Quantitative immunological studies of the albumins of several species of fire bellied toads, genus Bombina.

1. Rabbit antisera against purified serum albumin of Bombina bombina were used to study relationships between B. bombina, B. variegata, and B. orientalis. 2. Quantitative micro-complement fixation tests indicated the albumins of B. bombina from central Poland and Bulgaria were indistinguishable. The albumins from several populations of B. variegata differed very slightly from that of B. bombina. The albumin of B. orientalis was quite distinct from that of B. bombina. 3. Using albumin as a molecular clock, we estimated B. bombina and B. variegata diverged within the last million years, whereas the B. orientalis lineage diverged roughly 10-12 mil yr ago.     

Slow evolution of transferrin and albumin in birds according to micro-complement fixation analysis

Summary Rabbit antisera were prepared to purified ovotransferrin from chicken (order Galliformes) and red-winged blackbird (order Passeriformes) and to purified serum albumin from chicken and rhea (order Rheiformes). Quantitative microcomplement fixation was used to compare these proteins immunologically with those of representatives of all 27 orders of birds. The average interordinal immunological distances were 123 units for transferrin and 53 units for albumin.Extensive intraordinal comparisons of transferrin among 51 species within the order Galliformes and 33 species within the order Passeriformes were also carried out. Values ranging from 0–75 immunological distance units were found within each order.Rabbit antisera to purified alligator albumin were also prepared and shown to react with representatives of all 27 orders of birds, the average immunological distance being 166 units.When the data presented here are considered in relation to the fossil record of birds, it appears that transferrin and albumin have evolved more slowly in birds than in other vertebrates. If prevailing interpretations of the fossil record are correct, transferrin has evolved 2–4 times as fast in mammals and snakes as in birds, while serum albumin has evolved about 3 times as fast in mammals, iguanids, crocodilians, and frogs as in birds. Published immunological and sequence comparisons of lysozyme and cytochromec are also consistent with a slower rate of evolution in birds than in other vertebrates. The implications of a general slowdown in the evolution of bird proteins are discussed.This research was supported in part by grants from the National Science Foundation and the National Institutes of Health to A. C. Wilson and fellowship awards to A. H. Brush and R. A. Nolan (from NIH) and A. C. Wilson (from the Guggenheim Foundation). A preliminary account of part of this work was presented at the International Congress of Systematic and Evolutionary Biology in Boulder, Colorado, on August 9, 1973.The following abbreviations are used in this work: RWBB = red-winged blackbird; OT = ovotransferrin; EW = egg white; TE = tissue extract; ND = not done; MY = million years.     

Phylogenetic relationships within the New Zealand frog genus Leiopelma — immunological evidence

Abstract

Albumin evolution is examined within the anuran genus Leiopelma using the quantitative immunological technique of micro-complement fixation (MC'F). The albumins of L. archeyi and L. hamiltoni are relatively similar to each other, yet both differ greatly from the albumin of L. hochstetteri. Using albumin as a molecular clock, we estimate that L. hochstetteri diverged from the lineage leading to L. archeyi and L. hamiltoni during the Miocene, about 15 million years ago. The divergence of L. archeyi and L. hamiltoni appears to have been a Pliocene event (about 3 million years ago).     

Phylogenetic relationships and molecular evolution in uropeltid snakes (Serpentes: Uropeltidae): allozymes and albumin immunology

