Lower Vertebrate Species Research Articles

Distribution of GABA-like immunoreactivity in myenteric plexus of carp, frog and chicken

The distribution of GABA-like immunoreactivity was studied by means of indirect immunocytochemical methods in some lower vertebrate species (carp, frog, chicken). An immunoreactive network was revealed in the myenteric plexus of the alimentary canal of carp. GABA-positive nerve cells were attached closely to the fibres in the stomach. In other gut regions immunostained neurons were less frequent. Immunoreactive fibres often formed baskets on the surfaces of immunonegative neurons along the whole length of the alimentary canal. The number of immunopositive nerve fibres and pericellular baskets seemed to be lower in the mid- and hingut than in the foregut region. A similar distribution of GABA-immunoreactivity was revealed in the frog myenteric plexus. The ganglionated foregut region possessed a relatively dense GABAergic innervation. This part of the gut contained immunostained nerve cells and fibres, while the mid- and hindgut possessed only a scanty fibre system. Chicken exhibited an extensive immunoreactive plexus for GABA, although the GABA-stained perikarya were restricted mainly to the duodenum. Further regions of the small intestine were poor in immunoreactive cell bodies, which suggests a segmental origin and arrangement of GABAergic innervation within the plexus. In all three species studied, GABA-positive fibres run into the circular muscle layer. The varicosity suggests their influence on the movement of the smooth muscles through modifying the transmitter release of neighbouring terminals.

Autoradiographic analysis of the distribution of vasoactive intestinal peptide binding sites in the vertebrate central nervous system: a phylogenetic study

The distribution of vasoactive intestinal peptide (VIP) binding sites in the brain of several vertebrate species was examined by in vitro autoradiography on slide-mounted sections. This study included fish, frog, snake, pigeon, rat, mouse, guinea pig, cat and monkey brain. A fully characterized, monoiodinated form of vasoactive intestinal peptide (M- 125I-VIP), which maintains the biological activity of the native peptide in the central nervous system (CNS), was used throughout the study. Among the lower vertebrate species, no significant specific binding was found in the fish brain, whereas in the frog and snake brain, specific VIP binding sites were observed, mainly in the telencephalon. In the pigeon brain, high densities of VIP binding sites were localized in the hyperstriatum, neostriatum, archistriatum, hippocampal area, dorsolateral cortical area and in the optic tectum. Ectostriatum and paleostriatum augmentatum displayed lower densities of specific binding. In mammals, the highest concentrations of VIP binding sites were observed in the rodent brain. In the rat, mouse and guinea pig brain, high densities were detected in the olfactory bulb, external layers of the cerebral cortex, dentate gyrus, midline thalamic nuclei, geniculate nuclei, some hypothalamic nuclei, superior colliculus coeruleus. Intermediate densities were found in amygdala, caudate-putamen, septum and nucleus accumbens, CA1–CA3 fields of the hippocampus and central gray. The cerebellum of these species presented high densities of VIP binding sites, with species to species differences in their localization. The non-specific binding was, however, increased in the rodent cerebellum. Lower densities of VIP binding sites were observed in the cat and monkey CNS. In these two species, the non-specific binding was considerably higher than in the lower mammals brain. In the cat and monkey brain, as in the lower mammals, the highest densities were revealed in the neocortex, dentate gyrus, thalamic nuclei and some midbrain structures including substantia nigra and locus coeruleus. In all the species studied, the white matter was never labeled with M- 125I-VIP. This study suggests that VIP binding sites appear relatively early in the evolution of the vertebrate CNS. The most important densities of specific VIP binding sites are observed in the pigeon and rodent brain, whereas the cat and monkey present a marked increase in non-specific binding. It is interesting to note that the distribution of VIP binding sites as revealed by autoradiography is quite conservative in terms of evolution and indicates an association, although non-exclusive, of VIP receptors with brain regions involved in the processing of specific sensory inputs.

Eleven distinct VH gene families and additional patterns of sequence variation suggest a high degree of immunoglobulin gene complexity in a lower vertebrate, Xenopus laevis.

Lower vertebrate species, including Xenopus laevis, exhibit restricted antibody diversity relative to higher vertebrates. We have analyzed more than 180 VH gene-containing recombinant clones from an unamplified spleen cDNA library by selective sequencing of JH and CH positive clones following iterative hybridization screening with family-specific VH probes, 11 unique families of VH genes, each associated with a unique genomic Southern blot hybridization pattern, are described and compared. Considerable variation in the number of hybridizing components detected by each probe is evident. The nucleotide sequence difference between VH families is as great as, if not more than, that reported in other systems, including representatives of the mammalian, avian, and elasmobranch lineages. Some Xenopus Ig gene families encode alternative amino acids at positions that are otherwise invariant or very rarely substituted in known Igs. Furthermore, variations in complementarity determining region sequences among members of the same gene family and high degrees of DH and JH region complexity are described, suggesting that in at least this lower vertebrate species, the diversity of expressed Ig VH genes is not restricted.

An elasmobranchian somatostatin: Primary structure and tissue distribution in Torpedo marmorata

Extracts of brain, stomach, pancreas, and intestine from Torpedo marmorata, an elasmobranchian cartilaginous fish, contained somatostatin-like immunoreactivity. Gel filtration studies demonstrated that material with the elution volume of somatostatin-14 was the only component detected in all tissue extracts. This result contrasts with the situation in mammals where prosomatostatin is processed to multiple molecular forms in a tissue-specific manner. Somatostatin from pancreas and gut was purified to homogeneity and amino acid sequence analysis indicated that T. marmorata somatostatin from both tissues has the same structure as somatostatin-14 isolated from the higher vertebrates. Further examination of other lower vertebrate species is required in order to test the hypothesis that the ability to regulate the production of multiple forms of a regulatory peptide from a single precursor molecule developed only relatively late in evolution.

986 THE CELL SURFACE MOLECULE p24 IS HIGHLY CONSERVED ON VARIOUS TISSUES IN SEVERAL MAMMALIAN SPECIES

The cell surface molecule p24 (human leukocyte differentiation antigen CD9) appears to be broadly distributed on primate (human and non-human) tissues by serologic analysis. The present study was designed to extend these serologic observations in lower vertebrate species and correlate serology by indirect immunofluorescence (IF) with structural analysis of p24 using lactoperoxidase 125I labelled cells subjected to NP40 lysis, SDS-PAGE and autoradiography. Monoclonal antibody BA-2, which identifies an epitope of p24, reacted by IF with human platelets and fibroblasts, the MDCK (canine kidney epithelial), and BSC-1 (African green monkey kidney epithelial) cell lines, but not with bone marrow or mesonephrons of Rana pipiens. Immunochemical analysis identified p24 by identical migration in SDS-PAGE in each aforementioned cell type except Rana pipiens, which was not tested. BA-2 staining of normal canine kidney was compared with BA-2 staining of normal human kidney. Homologous reactivity was found with epithelium of Bowman's capsule and distal tubule and with arterial smooth muscle. In dog, but not human kidney, BA-2 reacted with mesangium. In human, but not dog, BA-2 identified endothelium. Together, these results indicate that p24 is highly conserved in apparent molecular weight and in discrete cell types of mammalian species dating back at least 70 × 106 years.

Effects of synthetic mammalian thyrotrophin releasing hormone, somatostatin and dopamine on the secretion of prolactin and growth hormone from amphibian and reptilian pituitary glands incubated in vitro.

Pituitary glands of grassfrog (Rana pipiens), bullfrog (Rana catesbeiana), clawed toad (Xenopus laevis) and two species of terrapin (Chrysemys picta and Pseudemys scripta) were incubated in medium containing hypothalamic extract (HE), thyrotrophin releasing hormone (TRH), somatostatin, dopamine, or combinations of these treatments. Prolactin and GH concentrations in the medium were determined by densitometry after polyacrylamide-gel electrophoretic separation. Hypothalamic extract stimulated secretion of both hormones in all species tested. Thyrotrophin releasing hormone stimulated secretion of prolactin and GH, showing a biphasic pattern of response. Dopamine had little effect alone, but inhibited HE- and TRH-stimulated release of prolactin, but not GH, in both amphibia and reptiles. Somatostatin by itself had no apparent effect on release of hormones, but it inhibited HE- and TRH-stimulated release of GH from both amphibian and reptilian pituitary glands. These results indicate that factors affecting mammals and birds also interact in the regulation of secretion of prolactin and GH in lower vertebrate species.

Protein polymorphisms, segregation in genetic crosses and genetic distances among fishes of the genus Xiphophorus (Poeciliidae).

The products of 49 protein-coding loci were examined by starch gel electrophoresis for populational variation in six species of Xiphophorus fishes and/or segregation in intra-and interspecific backcross and intercross hybrids. Electrophoretic variation was observed for 29 of the 35 locus products in a survey of 42 population samples. The highest frequency of polymorphic loci observed in noninbred populations was 0.143. After ten or more generations of inbreeding, all loci studied were monomorphic. Inbred strains generally exhibited the commonest electrophoretic alleles of the population from which they were derived. An assessment of genetic distances among Xiphophorus populations reflected classical systematic relationships and suggested incipient subspeciation between X. maculatus from different drainages as well as several species groups. Thirty-three loci were analyzed with respect to segregation in hybrids. The goodness of fit of segregations to Mendelian expectations at all loci analyzed (except loci in linkage group I) is interpreted as evidence for high genetic compatibility of the genomes of Xiphophorus species. It is anticipated that these data will result in a rapid expansion of the assignment of protein-coding loci to linkage groups in these lower vertebrate species.

Antibody response in Heterodontus.

Appropriately selected phylogenetic models are capable of providing insight into genetic mechanisms which may have become obscured during the passage of evolutionary time. In higher vertebrates a complex multigenic family encodes immunoglobulin-variable regions. The mechanisms involved in the expansion of the gene family and the stable maintenance of large numbers of individual genes presently are not understood. By defining the nature of antibody diversity in lower vertebrate species, it may be possible to approach such issues at a more fundamental level. Analyses of the immunoglobulins in Heterodontus francisci (horned shark), a representative phylogenetically primitive elasmobranch, indicate that this species may represent a useful developmental model.

Effects of spirometrid plerocercoids on several species of lower vertebrates

1. 1. The effects of the plerocercoid growth factor on several species of lower vertebrates were tested. 2. 2. There was little effect on growth or fattening in either fish or frogs. 3. 3. Plerocercoid infections and to a lesser degree worm homogenate injections markedly stimulated growth but not fattening in lizards.

Active sites of turtle and duck low molecular weight antibody to 2′4′dinitrophenol

Low mol. wt antibody directed to the dinitrophenol (DNP) grouping has been raised in both turtles and ducks by repeated challenge with DNP- Brucella abortus. Antibody was purified from the serum of both species by a combination of affinity chromatography and gel filtration. Purified antibody, upon interaction with hapten, induced a shift in the visible absorption spectra of the hapten suggestive of the participation of tryptophan in the active sites of the two antibody forms analyzed. Affinity labeling of the two antibody forms was effected with the hapten analog, meta nitrobenzene diazonium fluoroborate. Examination of the diazo spectra of the labeled antibody suggested the participation of tyrosine in the active site of both turtle and duck antibody and the participation of at least one other residue in the active site of the turtle antibody. Affinity label was shown to distribute in different ratios on the heavy and light chains of the two antibody forms. The studies indicate basic similarities between the active sites of antibodies derived from two lower vertebrate species and several previously characterized mammalian species.

N-terminal sequences of immunoglobulin heavy and light chains from three species of lower vertebrates.

IT has been established that immunoglobulin heavy (H) and light (L) chains have a variable amino terminal region of about 110–120 residues1–9. This region presumably makes up the antibody binding site and is responsible for the high degree of antigenic specificity that is a characteristic feature of these molecules. In spite of the growing body of amino-acid sequence data on immunoglobulin H and L chains, the genetic mechanisms by which an almost infinite array of specific antibodies can be expressed in response to antigenic stimulation remain to be established. It has been proposed, as one possible explanation, that the genes coding for the immunoglobulin polypeptide chains, are present in the germ line10–11. This study was prompted by this concept, as well as the structural similarities that have been demonstrated among various H and L chains, suggesting that genes coding for these chains evolved from a common ancestral gene12,13. We have investigated immunoglobulins from three lower vertebrates in the hope that sequence data of the variable region of these chains will provide an insight into the genetic mechanisms responsible for antibody variability.

Further observations on uptake of S35-labelled sulfate by renal tissue in vitro.

Additional studies have been made of the accumulation of S(35) by renal cortical tissue incubated in media containing radiosulfate. This process was found to occur in several mammalian species in addition to the rat, but was not observed as a significant occurrence in three species of lower vertebrates. In the case of rat renal tissue, S(35) uptake was found to be sensitive to the pH and osmolar concentration of the medium. The character of the anions present in conjunction with K(+) affected it as well. Various factors known to be related to in vitro accumulative processes, as well as to renal sulfate reabsorption by the intact dog, were tested on rat kidney cortex to assess the effect on radiosulfate uptake. In general, all substances tested (amino acids, metabolic intermediates, ATP, metabolic inhibitors, competitive inhibitors for PAH accumulation in vitro) were found to lessen S(35) uptake, or to be without effect upon it. The one striking exception was phlorhizin, which enhanced markedly S(35) uptake in vitro, as it does sulfate reabsorption in vivo. Some implications of these findings have been discussed.