Pepsinogen-like immunoreactivity among vertebrates: occurrence of common antigenicity to an anti-chicken pepsinogen antiserum in stomach gland cells of vertebrates

Stomachs of 14 species selected from five classes of vertebrate were surveyed concerning the reactivity to an anti-adult chicken pepsinogen antiserum (anti-ACPg) with indirect immunofluorescence method. Gland cells of all these stomachs showed reactivity to the antiserum. Crude extract of stomachs from five representatives of mammals, birds, amphibians and fish showed peptic activity (at pH 2.2) of which 70-90% were pepstatin-sensitive. Zymogram and immunoblotting of crude extract revealed that the anti-ACPg-reactive proteins have peptic activity. Molecular weights of anti-ACPg-reactive proteins determined by immunoblotting coincided with the values of purified pepsinogens previously reported for these animals. These results indicate that pepsinogens have been conserved well during vertebrate evolution.     

Presence of immunoreactive corticotropin-releasing hormone and cortisol molecules in invertebrate haemocytes and lower and higher vertebrate thymus

Corticotropin-releasing hormone- and cortisol-like molecules are present in the haemocytes of different molluscan species and in the epithelial cells, interdigitating cells and macrophages -- but not in the lymphocytes -- of fish, frog, chicken and rat thymus. Taking into account the fact that other pro-opiomelanocortin-derived peptides, such as adrenocorticotropin hormone, are present in the haemocytes and thymus of the same species, these results complete the list of stress mediators present in molluscan haemocytes and further support the hypothesis that, although the prototype stress response we have demonstrated in invertebrates is concentrated in a single cell, i.e. the haemocyte, it is similar to the response seen in vertebrates. Moreover, the data presented here are compatible with the hypothesis that an evolutionary, conserved stress response can occur locally with a single organ, e.g. the thymus, in which all the main mediators of this biological response, such as corticotropin-releasing hormone, adrenocor ticotropin hormone and glucocorticoids, are present. The implications of these findings for the physiology of thymus and stress response may be far reaching. © 1998 Chapman & Hall     

Distribution patterns of neurotensin-like immunoreactive cells in the gastro-intestinal tract of higher vertebrates

Summary The endocrine system of the gastro-intestinal tract of selected species representing the five higher vertebrate classes was investigated with reference to occurrence and distribution of neurotensin-like immunoreactive cells. Using antibodies against C-terminal and N-terminal fragments of neurotensin and against the C-terminal sequence of xenopsin it was demonstrated that the intestine of all species studied contains endocrine, neurotensin-like immunoreactive cells. However, large differences in localization and frequency of these neurotensin-like immunoreactive cells were found. Except for a teleostean fish, neurotensin-like immunoreactive cells in the gastro-intestinal tract were more frequent in non-mammalian vertebrates than in mammals. In contrast to mammals, where the highest density of neurotensin-like immunoreactive cells was present in the ileal mucosa, in the non-mammalian vertebrates studied the corresponding cells were most abundant in the pyloric-duodenal junction. The exact mapping of neurotensin-like immunoreactive cells is presented throughout the entire gastro-intestinal tract of six species (Rattus, Coturnix, Lacerta, Rana, Xenopus, Carassius) including a quantitative evaluation of sequential serial sections.     