Multilocus electrophoretic methods and microcomplement fixation comparisons of serum albumin are used to assess phylogenetic relationships among species of uropeltid snakes, to infer aspects of their population biology and biogeography, and to evaluate their relationships to other primitive snakes (Henophidia). There is very good agreement between phylogenetic inferences derived from the electrophoretic data and those derived from the albumin immunological data. Protein variation detected by electrophoresis is relatively high among 17 operational taxonomic units (OTUs) examined. The mean number of alleles per locus (5.1 across all OTUs), levels of polymorphism (25% of loci), and heterozygosity (4–6%), are typical of, or greater than, values reported for other snakes. Species of uropeltids are genetically highly differentiated, as measured by genetic distances (lowest interspecific Nei's unbiased genetic distances, 0.22-0.27 among several Sri Lankan species; 2.3 between Teretrurus of India and other uropeltines). The phylogenetic tree most consistent with both the immunological and electrophoretic data shows uropeltines from Sri Lanka to be monophyletic, but the Indian species are paraphyletic with respect to those from Sri Lanka. Rhinophis travancoricus of India is inferred to be the sister taxon to the Sri Lankan radiation. As the genera are presently understood, neither Rhinophis nor Uropeltis appears to be monophyletic. A biogeographic scenario derived from the phylogenetic hypothesis suggests an early diversification of uropeltids in India, followed by a single invasion into the lowlands of Sri Lanka. Subsequent evolution on Sri Lanka resulted in occupation of montane biotopes. Cylindrophis is the sister group to uropeltines and is considered a member of the Uropeltidae. The immunological data indicate no phylogenetic association between uropeltids and other ‘anilioid’ taxa, specifically Anilius, Loxocemus or Xenopeltis, although we cannot rule out a very remote relationship. We specifically reject the hypothesis that uropeltines and scolecophidians form a clade relative to henophidians. High levels of genetic variation and a trend toward negative F_IS values for polymorphic loci in three populations suggest generally large effective population sizes and outbreeding in these species. The niche-width variation hypothesis for allozyme loci is not supported by the uropeltid data. In comparison to other vertebrates, the relationship between Nei's genetic distance and albumin immunological distance in uropeltids suggests either conservative albumin evolution or strong differentiation at electrophoretic loci.     

Species specificities of l-glutamic acid decarboxylase: Immunochemical comparisons

The relationship of catfish brain l-glutamic acid decarboxylase (GAD) with those from other species were examined by comparing the extent of their interactions with antibodies against catfish brain GAD. Several immunochemical methods, such as immunodiffusion, inhibition of enzyme activity and microcomplement fixation tests were employed for these studies. Antibodies against GAD from catfish brain were observed to cross-react with the enzymes from goldfish, frog, chick and turtle. Rabbit and bovine GAD did not cross-react with these antibodies. In the presence of the antibodies, enzymes from goldfish, frog. Drosophila, chick and crayfish were inhibited 27, 46, 32, 38 and 57% respectively. Enzymes from all the other species examined were not affected at all. Microcomplement fixation tests showed that goldfish, frog and chick GAD were quite similar in immunoreactivities. These immunochemical studies demonstrated that GAD from lower vertebrates such as chick, goldfish and frog and some invertebrates such as Drosophila and crayfish are closely related to catfish GAD. In contrast, mammalian GAD shows little or no cross-reactivity with antibodies against catfish GAD.     

Lungfish albumin is more similar to tetrapod than to teleost albumins: purification and characterisation of albumin from the Australian lungfish, Neoceratodus forsteri

Lobe-finned fish, particularly lungfish, are thought of as the closest extant relatives to tetrapods. Albumin, the major vertebrate plasma protein, has been well studied in tetrapods, but there exists no comparative study of the presence and characteristics of albumin in lobe-finned fish versus other vertebrates. There is a controversy over the presence of albumin in fish, although it is present in salmonids and lamprey. The presence of albumin in lungfish has also recently been documented. We identified albumin in plasma of the Australian lungfish, Neoceratodus forsteri, using a combination of agarose gel electrophoresis, [(14)C]palmitic acid binding and SDS-PAGE. Lungfish albumin was purified using DEAE-ion exchange chromatography, and has a mass of 67 kDa, is present at approximately 8 g/L in plasma and like other fish albumins, does not bind nickel. However, like tetrapod albumins, it is not glycosylated. N-terminal and internal peptide sequencing generated 101 amino acids of sequence, which showed a high degree of identity with tetrapod albumins. Despite the similarity in sequence but congruent with the evolutionary distances separating them, lungfish albumin did not cross-react with anti-chicken or anti-tuatara A albumin antisera. Lungfish albumin has characteristics more akin with tetrapod albumin and less like those of other fish.     

Identification of a 130-kDa albumin in tuatara (Sphenodon) and detection of a novel albumin polymorphism

Electrophoretic, immunochemical, and protein sequence analyses were performed on plasma albumin of the tuatara (Sphenodon), a rare reptile endemic to New Zealand. The analyses revealed that, unlike other terrestrial vertebrates, tuatara do not seem to possess a 60- to 75-kDa plasma albumin. The common form of plasma albumin in this genus has an apparent molecular mass of 130 kDa, making it by far the largest albumin reported for any terrestrial vertebrate. Starch gel electrophoresis of samples from tuatara on 24 of the 30 islands inhabited by this genus resolved two forms of the 130-kDa albumin (albumins A and C). A third albumin of approximately 170 kDa (albumin B), reflecting a novel alloalbuminemia, was found in tuatara in three geographically isolated populations. Albumin A appears to be restricted to populations at the southern extremity of the tuatara's distribution, while albumin C was found in all but four (southern) populations. Possible explanations for the origin and distribution of these albumins are discussed.     

STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti-chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea-polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ-nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi , 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.     

Immunofluorescence as an Aid in the Early Diagnosis of Trichinosis

The established serological tests for trichinosis are often negative during the period when laboratory investigation is most likely to be useful.Another serological test, the immunofluorescence test, appears to be more promising in this respect. The results were based on studies involving experimental animals and human patients. In two rabbits orally infected with Trichinella spiralis larvae, antibodies were demonstrable by immunofluorescence on the fourth day after infection, by complement fixation on the eighth and tenth days, and by the precipitin test on the thirteenth and twenty-eighth days, respectively. In three human cases the immunofluorescence antibody test was positive two weeks (the earliest blood samples available) after onset, while precipitin and complement fixation tests did not become positive until the end of the fourth week. The immunofluorescence test thus becomes positive at least two weeks earlier than the other two, a factor which undoubtedly increases its value in diagnosis.     

Studies of relationship among terrestrial Pseudomonas,Alcaligenes, and enterobacteria by an immunological comparison of glutamine synthetase

Antibody to purified glutamine synthetase from Escherichia coli was prepared and used for an immunological comparison of glutamine synthetases from species of Salmonella, Citrobacter, Enterobacter, Serratia, Proteus, Erwinia, Aeromonas, Pseudomonas, Acinetobacter, Xanthomonas, Alcaligenes, and Paracoccus. The results of Ouchterlony double diffusion experiments and quantitative microcomplement fixation studies indicated that the amino acid sequence of this enzyme was highly conserved in different organisms. The order of relationship to E. coli was found to be similar to that derived from immunological investigations of other enzymes. In addition, congruence was observed between ribosomal RNA homology and the results of the microcomplement fixation experiments. The results also suggested that some species of Alcaligenes were more closely related to species of Pseudomonas than to each other. Immunological comparisons of glutamine synthetases appear to be very useful for the elucidation of relationships among distantly related species and genera.Non-Standard Abbreviations GS glutamine synthetase - rRNA ribosomal RNA - ImD immunological distance     

Soluble Antigens for Immunofluorescence Detection of Histoplasma capsulatum Antibodies

Soluble antigens from Histoplasma capsulatum in the mycelial and yeast phase were purified by gel filtration, fixed onto paper discs, and employed in an indirect immunofluorescence procedure to detect antibody in sera from individuals infected with H. capsulatum. The elution patterns of crude histoplasmin passed through Sephadex G-200 revealed two minor peaks of protein showing immunofluorescence, complement fixing, and precipitating-antigen activity. A large peak containing the pigment and other low molecular weight materials showed no serological activity. A polysaccharide antigen obtained from fragmented, deproteinized yeast-phase cells was reactive in the fluorescent-antibody test but showed no antigen activity in complement fixation or precipitin tests. Although certain sera from culturally proven cases of blastomycosis, coccidioidomycosis, and cryptococcosis reacted with the purified Histoplasma antigens, preliminary evaluation indicated that the immunofluorescence technique may be of value as a screening procedure for the serodiagnosis of histoplasmosis.     

Albumin evolution in polyploid species of the genusXenopus

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.     

Physical properties of some snake plasma albumins

The albumin-like proteins were purified from the plasma of three terrestrial elapids and two sea snakes. The albumin-like fraction averaged 25% (range: 21-30%) in concentration of the total plasma proteins as determined by densitometer. The physical properties of the albumin-like proteins purified from these snakes were compared. These properties, e.g. electrophoretic mobility, isoelectric point, extinction coefficient, and molecular weight, were shown to be strikingly similar to those of human plasma albumin. The physical properties of the plasma albumins of the snakes studied are similar to one another. This similarity does not explain our previous observation that Naja albumin is considerably remote immunologically from those of other elapids (Mao et al., 1983).     

