Wallaby serum amyloid A protein: cDNA cloning, sequence and evolutionary analysis.

A serum amyloid A (SAA) clone was isolated from a Tammar wallaby cDNA library, the most distantly related mammalian species for which an SAA has been described to date. The clone predicts a premolecule of 127 amino acids with good homology to other mammalian SAAs, and consists of an 18 residue leader peptide and a mature protein of 109 amino acids. Evolutionary analysis at both the protein and nucleotide level indicate that the wallaby SAA clone clusters with the acute phase SAAs. However, as the SAA superfamily has undergone concerted evolution it is not possible to determine at this point which acute phase SAA it is most like. The grouping of wallaby SAA inside the acute phase SAA cluster demonstrates that at least some of the duplication events giving rise to multiple acute phase genes occurred prior to the divergence of the eutherian and metatherian mammals.

Binding of thyroxine to pig transthyretin, its cDNA structure, and other properties.

Thyroxine binding to proteins in pig plasma during electrophoresis was observed in the albumin, but not in the prealbumin and post-albumin regions. Transthyretin could be identified in medium from in vitro pig choroid plexus incubations by size and number of subunits and a very high rate of synthesis and secretion. Its electrophoretic mobility was intermediate between that of thyroxine-binding globulin and albumin. It bound thyroxine, retinol-binding protein, anti-(rat transthyretin) antibodies and behaved similarly to transthyretins from other vertebrate species when plasma was extracted with phenol. Inhibition experiments with the synthetic flavonoid F 21388, analysing the binding of thyroxine, suggested that transthyretin is not a major thyroxine carrier in the bloodstream of pigs. Cloning and sequencing of transthyretin cDNA from both choroid plexus and liver showed that the same transthyretin mRNA is expressed in pig choroid plexus and liver. The amino acid sequence derived from the nucleotide sequence revealed that pig transthyretin differs from the transthyretins of all other studied vertebrate species by an unusual C-terminal extension consisting of the amino acids glycine, alanine and leucine. This extension results from the mutation of a stop codon into a codon for glycine. The unusual C-terminal extensions do not seem to interfere with the access of thyroxine to its binding site in the central channel of transthyretin.

The cerebral expression of plasma protein genes in different species.

The cerebrospinal fluid (CSF) contains the same proteins as blood plasma, but with a different pattern of concentrations. Protein concentrations in CSF are much lower than those in blood. CSF proteins are derived from blood or synthesized within the brain. The choroid plexus is an important source of CSF proteins. Transthyretin is the protein most abundantly synthesized and secreted by choroid plexus. It determines the distribution of thyroxine in the cerebral compartment. Synthesis of transthyretin first evolved in the brain, then later it became a plasma protein synthesized in the liver. Other proteins secreted by choroid plexus are serum retinol-binding protein, transferrin, caeruloplasmin, insulin-like growth factors, insulin-like growth factor binding proteins, cystatin C, alpha 1-antichymotrypsin, alpha 2-macroglobulin, prothrombin, beta 2-microglobulin and prostaglandin D synthetase. Species differences in expression of the genes for these proteins are outlined, and their developmental pattern, regulation and roles in the cerebral extracellular compartment are discussed.

Wallaby transthyretin.

A cDNA library was constructed from liver RNA of the Australian diprotodont marsupial Macropus eugenii, the Tammar wallaby. A cloned full-length transthyretin cDNA was sequenced. The derived amino-acid sequence showed 68% overall similarity to that of human transthyretin, with 86% similarity in the thyroxine binding site. Comparisons of nucleotide and amino acid sequences from several vertebrate species indicated that the greatest differences were in the region corresponding to the disordered N-terminus of mature human transthyretin. The evolutionary trees deduced from parsimony analyses of amino acid and nucleotide sequences of transthyretins, are consistent with that derived from fossil records.

Regulation of apolipoprotein gene expression and plasma high-density lipoprotein composition in experimental nephrosis.

Hepatic and intestinal RNA levels were measured in rats made nephrotic by injection of puromycin aminonucleoside (PAN). The following increases in hepatic RNA levels, relative to controls, were measured: poly A+ (1.2), ribosomal (1.2), mRNA levels for transferrin (1.8), albumin (3.8) apolipoprotein (apo)E (2.3), apoB (2.5), apoA-II (1.9) and apoA-I (6.1). Increases of 1.5- to 2.2-fold in hepatic mRNA levels for albumin, apoA-II, apoB and apoE were measured in pre-nephrotic animals killed before the onset of proteinuria. Intestinal RNA levels in pre-nephrotic and nephrotic animals were not significantly different from control values. Transcription of the hepatic apoA-I gene increased 1.8-fold in nephrotic animals compared to controls. Immunological detection of apolipoproteins in high-density lipoproteins (HDL) separated by gradient gel electrophoresis indicated an increase in apoA-I and a decrease in apoA-IV and apoE containing HDL particles in nephrosis. To simulate the effects of increased apoA-I gene expression, human apoA-I was added to rat plasma in vivo and in vitro. ApoE was displaced from HDL by increased concentration of apoA-I. The results indicate that relatively small changes in apoA-I levels in the serum lead to significant changes in the apolipoprotein composition of HDL.

Colonization of rat molar teeth by mutans streptococci with different salivary agglutination characteristics.

The oral implantation of salivary agglutination-positive and -negative mutans streptococci was studied using streptomycin resistant (StrR) organisms. StrR Streptococcus mutans strains Ingbritt and NCTC 10449 are agglutinated by rat saliva and the StrR strains Streptococcus sobrinus 6715-13 and Strep. mutans GS5 are not. Four groups of Sprague-Dawley rats were inoculated orally with each organism (one per group) and fed a sucrose diet. A further two groups of animals were similarly inoculated with either the agglutination-positive Strep. mutans Ingbritt or the agglutination-negative Strep. sobrinus 6715-13 and fed a glucose diet. StrR streptococci were recovered from smooth-surface dental plaque of all animals on the sucrose diet with no significant difference in the recovery of agglutination-positive Strep. mutans strains Ingbritt and NCTC 10449 and agglutination-negative Strep. mutans GS5. However, the recovery of agglutination-negative Strep. sobrinus 6715-13 from smooth-surface plaque of animals on either the sucrose or the glucose diets was significantly lower than that of the other strains. Agglutination-positive Strep. mutans Ingbritt colonized smooth enamel surfaces of animals on the sucrose and the glucose diets in numbers that were not significantly different. However, the colonization of such surfaces by agglutination-negative Strep. sobrinus 6715-13 was significantly enhanced by the sucrose diet. Agglutination-positive and -negative StrR mutans streptococci were recovered from fissure plaque of all inoculated sucrose-fed animals in numbers that were not significantly different. Successful colonization of smooth enamel surfaces by the StrR streptococci resulted in increased smooth-surface caries.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a rat salivary sialoglycoprotein complex which agglutinates Streptococcus mutans.

Rat saliva agglutinated Streptococcus mutans Ingbritt and NCTC 10449 and Streptococcus sanguis NCTC 7864 but not S. mutans NCTC 10921, GS 5, or LM 7, Streptococcus sobrinus 6715-13 or OMZ 65, or Streptococcus cricetus HS 6, as measured turbidometrically. The specificity of agglutination by rat saliva was the same as that by human saliva. Agglutination was associated with a mucin complex (rat salivary agglutinin complex [rS-A]) of sulfated sialoglycoproteins, with a trace of associated lipid and an apparent Mr of 1.6 X 10(6), isolated by gel-filtration Fast Protein Liquid Chromatography. The complex was dissociated in a high-ionic-strength buffer containing 6 M urea and then fractionated by gel filtration and anion-exchange Fast Protein Liquid Chromatography into four sulfated sialoglycoprotein components, designated rS-A-1Q1, rS-A-1Q2, rS-A-1Q3, and rS-A-2, with rS-A-1Q2 being polydisperse through differential glycosylation of the polypeptide backbone. The dissociation destroyed agglutination activity. The polypeptide backbones contained up to 42% serine plus threonine and up to 40% glycine plus alanine plus proline plus valine. The carbohydrate moiety of the rS-A sialoglycoproteins consisted of N-acetylgalactosamine, sialate, galactose, fucose, N-acetylglucosamine, and small amounts of mannose, with the predominant sugar being N-acetylgalactosamine. Agglutination was inhibited by 1 mM EDTA but was restored by 1.5 mM CaCl2. Agglutination was also inhibited by 5 mM CaCl2; nonimmune sera; cationic polymers; and wheat germ, lentil, soybean, and peanut lectins. However, agglutination was not affected by lipoteichoic acid, various anionic proteins, or various sugars. Neuraminidase treatment of rS-A did not affect activity, but tryptic digestion of S. mutans did prevent agglutination. The results are consistent with calcium bridging the negative groups within the rS-A complex and allowing the approach of rS-A to the bacterial cell surface to effect a specific conformational attachment.