The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. Highresolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Among eukaryotes, peptidoglycan is found only in cyanellecontaining organisms. These consist of about 12 different species and have been grouped under the denomination glaucocystophytes (29). Recently, this has been corroborated by 18S rRNA-derived phylogenetic analysis (5). Cyanophora paradoxa is the only member of this group which can be grown easily; therefore, it is the best investigated one. The round cyanelles of this obligatorily photoautotrophic protist were originally regarded as endosymbiotic cyanobacteria for morphological reasons, such as thylakoid structure (13), carboxysomes (31), and a lysozyme-sensitive peptidoglycan sacculus (37). With the onset of molecular biological investigations of C. paradoxa (18,32), it became clear that the cyanelle genome is smaller than the genomes of unicellular free-living cyanobacteria by a factor of about 25. Thus, cyanelles in fact represent the plastids of C. paradoxa, having retained the peptidoglycan layer which is crucial for division, as indicated by the inhibitory effects of ␤-lactam antibiotics (4, 29). The peptidoglycan layer is located between the organelle membrane and an outer membrane of unknown origin and has a thickness of about 7 nm (30).Investigations of cyanelle peptidoglycan synthesis and degradation have shown that cyanelles apparently harbor a complete set of enzymes involved in these processes. Seven penicillin-binding proteins ranging from 110 to 35 kDa have been identified in the cyanelle envelope by labelling with a radioactive derivative of ampicillin (4). Indirect evidence has been obtained for periplasmic localization of DD-and LD-carboxypeptidases and DD-endopeptidase, enzymes capable of hydrolyzing defined bonds in peptidoglycan (36). The biosynthesis of the UDP-N-acetylmuramyl pentapeptide precursor of peptidoglycan has been shown to occur in the cyanelle stroma (36). The cyanelle genome (135.6 kb) of one of the two known isolates of C. paradoxa, strain LB555-UT...

Neuroendocrine tumors may present with pseudoallergic reactions like diarrhea and idiopathic anaphylaxis. Here we present the P-STS human ileal neuroendocrine cell line as a model cell line for these tumors. Neuroendocrine markers and changes in cytoplasmic calcium concentration ([Ca2+]i) in response to several possible activators of 5-hydroxytryptamine (5-HT) release were analyzed. P-STS cells still expressed chromogranin A and synaptophysin after 2 years of culture. Tryptophan hydroxylase 1 mRNA and a low amount of 5-HT were also detected. Acetylcholine (ACh) caused a rise in [Ca2+]i. Somatostatin inhibited, whereas histamine (HA) but not the HA receptor ligand betahistine enhanced activation by ACh. The [Ca2+]i response to ACh/HA was inhibited by the HA receptor H3 (H3R) agonist methimepip and by the antidepressant imipramine. Further [Ca2+]i response studies indicated the presence of H4Rs and of a functional calcium sensing receptor. High or low affinity IgE receptor protein or mRNA were not detected. Taken together, neuroendocrine markers and response to intestinal neurotransmitters approve the P-STS cell line as a valuable model for enterochromaffin cells. Enhancement of their ACh-induced pro-secretory response by HA, with a role for H3R and H4R, suggests an amplifying role of neuroendocrine cells in allergen-induced diarrhea or anaphylaxis.

In Cyanophora paradoxa photosynthetic organelles termed cyanelles perform the functions of chloroplasts in higher plants, while the structural and biochemical characteristics of the cyanelle are essentially cyanobacterial. Our interest in studying the evolutionary relationship between cyanelles and chloroplasts led us to focus on cyanelle-encoded genes of the translational apparatus, specifically genes equivalent to those of the bacterial S10 and spc operons. The structure of a large ribosomal protein gene cluster from cyanelle DNA was characterized and compared with that from plastids and bacteria. Sequences of the following cyanelle genes encompassing 4.8 kb are reported here: 5'-rpl22-rps3-rpl16-rps17-rpl14-rpl5-rps8-rpl6-rpl18- rps5-3'. Cyanelles contain five more ribosomal protein genes than do higher plant chloroplasts and four more genes than Euglena gracilis plastids in the S10/spc region of this gene cluster. The gene encoding rpl36 is absent, in contrast to the case in other plastid DNAs. These genes, including the previously characterized genes rpl3, rpl2 and rps19, are transcribed as a primary transcript of approximately 7500 nucleotides. The occurrence of transcripts smaller than this presumptive primary transcript suggests that it is processed into defined segments. Transcription terminates 3' of rps5 where a 40 bp hairpin with one mismatch (-42.2 kcal) may be folded. Immediately downstream of rps5 an open reading frame, ORF492, is contained on a separate transcript. A comparison of gene content, operon structure and deduced amino acid sequence of the genes in the S10 and spc operons from different organisms supports the notion that cyanelles are intermediary between known plastids and cyanobacteria.

Cyanelle peptidoglycan from the glaucocystophyte algae Glaucocystis nostochinearum and Cyanoptyche gloeocystis was investigated by high-performance liquid chromatography of muropeptides, supported by matrixassisted laser desorption-ionization mass spectrometry. The peptidoglycans of both species are modified with N-acetylputrescine, as has been demonstrated for cyanelle peptidoglycan of Cyanophora paradoxa.