Connexin43 orthologues in vertebrates: phylogeny from fish to man

The gap junction protein connexin43 (Cx43) is widely expressed in all vertebrate species; however, in ventricular myocardium, Cx43 expression is restricted to mammalian species only, where it provides the molecular correlate for both electrical conduction and synchronization of the repolarization process. The evolutionarily late appearance of Cx43 in the heart suggests physiological adaptation to euthermia with its concomitant demands related to increased cardiovascular output. We tested to what extent mammalian Cx43 differs from that of non-mammalian vertebrates and whether Cx43 from hibernating species contains specific sequence characteristics which could be attributed to their non-isothermal life cycle. We cloned the complete coding region of Cx43 from the African green monkey, European hedgehog (hibernator), Russian dwarf hamster, rabbit, European ground squirrel (hibernator) and pig. After sequencing, these were compared to 12 full-length Cx43 sequences present in GenBank (3 fish, 2 frogs, chicken and 6 mammals amongst which there was one other hibernator). Overall identity ranged from 68.7% to 97.7% at the nucleotide level and from 71.6% to 99.7% at the amino acid level. The phylogeny of Cx43 mirrors the general phylogenetic histories of the investigated species to a large extent. From 382 amino acids there were only 6 specific for mammals. There were no substitutions specific for hibernators. In conclusion, mammalian Cx43 is characterized by 6 specific amino acids, and no obvious differences between non-hibernating and hibernating mammals were observed.Edited by D. Tautz     

Presence of immunoreactive neurophysins of a higher molecular weight in addition to the 10.000 form in the ovine pineal gland

Using an aqueous extraction followed by ultrafiltration through Amicon Diaflo membranes, two ovine pineal fractions were obtained, which contain immunoreactive neurophysin. The presence of neurophysin was monitored by radioimmunoassay, employing an antiserum raised against pituitary bovine neurophysin and selected because it reacts with neurophysins of many other mammals. From 50 g of wet ovine pineal glands 552 micrograms of immunoreactive neurophysins were obtained. About 5% of these immunoreactive neurophysins are eluted from three different Sephadex columns with an elution volume corresponding to Mr above 10,000 between bovine serum albumin and pituitary neurophysin. The remaining 95% of ovine immunoreactive pineal neurophysin (Mr 10,000) shares immunological and physico-chemical properties with highly purified bovine pituitary neurophysin used as a reference. From the results of gel filtration and affinity chromatography on LVP-Sepharose it was concluded that ovine pineal gland may contain a neurophysin precursor molecule in addition to the neurophysin Mr 10,000.     

Termination of pregnancy in mice with antiserum to chicken riboflavin-carrier protein

The importance of riboflavin-carrier protein (RCP) in the maintenance of pregnancy in mice has been studied. Selective passive immunoneutralization of the maternal RCP resulted in fetal death and resorption. Six hours after chicken RCP antiserum treatment, the following observations were made: there was profuse vaginal bleeding in all the animals, a 60% reduction in embryonic ornithine decarboxylase (ODC) activity, a 70% reduction in the maternal progesterone levels, and a 50% reduction in the 14C-riboflavin uptake by the embryo. The above observations are indicative of fetal distress and resorption. By 24 h after treatment, there was 100% resorption of fetuses and the mouse progesterone levels dropped to 20% of untreated or normal rabbit serum (NRS)-treated values. Cytological studies of the fetal liver revealed the classical signs of cellular degeneration in hepatocytes as well as hematopoietic cells. The effect was apparent as early as 1 h after antiserum administration. The erythroid aplasia supports the biochemical evidence that fetal demise is due to preferential riboflavin deficiency of the fetus.     

Presence of ACTH and beta-endorphin immunoreactive molecules in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) and their possible role in phagocytosis

The presence of ACTH and beta-endorphin immunoreactive molecules in the cell-free hemolymph and in the hemocytes of the freshwater snail Planorbarius corneus were demonstrated by immunocytochemistry and RIA tests. Only spreading phagocytic hemocytes were positive, in contrast with other hemocytes devoid of phagocytic activity, i.e., round hemocytes. These data were confirmed by flow cytometry. Another cell type with marked phagocytic activity, i.e., digestive cells of digestive gland, were also positive to anti-ACTH. Corticotropin-releasing factor immunoreactive molecules were found in the cell-free hemolymph and hemocytes, by RIA. Our data suggest that cells with phagocytic activity, the oldest immune response, may represent a suitable model to unravel the tangled web of the common ancestor of the immune and the neuroendocrine systems.     

