Temporal and spatial trends in prey composition of wahoo Acanthocybium solandri: a diet analysis from the central North Pacific Ocean using visual and DNA bar-coding techniques.

A diet analysis was conducted on 444 wahoo Acanthocybium solandri caught in the central North Pacific Ocean longline fishery and a nearshore troll fishery surrounding the Hawaiian Islands from June to December 2014. In addition to traditional observational methods of stomach contents, a DNA bar-coding approach was integrated into the analysis by sequencing the cytochrome c oxidase subunit 1 (COI) region of the mtDNA genome to taxonomically identify individual prey items that could not be classified visually to species. For nearshore-caught A. solandri, juvenile pre-settlement reef fish species from various families dominated the prey composition during the summer months, followed primarily by Carangidae in autumn months. Gempylidae, Echeneidae and Scombridae were dominant prey taxa from the offshore fishery. Molidae was a common prey family found in stomachs collected north-east of the Hawaiian Archipelago while tetraodontiform reef fishes, known to have extended pelagic stages, were prominent prey items south-west of the Hawaiian Islands. The diet composition of A. solandri was indicative of an adaptive feeder and thus revealed dominant geographic and seasonal abundances of certain taxa from various ecosystems in the marine environment. The addition of molecular bar-coding to the traditional visual method of prey identifications allowed for a more comprehensive range of the prey field of A. solandri to be identified and should be used as a standard component in future diet studies.

Some properties of antisera to serum amyloid A protein (SAA): inhibition of precipitation by complexing of SAA to albumin.

Three potent rabbit antisera to human serum amyloid A protein (SAA) appear to be directed exclusibely to the carboxy terminal region not shared with the tissue amyloid A protein. Since binding to albumin completely blocks the reaction of these antisera with the antigen, and since SAA exists in serum complexed to albumin, the anti-SAA cannot be used to detect or quantitate this serum component. The possibility that similar problems will be encountered with immunoassays for molecules that exist complexed to other proteins is discussed.

Immunologic studies of water-soluble human amyloid fibrils. Comparative studies of eight amyloid preparations.

Eight preparations of soluble amyloid and degraded amyloid (DAM) were compared immunologically. Unlike amyloid fibrils, six of eight preparations of DAM proved to be relatively strong immunogens. Antisera to DAM reacted weakly or not at all with normal human serum or extracts of normal tissues, but were specifically reactive with amyloid fibrils or DAM. Comparative studies of DAM'S from eight different subjects showed some degree of cross-reactivity among them, yet demonstrated that they were not identical. Similar conclusions were obtained by quantitative precipitin and complement fixation analyses. Comparison of the amyloid fibrils with the homologous DAM by complement fixation and absorption studies demonstrated the existence in DAM of antigenic determinants that were lacking or inaccessible in the native fibrils. A search for amyloid precursors and antibodies to amyloid in the sera of 12 patients proved unsuccessful.

Degradation of serum amyloid A protein by surface-associated enzymes of human blood monocytes.

Peripheral blood monocytes incubated in a serum-free medium degraded serum amyloid A (SAA) protein along three pathways. Of 20 normal subjects, 8 degraded SAA completely with no detectable intermediates. Eight subjects transiently produced an amyloid A (AA)-like intermediate which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) with tissue AA protein and reacted with antisera to AA, whereas four subjects yielded a persistent AA-like intermediate on PAGE. This group also failed to degrade tissue AA protein. Cells from 10 patients with amyloidosis fell into the second group. The responsible enzymes appear to be serine proteases because they are inhibited by disopropyl fluorophosphate. They were not affected by epsilon-amino caproic acid, L-1-tosylamide-2-phenylethyl chloromethyl ketone, or N-alpha-p-tosyl-L-lysine chlormethyl ketone. It appears possible that the enzymes are associated with the outer membrane of the cell because only a small fraction of the activity is secreted into the medium and because enzyme activity remains after fixation of the cells with glutaraldehyde which completely stops phagocytosis. Perhaps differences in patterns of proteolysis may play a role in the predisposition to amyloidosis.

Immunologic studies of the major nonimmunoglobulin protein of amyloid. I. Identification and partial characterization of a related serum component.

Antisera have been prepared against the major nonimmunoglobulin component of secondary and familial Mediterranean fever (FMF) associated amyloid which has been called A component or acid soluble fraction (ASF). The antisera were shown to be monospecific for ASF by precipitation of (125)I-labeled antigen and gave a reaction of identity with four different ASF preparations. The antisera were able to detect a circulating component in human serum that migrated in the alpha1-globulin region. This circulating component gave a line of identity with degraded ASF by double immunodiffusion. 57 normal sera and 89 sera from patients with diseases known to be frequently associated with amyloidosis were tested by immunodiffusion for the circulating ASF component. 7% of normal sera and 50-80% of the pathologic sera had elevated amounts of this component. Absorption studies showed that all normal sera probably have small amounts of this component while cord sera do not have detectable amounts. This component was partially purified and was shown to be slightly larger than albumin. The relation of the circulating component to the acid soluble fraction of amyloid is discussed.

Structural studies of human gamma-G-myeloma proteins of different antigenic subgroups and genetic specificities.

1. Peptide maps of Fc fragments or heavy chains of 36 G myeloma proteins and two "heavy chain disease" proteins belonging to the four gamma-chain subgroups revealed very striking similarities between them. However differences in a few peptides were noted. This was most pronounced for the Ge(gamma(2)d) subgroup which lacked three peptides characteristic of the other three subgroups. While Fc fragments from different proteins belonging to the same subgroup appeared very similar, minor differences in addition to those based on currently recognized Gm factors were occasionally noted. 2. Fc fragments from Gm(a+) We(gamma(2)b) proteins had a peptide previously shown to be characteristic of normal Gm(a+) gammaG-globulins. Fc fragments from Gm(a-) molecules belonging to the We(gamma(2)b), Vi(gamma(2)c), or Ne(gamma(2)a) subgroups, whether Gm(b+), Gm(f+), or Gm(-), had the peptide previously identified in Gm(b+f+) normal gammaG-globulin. This "non-a" peptide was absent in peptide maps from Gm(-) molecules of the Ge(gamma(2)d) subgroup which contained instead another peptide with the same electrophoretic mobility but migrating slightly further on chromatography. 3. Both the "a" and "non-a" peptides were pentapeptides having three amino acids in common, and differing in the other two. The "a" peptide contained one residue of lysine, aspartic acid, threonine, leucine, and glutamic acid. The "non-a" peptides prepared from Gm(b+), Gm(f+), and Gm(-) proteins were identical and contained one residue of lysine, threonine, and methionine sulfone, and two residues of glutamic acid. 4. Several possible mechanisms for the origin of these differences, and their possible role in serologic specificity are discussed.

A variant of prealbumin from amyloid fibrils in familial polyneuropathy of Jewish origin.

Amyloid fibrils were isolated from spleen and thyroid obtained at autopsy from one patient (S.K.O.) of Jewish origin with familial amyloidotic polyneuropathy. Gel filtration on Sephadex G100 after solubilization in 5 M guanidine HCl yielded three major components with 14,000, 9,000, and 5,000 mol wt, respectively. The two larger components shared antigenic determinants with human prealbumin. Amino acid analysis and amino terminal sequence studies revealed the 14,000-mol wt protein to be an intact prealbumin subunit. The 9,000-mol wt fragment obtained in highest yield encompassed the region from position 49-127 and the 5,000 mol wt fraction encompassed the amino terminal of prealbumin (position 1-48). An amino acid substitution (Gly/Thr) was detected at position 49, where enzymatic cleavage occurred. Thus, several prealbumin-derived fragments, predominantly the carboxyl end, constitute the amyloid fibrils in a heredofamilial amyloidosis syndrome of dominant inheritance.

Physical, chemical, and ultrastructural studies of water-soluble human amyloid fibrils. Comparative analyses of nine amyloid preparations.

Amyloid fibrils were isolated from the tissues of nine patients with amyloidosis in a state of high purity by homogenization of the tissue followed by extraction with distilled water. Physical, chemical, and ultrastructural studies suggest that amyloid fibrils from different individuals resemble each other, but are not identical. In tissue sections as well as by negative staining of isolated fibrils, morphologic variations were observed. Among the isolated fibrils at least three types were noted. The majority resembled those described previously. However, one subject had two types of fibrils which differed in size and appearance. Most of the preparations sedimented as a single component with a sedimentation coefficient of 45-50S or as a larger polymer. However, two of the preparations had sedimentation coefficients of 8-9S, and a third one had a major 95S component and a minor 9S fraction. While the preparations of amyloid were not sufficiently pure for amino acid analyses, peptide maps demonstrated differences among amyloid preparations from different subjects. The amyloid fibrils in their native state proved to be remarkably resistant to digestion by a number of proteolytic enzymes. Several chemical methods were tried to produce smaller subunits. Of these, the most successful one was the use of 0.1 M NaOH which yielded a smaller, soluble fraction with sedimentation coefficients ranging from 1.1 to 2.8S. Accompanying this degradation, there was little loss of peptides or carbohydrates. Based on the results of the chemical analyses, it is estimated that the subunit produced by sodium hydroxide had a molecular weight of approximately 35,000-40,000.

Synthesis and assembly of immunoglobulins by malignant human plasmacytes. I. Myelomas producing gamma-chains and light chains.

Three basic patterns of gamma-globulin synthesis are described in malignant human plasmacytes: extreme unbalanced synthesis where only L chains are synthesized; unbalanced synthesis in which intact gammaG globulin and an excess of free L chains are synthesized and secreted; and balanced synthesis where H and L chains appear to be synthesized in equimolar amounts. Studies of the cellular products appear to reflect the biosynthetic processes of the cells in a more reliable fashion than does analysis of serum or urinary proteins. The absence of Bence Jones proteins from the urine does not necessarily indicate that free L chains are not being synthesized and secreted at the cellular level. Similarly, the completed globulin molecule secreted by malignant plasma cells may not be demonstrable by examination of serum. Patterns of globulin synthesis in human myelomatous tissues vary as do patterns of globulin synthesis in mouse plasmacytomas. Pulse-chase studies of the cells from one patient showed that a gammaG myeloma protein was assembled via an HL (half molecule) intermediate.

Synthesis and assembly of immunoglobulins by malignant human plasmacytes and lymphocytes. II. Heterogeneity of assembly in cells producing IgM proteins.

Bone marrow or lymph node cells from 10 patients whose sera contained large amounts of monoclonal IgM proteins were incubated with radioactive amino acids in short-term tissue culture. Samples of soluble cytoplasmic extracts and secreted material were examined by immunologic precipitation with specific antisera, acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and sucrose gradient centrifugation. In all samples studied, 8S IgM was the major intracellular precursor of the fully assembled 19S protein. Cells obtained from some patients contained little or no fully assembled 19S protein intracellularly; however, cells from most patients seemed to accumulate fully assembled 19S molecules intracellularly before secretion. Secreted material from both groups contained large amounts of 19S IgM. The differentiation between accumulating and nonaccumulating cells did not correlate with heavy or light chain antigenic type. Synthesis and assembly appeared to be identical in cells obtained from different anatomic sites in the same patient Studies carried out in one patient before and after therapy revealed no qualitative differences in the pathway of assembly and reflected only a decrease in the total number of immunoglobulin-producing cells.

Human monoclonal macroglobulins with specificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6-pyruvylated-D-galactose.

Two human IgM myeloma proteins, IgMWEA and IgMMAY, were found to react with agar and Klebsiella polysaccharides that contain pyruvylated D-galactose (DGal). Quantitative precipitin data and precipitin inhibition studies with methyl alpha- and beta-glycosides of 4,6-pyruvylated-D-galactose showed their combining sites to be different, although each was directed against the pyruvylated-D-Gal, one reacting most specifically with Klebsiella polysaccharides with terminal nonreducing beta-linked 2,4 pyruvylated-D-Gal, whereas the other reacted equally well with Klebsiella polysaccharides that contain 3,4 beta-linked and 4,6 alpha-linked terminal nonreducing pyruvylated-DGal. Inhibition studies showed that both sites are directed toward one of the two space isomers of 3,4- or 4,6-pyruvylated DGal, the form in which the methyl group of the pyruvate is equatorial, or endo, and its carboxyl group axial, or exo, to the plane of the acetal ring. Coprecipitation studies showed the combining site of IgMWEA to be located on an (Fab')2 fragment and not on the (Fc)5mu fragment. The monoclonal peak in the serum of IgMMAY was specifically precipitated by Klebsiella polysaccharide. Myeloma proteins with specificities of this type may occur with reasonable frequency in humans and may be a consequence of clonal expansion from inapparent infection, carrier states, or disease produced by various Klebsiella organisms.

Immunoglobulin M heavy chain disease: intracellular origin of the mu chain fragment.

Cells obtained from a patient with mu heavy chain disease synthesize a mu heavy chain fragment with a molecular weight of 55,000. The fragment is detected intracellularly after short labeling times and then is assembled inside the cell and secreted as a disulfide-linked polymer.

Molecular defect in a gamma-2 heavy chain.

The first gamma-2 (gamma2) heavy chain disease protein Gif has pyrroli-dinecarboxylic acid as its amino terminal residue, much of the Fd variable region, and an internal deletion of the heavy chain of about 100 residues corresponding to most of the Fd constant region. Normal sequence resumes with a glutamic acid residue at position 216 in the hinge region. This is the third gamma heavy chain disease protein where normal sequence resumes at the same position after the deletion.

Interaction of immunoglobulins with liposomes.

Liposomes were used as model targets to test the effect of immunoglobulins on biomembranes. Heat-aggregated immunoglobulins (Ig) exceeded native immunoglobulins in their capacity to release anions and glucose from model liposomes (either lecithin-dicetyl-phosphate-cholesterol or lecithin-stearylamine-cholesterol in molar ratios of 7:2:1). This interaction was not dependent upon the presence of cholesterol in the membrane. Mild heat-aggregation (10 min at 61.5 degrees C) increased the membrane-perturbing activity of certain Ig. Activity varied among classes and subclasses: IgG1 > pooled IgG > IgG4 > IgA1 > IgG3. IgG2, IgA2 and IgM were inert. Fc fragments of IgG were as active as IgG1, whereas Fab fragments were inactive. Prolonging aggregation to 60 min destroyed the activity of Ig. Membrane-activity could not be induced in non-Ig molecules (such as bovine serum albumin) by 10 or 60 min heat-aggregation. Density gradient centrifugation of IgG1 molecules indicated that membrane perturbing activity was associated with 15-20-s aggregates. Sepharose 4B chromatography demonstrated preferential interaction between cationic membranes and aggregated Ig, whereas anionic membranes interacted nonselectively with both native and aggregated Ig via salt-like interactions. One explanation for these data is that heat aggregation induces a conformational change in the Fc regions of certain Ig permitting them to interact with liposomes, presumably by enhancing their hydrophobic associations with membrane phospholipids.

Bence Jones proteins and light chains of immunoglobulins. Preferential association of the V lambda VI subgroup of human light chains with amyloidosis AL (lambda).

