Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the two precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY(SO3)DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV, APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.

SUMMARY In arthropods, a group of peptides possessing a–Y(SO3H)GHM/LRFamide carboxy-terminal motif have been collectively termed the sulfakinins. Sulfakinin isoforms have been identified from numerous insect species. In contrast, members of this peptide family have thus far been isolated from just two crustaceans, the penaeid shrimp Penaeus monodon and Litopenaeus vannamei. Here, we report the identification of a cDNA encoding prepro-sulfakinin from the American lobster Homarus americanus. Two sulfakinin-like sequences were identified within the open-reading frame of the cDNA. Based on modifications predicted by peptide modeling programs, and on homology to the known isoforms of sulfakinin, particularly those from shrimp, the mature H. americanus sulfakinins were hypothesized to be pEFDEY(SO3H)GHMRFamide (Hoa-SK I) and GGGEY(SO3H)DDY(SO3H)GHLRFamide (Hoa-SK II). Hoa-SK I is identical to one of the previously identified shrimp sulfakinins, while Hoa-SK II is a novel isoform. Exogenous application of either synthetic Hoa-SK I or Hoa-SK II to the isolated lobster heart increased both the frequency and amplitude of spontaneous heart contractions. In preparations in which spontaneous contractions were irregular, both peptides increased the regularity of the heartbeat. Our study provides the first molecular characterization of a sulfakinin-encoding cDNA from a crustacean, as well as the first demonstration of bioactivity for native sulfakinins in this group of arthropods.

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The hc2 human papillomavirus DNA test (HC2) is effective when screening women for cervical dysplasia, and it might be effective in the screening for anal dysplasia. Differences between the anal and the cervical canals could affect the test performance. This prospective study (n=292) measured the HC2 signal and in agreement with a histologic endpoint of high-grade dysplasia for anal specimens collected in various ways. Sensitivities were 91%, 85%, and 62% for specimens collected in a sample transport medium and a liquid-based cytology medium processed by Gyn or Non-Gyn protocol, respectively. HC2 sensitivity and specificity to predict high-grade anal dysplasia were similar for brush or swab specimen collections, but HC2 signal was 6 times higher with the brush. Specificity and sensitivity were similar whether the sample was collected first or after a cytology sample for brush or swab, but swab specimens at the second collection had an HC2 signal (mean) 48% lower than that of the first collection, and the swab cellularity was lower. The presence of maximum stool decreased the HC2 signal in anal swab specimens. Consensus polymerase chain reaction (PCR) confirmed that the 13 human papillomavirus probe types in HC2 were optimal for performance. HC2 could potentially be further investigated for use in screening anal dysplasia. A larger prospective study is indicated.