The bioluminescent Ca2+-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca2+-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC50 value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca2+-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.
Abstract: Tachykinin-like peptides have been identified in many vertebrate and invertebrate speci... more Abstract: Tachykinin-like peptides have been identified in many vertebrate and invertebrate species. On the basis of the data reviewed in this paper, these peptides can be classified into two distinct subfamilies, which are recognized by their respective sequence characteristics. All known vertebrate tachykinins and a few invertebrate ones share a common C-terminal sequence motif, -FXGLMa. The insect tachykinins, which have a common -GFX1GX2Ra C-terminus, display about 30% of sequence homology with the first group.Tachykinins are multifunctional brain/gut peptides. In mammals and insects, various isoforms play an important neuromodulatory role in the central nervous system. They are involved in the processing of sensory information and in the control of motor activities. In addition, members of both subfamilies elicit stimulatory responses on a variety of visceral muscles. The receptors for mammalian and insect tachykinins show a high degree of sequence conservation and their functional characteristics are very similar. In both mammals and insects, angiotensin-converting enzyme (ACE) plays a prominent role in tachykinin peptide metabolism.
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