Localization of cytokeratins in tissues of the rainbow trout: Fundamental differences in expression pattern between fish and higher vertebrates

Using a panel of antibodies against different cytokeratins in immunofluorescence microscopy on frozen tissue sections and two-dimensional gel electrophoresis of cytoskeletal proteins from these tissues, we have studied the tissue distribution of cytokeratins in a fish, the rainbow trout Salmo gairdneri. We have distinguished at least 14 different cytokeratin polypeptides in only a limited number of tissues, thus demonstrating the great complexity of the cytokeratin pattern in a fish species. The simplest cytokeratin pattern was that present in hepatocytes, comprising one type-II (L1) and two type-I (L2, L3) polypeptides that appear to be related to mammalian cytokeratins 8 and 18, respectively. Two or all three cytokeratins of this group were also identified in several other epithelial tissues, such as kidney. Epithelia associated with the digestive tract contained, in addition, other major tissue-specific cytokeratins, such as components D1-D3 (stomach, intestine and swim bladder) and B1 and B2 (biliary tract). With the exception of D1, all these polypeptides were also found in a cultured cell line (RTG-2). Epidermal keratinocytes contained D1 and six other major cytokeratins, termed E1-E6. The most complex cytokeratin pattern was that found in the gill epithelium. Surprisingly, antibodies specific for cytokeratins of the L1-L3 group also reacted with certain cell-sheet-forming tissues that are not considered typical epithelia and in higher vertebrates express primarily, if not exclusively, vimentin. Such tissues were (a) endothelia, including the pillar cells of the "gill filaments", (b) scale-associated cells, and (c) the ocular lens epithelium, and also several nonepithelial cell types, such as (d) fibroblasts and other mesenchymal cells, (e) chondrocytes, (f) certain vascular smooth muscle cells, and (g) astroglial cells of the optic nerve. The differences between the patterns of cytokeratin expression in this fish species and those of higher vertebrates are discussed. It is concluded that the diversity of cytokeratins has already been established in lower vertebrates such as fish, but that the tissue-expression pattern of certain cytokeratins has been restricted during vertebrate evolution. We discuss the value of antibodies specific for individual cytokeratin polypeptides as marker molecules indicating cell and tissue differentiation in fish histology, embryology, and pathology.     

Sequence and gene organization of the chicken mitochondrial genome. A novel gene order in higher vertebrates

The 16,775 base-pair mitochondrial genome of the white Leghorn chicken has been cloned and sequenced. The avian genome encodes the same set of genes (13 proteins, 2 rRNAs and 22 tRNAs) as do other vertebrate mitochondrial DNAs and is organized in a very similar economical fashion. There are very few intergenic nucleotides and several instances of overlaps between protein or tRNA genes. The protein genes are highly similar to their mammalian and amphibian counterparts and are translated according to the same variant genetic code. Despite these highly conserved features, the chicken mitochondrial genome displays two distinctive characteristics. First, it exhibits a novel gene order, the contiguous tRNA(Glu) and ND6 genes are located immediately adjacent to the displacement loop region of the molecule, just ahead of the contiguous tRNA(Pro), tRNA(Thr) and cytochrome b genes, which border the displacement loop region in other vertebrate mitochondrial genomes. This unusual gene order is conserved among the galliform birds. Second, a light-strand replication origin, equivalent to the conserved sequence found between the tRNA(Cys) and tRNA(Asn) genes in all vertebrate mitochondrial genomes sequenced thus far, is absent in the chicken genome. These observations indicate that galliform mitochondrial genomes departed from their mammalian and amphibian counterparts during the course of evolution of vertebrate species. These unexpected characteristics represent useful markers for investigating phylogenetic relationships at a higher taxonomic level.     

