Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.

We have investigated low molecular weight antibacterial proteins from the Cecropia moth, Hyalophoru cecropia. In addition to the previously described cecropins A and B, five new antibacterial proteins were discovered, the cecropins C, D, E and F, and the factor G. A scheme for the purification of these factors is presented.Cecropin D is a major cecropin, its amino acid sequence, WNPFKELEKVGQRVRDAVISAGPAVATVA-QATALAK, shows homology to cecropin A and B. Like these cecropins, cecropin D has a blocked C-terminal. The previously tentative C-terminal sequence of cecropin A is also confirmed. It is concluded that the three major cecropins, A, B and D, are products of three different genes that are derived from a common ancestor.The cecropins C, E and F were present in very low amounts, and thus their primary structures could not be fully elucidated. Cecropin C has an amino acid sequence that up to residue 37 is identical to the sequence of A, though it lacks the C-terminal blocking group. It may be a precursor or degradation product of cecropin A. The minor cecropin E shows a similar relation to cecropin D. Cecropin F has a single amino acid replacement (17 Asp+Asn) compared to cecropin D, and is probably a product of an allele that is present at a low frequency in the population. The primary structure of the factor G could not be determined, however its amino acid composition is different from that of the cecropins.All the major cecropins were found to be efficient against several gram-positive and gram-negative bacterial strains. N o significant difference was found between them in their activity against Escherichia coli, though against some less susceptible bacteria the most basic cecropins were more effective, the activity falling in the series B > A s D .Diapausing pupae of the Cecropia moth (Hyalophora cecropia) are an excellent model system for the study of humoral immunity in higher insects. A main advantage is that this system allows a selective activation of the genes for immunity; during the pupal diapause the rest of the genome remains in dormancy. The best way to activate the immune system of Cecropia pupae is by an injection with live nonpathogenic bacteria. The first response is phagocytosis, then follows the onset of synthesis of RNA and about 15 hemolymph proteins (for reviews, see [l] and [2]).Much of the potent antibacterial activity that appears in hemolymph was traced to two low molecular weight proteins, cecropins A and B [3], first referred to as the P9 proteins [4]. Cecropin-like substances were also demonstrated in seven other Lepidopteran insects [5]. At the same time it became clear that Cecropia contained several additional antibacterial factors, some of which could also be cecropins.We here describe the purification and characterization of five such factors, four of which are shown to be cecropins. A fifth minor antibacterial factor, compound G, was also isolated, but its identity is not yet established. Amino acid sequence data are given for all of the cecropins, but only for cecropin D...

Two novel sites of autophosphorylation were localized to the juxtamembrane segment of the human plateletderived growth factor (PDGF) $-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants were made in which Tyr579, Tyr581 or both were replaced with phenylalanine residues; the receptor mutants were stably expressed in porcine aortic endothelial cells. Compared with the wild-type receptor, the Y579F and Y581F mutants were less able to mediate association with and activation of the Src family tyrosine kinases. The ability of these phosphorylation sites to mediate directly the binding of the Src family proteins was also demonstrated by using phosphotyrosine-containing synthetic peptides representing the juxtamembrane sequence of the receptor. Both the Y579F and Y581F mutants were similar to the wild-type receptor with regard to their protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. A conclusive evaluation of the role of the Src family members in signal transduction could, however, not be made since our attempt to prevent completely the association by mutation of both Tyr579 and Tyr581, resulted in loss of kinase activity and was therefore not informative. The present data, together with previous observations, demonstrate a high degree of specificity in the interaction between different autophosphorylation sites in the PDGF $-receptor and downstream components in the signal transduction pathway.

Six closely related antibacterial proteins, attacins A‐F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A‐F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20‐23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino‐terminal sequences, attacins A‐D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram‐negative bacteria isolated from the gut of a silk worm but they did not act on other Gram‐positive and Gram‐negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.

Antibodies to citrulline-modified proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359–369; ARGLTGRPGDA) with or without arginines modified by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients' synovial fluid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising β-turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA.