Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor.     

Yersinia lead SUMO attack

Little is known about the mechanism by which Yops, proteins that Yersinia inject into the cytosol of macrophage, cause downregulation of the inflammatory response and diseases such as the plague. Now it appears that Yops are the first bacterial member of a new family of ubiquitin-like proteases.     

Sequential opening of IP(3)-sensitive Ca(2+) channels and SOC during alpha-adrenergic activation of rabbit vena cava

Alpha(1)-aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca(2+) oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release channels (IP(3)R channels) can prevent the initiation and abolish ongoing alpha(1)-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP(3)R channels, not to its slight inhibition of the L-type voltage-gated Ca(2+) channel and the sarco(endo)plasmic reticulum Ca(2+) ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca(2+) release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP(3)R-channel-mediated SR Ca(2+) release or store depletion. This is in accordance with its sensitivity to Ni(2+) and La(3+) (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP(3)R channels and the SOC are required for PE-mediated [Ca(2+)](i) oscillations and constriction of the rabbit IVC.     

The mechanism of excitation-contraction coupling in phenylephrine-stimulated human saphenous vein

The human saphenous vein (HSV) is the most widely used graft in coronary artery revascularization procedures and is susceptible to spasm perioperatively. The aim of this study is to elucidate the mechanism(s) of agonist-induced excitation-contraction coupling in this vessel. Isometric contraction experiments were combined with in situ smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) imaging by confocal microscopy of intact undistended HSV segments during activation with phenylephrine (PE; 50 microM). Stimulation with PE produced a sustained contraction. Preincubation with 5 microM nifedipine, a blocker of the L-type voltage-operated Ca(2+) channel, or 50 microM SKF-96365, a blocker of both the voltage- and receptor-operated channels, reduced force generation by 25-30%. Ca(2+) imaging revealed that PE elicited only a transient rise in [Ca(2+)](i), suggesting that Ca(2+) plays only a minor role. However, a requirement for basal Ca(2+) levels was demonstrated when PE contractions could not be maintained in Ca(2+)-free medium. In light of the transient Ca(2+) response, it appears that signals other than Ca(2+) must maintain the tonic contraction elicited by PE, such as those that sensitize the myofilaments to Ca(2+). Application of HA-1077 (a Rho kinase inhibitor) at the peak of the contraction completely abolished the plateau phase of the response, whereas application of genistein (a tyrosine kinase inhibitor) reduced this phase by approximately 50%. The foregoing results suggest that, whereas the transient Ca(2+) signal can contribute to the development of force, maintenance of the plateau phase of the PE contraction in the HSV is the result of myofilament Ca(2+) sensitization by Rho kinase and tyrosine phosphorylation. The elucidation of the mechanisms of excitation-contraction coupling in the HSV may be useful for the development of therapeutic strategies for the alleviation of vein graft spasm.     

Use of the Verrucomicrobia-specific probe EUB338-III and fluorescent in situ hybridization for detection of "Candidatus Xiphinematobacter" cells in nematode hosts

Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium "Candidatus Xiphinematobacter" in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.     

Functional evidence for D- and T-loop interactions in tmRNA

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs. Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342). Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.     

Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus

Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.