Albumin evolution in West Indian frogs of the genus Eleutherodactylus (Leptodactylidae): Caribbean biogeography and a calibration of the albumin immunological clock

Antisera to serum albumins from five West Indian species of the frog genus Eleutheroductylus were prepared, and the reciprocal immunological distances (IDs) obtained were used to provide a time frame for the evolution of this group in the West Indies. One-way IDS were obtained to 25 additional species within the genus, with emphasis on those from the West Indies. These immunological data support both a recent classification of Eleutheroductylus based on an analysis of slow-evolving allozyme loci, and the monophyly of the 17 native Jamaican species as indicated by a more comprehensive electrophoretic study. This is in contrast to the results of morphological studies supporting multiple invasions of Jamaica by Eleutherodactylus . Within the subgenus Euhyas , IDS ranged from6–27 between Jamaican species, whereas between species on different islands the range was29–67. The subgenus Syrrhophus in southern North America was found to be the sister group to the subgenus Euhyus , a western Caribbean clade. Pelorius , a subgenus restricted to Hispaniola, was found to be the sister group of the subgenus Eleutheroductylus in the West Indies. The largest IDs obtained for West Indian species were those between the two major groups, the subgenera Eleutheroductylus and Euhyas .
The albumin immunological clock for Eleutheroductylus was calibrated with three events in the geologic history of the Caribbean: the breakup of the proto-Antilles (65-75 million years before present [mybp]), the emergence of Jamaica (20-30 mybp), and the uplift of the Blue Mountains in Jamaica (5-10 mybp). Immunological distances corresponding to those events yield a calibration of 1 ID=0.60 million years (my), the same as that previously obtained for other groups of amphibians and thus supports the use of albumin immunological distance as a molecular chronometer in the genus Eleutherodactylus     

Immunological relationships of the South American frog genus Eupsophus (Leptodactylidae)

The agar-gel precipitin technique was used to examine the immunological relationships of the South American frog genus Eupsophus. This taxon shows similarities with the members of the Telmatobiini tribe and presents a characteristic precipitin band, here labelled as A.     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.     

Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 g protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 g gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.     

Common antigen between coxsackievirus A 16 and enterovirus 71.

Cross immunofluorescence revealed that coxsackievirus A 16 (CA 16) shared a common antigen with enterovirus 71 (E 71). The cross reactivity of these two serotypes was also examined by complement fixation test with purified virus preparations fractionated by sucrose density gradient centrifugation and two peaks of antigenicity were detected, one being type-specific and the other cross-reacting. The common antigen was heat-stable and attributable to empty capsids. Immuno-diffusion also revealed the common antigen. Infants without antibody to E 71 developed complement fixing and precipitin antibody to E 71 after recovery from hand, foot and mouth disease caused by CA 16.     

Comparative Studies of Antigens from Mycoplasma mycoides and Escherichia coli

Extracts of sonically disrupted Mycoplasma mycoides and Escherichia coli were fractionated by sucrose density gradient centrifugation. The presence of antigen in each of the fractions was determined by complement-fixation and agar-gel diffusion precipitin tests, in which cow, pig, and rabbit anti-M. mycoides sera and rabbit anti-E. coli serum were used. Fractions of M. mycoides, with a buoyant density of 1.225 or lower, fixed complement with cow and pig anti-M. mycoides sera. These fractions also formed precipitin lines with pig antiserum. Fractions in the buoyant density range of 1.10 to 1.20 fixed complement with rabbit anti-E. coli serum, but precipitin lines were not formed. All E. coli fractions fixed complement and gave precipitin lines with homologous serum. But fractions in the buoyant density range of 1.10 to 1.20 had minimal complement fixation with heterologous M. mycoides sera. The cross-reacting antigens in M. mycoides and E. coli had a buoyant density of 1.10 to 1.20; the specific antigens were isolated from M. mycoides at a buoyant density of 1.08 to 1.02.     

The effect of ligand presaturation on the interaction of serum albumins with an immobilized Cibacron Blue 3G-A studied by affinity gel electrophoresis.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.