The evolution of pepsinogen C genes in vertebrates: duplication, loss and functional diversification

Background

Aspartic proteases comprise a large group of enzymes involved in peptide proteolysis. This collection includes prominent enzymes globally categorized as pepsins, which are derived from pepsinogen precursors. Pepsins are involved in gastric digestion, a hallmark of vertebrate physiology. An important member among the pepsinogens is pepsinogen C (Pgc). A particular aspect of Pgc is its apparent single copy status, which contrasts with the numerous gene copies found for example in pepsinogen A (Pga). Although gene sequences with similarity to Pgc have been described in some vertebrate groups, no exhaustive evolutionary framework has been considered so far.

Methodology/Principal Findings

By combining phylogenetics and genomic analysis, we find an unexpected Pgc diversity in the vertebrate sub-phylum. We were able to reconstruct gene duplication timings relative to the divergence of major vertebrate clades. Before tetrapod divergence, a single Pgc gene tandemly expanded to produce two gene lineages (Pgbc and Pgc2). These have been differentially retained in various classes. Accordingly, we find Pgc2 in sauropsids, amphibians and marsupials, but not in eutherian mammals. Pgbc was retained in amphibians, but duplicated in the ancestor of amniotes giving rise to Pgb and Pgc1. The latter was retained in mammals and probably in reptiles and marsupials but not in birds. Pgb was kept in all of the amniote clade with independent episodes of loss in some mammalian species. Lineage specific expansions of Pgc2 and Pgbc have also occurred in marsupials and amphibians respectively. We find that teleost and tetrapod Pgc genes reside in distinct genomic regions hinting at a possible translocation.

Conclusions

We conclude that the repertoire of Pgc genes is larger than previously reported, and that tandem duplications have modelled the history of Pgc genes. We hypothesize that gene expansion lead to functional divergence in tetrapods, coincident with the invasion of terrestrial habitats.     

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus

Summary The wall structure of arteriovenous anastomoses in the rabbit ear was investigated. (1) Clusters of epithelioid smooth muscle cells form 3–4 longitudinally oriented plicae. The channel shows a single, irregularly outlined lumen, and its wall is very thin between adjacent plicae. (2) Endothelial cells covering the plicae protrude into the lumen, thus suggesting active contraction or shortening of the plicae. (3) The tunica adventitia is composed of 4–6 sheaths of flat fibroblasts, which may serve as a barrier to prevent loss of neurotransmitters. Processes of some of the fibroblasts also extend into the tunica media. (4) The tunica media is composed of an outer circular layer of typical smooth muscle cells, and an inner longitudinally running plica of ramified smooth muscle cells. Wide intercellular spaces between these ramified cells are filled with collagen fibrils, microfibrils, amorphous intercellular substances, and fibroblasts. Fibroblasts form close membrane contacts with each other, and with the smooth muscle cells. (5) Fibroblasts and other connective tissue components may function as an elastic support during active motility of the anastomotic channel.     

Isolation of a novel ras gene from Trichomonas vaginalis: a possible evolutionary ancestor of the Ras and Rap genes of higher eukaryotes.

The Ras subfamily proteins are small, monomeric GTP-binding proteins with vital roles in regulating eukaryotic signal transduction pathways. Gene duplication and divergence have been postulated as the mechanism by which such family members have evolved their specific functions. A cDNA clone of TvRsp was isolated and sequenced from a cDNA expression library of the primitive eukaryote Trichomonas vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified by PCR and sequenced. Sequence analysis suggested that TvRsp was an intronless gene. This gene encoded a protein of 181 amino acids and contained the 5 conserved G domains that designated it as a Ras or Rap subfamily member. However, the deduced amino acid sequence shared only 34%-37% overall identity with other Ras subfamily members of different species, and the presence of motifs characteristic of both the Ras and Rap families of GTPase confused the familial classification of this gene. Phylogenetic analysis showed its origins at the divergence point of the Ras/Rap families and suggested that TvRsp was a possible evolutionary ancestral gene of the ras/rap genes of higher eukaryotes. This information was of importance not only from the perspective of understanding the evolution and diversity of eukaryotic signal transduction pathways but also in providing a framework by which to understand protein processing in the growth and differentiation of single-celled microorganisms.     

