We have isolated and characterized cDNAs from Hydra which encode antistasin, a potent inhibitor of factor Xa in the vertebrate blood clotting cascade. Hydra antistasin is expressed in gland cells and represents a major class of transcripts from Hydra 's head. Sequence analysis revealed that Hydra antistasin contains 6 internal repeats of a 25–26 amino acid sequence with a highly conserved pattern of 6 cysteine and 2 glycine residues identical to that in leech antistasin. Conservation of antistasin in a lower metazoan provides a potential link between the vertebrate and invertebrate coagulation systems.
ABSTRACT Here we present the cloning, expression and immunocytochemical localization of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clones were identified by in situ hybridization to differentiating nematocytes. Sequencing of a full-length clone revealed the presence of an N-terminal signal peptide, suggesting that the mature protein is sorted via the endoplasmic reticulum to the post-Golgi vacuole in which the nematocyst is formed. The N-terminal region of spinalin (154 residues) is very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly as pairs. For both regions a polyglycine-like structure is likely and this may be stabilized by hydrogen bond-mediated chain association. Similar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues) and an acidic region (9 glutamates and 9 aspartates out of 32 residues). Western blot analysis with a polyclonal antibody generated against a recombinant 19 kDa fragment of spinalin showed that spinalin is localized in nematocysts. Following dissociation of the nematocyst’s capsule wall with DTT, spinalin was found in the insoluble fraction containing spines and the operculum. Immunocytochemical analysis of developing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogenesis to form spines on the external surface of the inverted tubule and the operculum.
Zusammenfassung Die β‐Carotinverteilung innerhalb der verschiedenen Lipoproteinfraktionen im Serum von Milchkühen mit verschieden hohen β‐Carotinspiegeln im Blut und während der Kolostralphase wurde untersucht. In allen Fällen lief eine Vitamin‐A‐Bestimmung parallel. Über 90% des β‐Carotins erschienen in der “high density lipoprotein”‐Fraktion. In der “very low density lipoprotein”‐ und “low density lipoprotein”‐Fraktion fanden sich 6–9%. Rasse oder Fütterung hatten keinen Einfluß auf die β‐Carotinverteilung. Auch während der Kolostralphase konnten keine Abweichungen gefunden werden. Schwankungen der β‐Carotinkonzentration in der Milch gingen nicht mit Verschiebungen in der β‐Carotinverteilung im Serum einher. Vitamin A blieb nach Abtrennung der Lipoproteinfraktionen jeweils vollständig im Serumtest erhalten und zeigte damit eine vom β‐Carotin völlig unabhängige Transportweise. Summary Distribution of β‐Carotene among the Lipoproteins in Serum of Dairy Cows The distribution of β‐carotene among the various lipoprotein fractions in the serum of dairy cows with different levels of β‐carotene in blood and during the colostral period was investigated. In all cases vitamin A was determined simultaneously. More than 90% of the β‐carotene appeared in the high density lipoprotein fraction, while in the “very low” and “low” density lipoprotein fraction 6–9% of β‐carotene was found. Breed and feed had no influence on the β‐carotene distribution. During the colostral period also, no deviations were found. Differences in the β‐carotene concentration in milk were not accompanied by changes in the β‐carotene distribution. The total amount of vitamin A in blood after separation of the lipoproteins was all found in the serum residue. This again demonstrates the completely different mode of transport of β‐carotene from that of vitamin A. Résumé Répartition du β‐carotène sur les lipoprotéins sériques chez des vaches laitières On a examiné la répartition du β‐carotène dans les différentes fractions lipoprotéiniques sériques chez des vaches laitières avec des taux de β‐carotène différents dans le sang et pendant la phase colostrale. Une détermination de vitamine A a été faite parallèlement. Plus de 90% de β‐carotène est apparu dans la fraction «high density lipoprotein». On a trouvé 6–9% dans les fractions «very low density et low density lipoprotein». La race ou l'alimentation n'ont pas eu d'influence sur la répartition du β‐carotène. Il n'y a pas eu de déviation durant la phase colostrale. Les variations de la concentration du β‐carotène dans le lait ne vont pas de pair avec des décalages dans la répartition du β‐carotène du sérum. La vitamine A est toujours restée complètement dans le reste du sérum après une séparation des fractions lipoprotéiniques et a montré ainsi un mode de transport tout à fait indépendant du β‐carotène. Resumen La distribución de la carotina β entre las lipoproteínas séricas en las vacas lecheras Se investigó la distribución de la carotina β dentro de las diversas fracciones lipoproteicas en el suero sanguíneo de vacas lecheras con niveles diferentes de carotina β en sangre y durante la fase calostral. En todos los casos se determinó simultáneamente la vitamina A. Más del 90% de la carotina β apareció en la fracción de «lipoproteína de densidad elevada». En las fracciones de «densidad muy baja» y lipoproteína de «densidad baja» se halló un 6–9%. La raza y la alimentación no ejercieron ningún influjo sobre la distribución de la carotina β. Tampoco durante la fase calostral se pudieron encontrar divergencias. Las diferencias en la concentración de carotina β en la leche no iban acompañadas por desvíos en la distribución de carotina β en el suero sanguíneo. La vitamina A se halló en su totalidad en el resto sérico tras la separación de las lipoproteínas. Con lo cual se demostró que la vitamina A y la carotina β disponen de medios de transporte completamente independientes.