The first step in the activation of chicken pepsinogen is similar to that of prochymosin.

Chicken pepsinogen was incubated at pH2.5 with pepstatin. The zymogen activated itself by a sequential mechanism and an intact peptide derived from residues 1-26 in the protein was released in the first step. This peptide was found to inhibit the milk-clotting activities of pig and chicken pepsins and calf chymosin but to different extents.     

Termination of pregnancy in mice following antiserum administration to modified chicken riboflavin carrier protein.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards conformational epitopes and antibodies to disulphide bond reduced carboxymethylated RCP (RCM-RCP) are towards sequential epitopes. The major cyanogen bromide (CNBr) fragments and tryptic fragments of RCM-RCP interact with both antiserum to RCM-RCP and RCP. Passive immunization of pregnant mice with antibodies to RCM-RCP results in bioneutralization, leading to termination of pregnancy. Recently, a major tryptic fragment of RCM-RCP (24 +/- 2 kd) which could assume conformation at the antibody combining site of native RCP, obtained following mild trypsinization has been identified [Natraj et al. J. Biosci, 15 (1990) 341]. Rabbit antibodies to RCM-RCP treated with trypsin generated antibodies of low titer which interacted with RCM-RCP as well as RCP. The interaction of this antibody with RCP was of high affinity and could be displaced with RCP. The bioneutralizing ability of the antibody was demonstrated by its ability to cause termination of pregnancy in mice.     

Transient expression of megakaryocyte-derived protein immunoreactive with an antiserum to cartilage oligomeric matrix protein in developing rat liver

We analyzed a megakaryocyte-derived protein immunoreactive with an antiserum to cartilage oligomeric matrix protein (COMP) in the developing rat liver. Staining with the anti-COMP antiserum in the developing rat liver increased during embryogenesis, and was strongest in the livers of 17-day-old embryos. However, staining in the liver was not detected at eight days after birth or thereafter. The stained cells were found to be megakaryocytes. We partially purified the protein showing cross-reaction with the antiserum to COMP from a megakaryocyte-rich cells fraction in 17-day-old embrionic rat livers. The molecular weight of this protein (approximately 95 kDa) was close to the molecular weight of COMP (105 kDa). Amplification of an RT-PCR fragment (225 bp) corresponding to part of COMP mRNA was detected in cartilage, but not in megakaryocytes of fetal liver or bone marrow. Based on these results, the fetal rat liver megakaryocyte-derived protein that reacted with the antiserum against COMP was thought to contain a common epitope with COMP from cartilage, but to be a different protein from COMP.     

Phylogenetic dating and characterization of gene duplications in vertebrates: the cartilaginous fish reference

Vertebrates originated in the lower Cambrian. Their diversification and morphological innovations have been attributed to large-scale gene or genome duplications at the origin of the group. These duplications are predicted to have occurred in two rounds, the "2R" hypothesis, or they may have occurred in one genome duplication plus many segmental duplications, although these hypotheses are disputed. Under such models, most genes that are duplicated in all vertebrates should have originated during the same period. Previous work has shown that indeed duplications started after the speciation between vertebrates and the closest invertebrate, amphioxus, but have not set a clear ending. Consideration of chordate phylogeny immediately shows the key position of cartilaginous vertebrates (Chondrichthyes) to answer this question. Did gene duplications occur as frequently during the 45 Myr between the cartilaginous/bony vertebrate split and the fish/tetrapode split as in the previous approximately 100 Myr? Although the time interval is relatively short, it is crucial to understanding the events at the origin of vertebrates. By a systematic appraisal of gene phylogenies, we show that significantly more duplications occurred before than after the cartilaginous/bony vertebrate split. Our results support rounds of gene or genome duplications during a limited period of early vertebrate evolution and allow a better characterization of these events.