An antiserum prepared against a lambda-Bence Jones protein from a patient (SUT) who had multiple myeloma and amyloidosis had specificity for lambda-light chains of the chemically defined variable (V) region lambda-chain subgroup lambda VI. Sequence analyses of protein SUT and of five other lambda-light chains recognized immunologically as of the V lambda VI subgroup revealed that all six proteins had the N-terminal sequence characteristic for prototype lambda VI proteins. The isotypic nature of the V lambda VI subgroup was demonstrated immunochemically: lambda VI molecules were detected among light chains isolated from the IgG proteins of each of 12 normal individuals and lambda VI antigenic determinants were also detectable on the intact IgG proteins. The frequency of lambda VI molecules among lambda-type light chains is estimated to be approximately 5% based on the finding that 5 of 91 lambda Bence Jones proteins were of the V lambda VI subgroup. Proteins of the V lambda VI subgroup, in contrast to those of the other five chemically-classified lambda chain subgroup, appear to be preferentially associated with the amyloid process as evidenced by the fact that all six lambda VI proteins were obtained from patients with amyloidosis AL and, in addition, 5 of 42 lambda-type monoclonal immunoglobulins from patients with primary or myeloma-associated amyloidosis were classified by immunodiffusion analyses as having lambda VI-type light chains.

Variation with age and disease of an amyloid A protein-related serum component.

Using the radioactively-labeled alkaline-degraded acid-soluble fraction of amyloid ([ 125I ]DAA), we developed a radioimmunoassay for the previously described amyloid-related component of the human serum (SAA). Screening the sera of 228 normal individuals and of 297 patients with a variety of illnesses, we found that SAA is a component of all human sera, including cord blood (mean 94 plus or minus 57 ng/ml). The concentration of this component increases significantly with the aging process, reaching very high levels in the eighth and nine decades. It is also elevated in all cases of amyloidosis (except for those associated with nephrotic syndrome) as well as in many patients with myeloma, macroglobulinemia, lymphoma, carcinoma, rheumatoid arthritis, and tuberculosis. A marked increase was noted in the early stages of a variety of acute inflammatory and infectious states with a return to normal levels paralleling clinical improvement and faster than the erythrocyte sedimentation rate. The possible implications of this component in the genesis of amyloid and in the immune process are discussed.

The amino acid sequence of a major nonimmunoglobulin component of some amyloid fibrils.

The complete amino acid sequence of a protein, acid soluble fraction, (ASF) which constitutes up to 50% of amyloid fibrils from a patient with familial Mediterranean fever has been obtained. Partial amino acid sequences of three other proteins from patients with secondary amyloidosis were identical in the regions studied except for an alanine-valine interchange in one. The ASF contains no cysteine, does not resemble any known immunoglobulin, and has not been detected as yet in myeloma-associated amyloid.

Some effects of the administration of endotoxin in mice. Specific cleavage of serum albumin by an acid protease and the generation of amyloid serum component.

Endotoxin has been shown to induce amyloidosis in mice and to result in the appearance in serum of large amounts of amyloidrelated protein (SAA). After injection of 300 mug lipopolysaccharide Escherichia coli, SAA behaves as an acute phase reactant with levels reaching a peak of >600 mug/ml at 18-22 h and returning to base line (<50 mug/ml) by 48 h in each of four strains tested; only the endotoxin-resistant C3H/HeJ strain showed a smaller response. Lesser, though significant, elevations were also found after subcutaneous injection of 25 mg of casein, bovine serum albumin, ovalbumin, or monomeric immunoglobulin G, whereas pyrogen-free human serum albumin/U. S. Pharmacopeia failed to raise SAA levels. SAA generation may thus be a result of endotoxin contamination of these protein preparations. Also present in equivalent amounts in acidified serum from endotoxin-treated mice, but barely detectable in control sera, was a 3,000-dalton molecule whose amino acid sequence is identical to the amino terminal 24 residues of mouse albumin. The appearance of SAA and the amino terminal albumin fragment after endotoxin were unaffected by pretreatment with cobra venom factor, and equivalent levels were found in C5-deficient mice. Pretreatment with pepstatin in vivo, or before acidification in vitro, prevented the appearance of the albumin fragment but had no effect on the appearance of SAA, whereas leupeptin and antipain did not affect the appearance of either SAA or the albumin fragment. These studies suggest that the generation of SAA after endotoxin administration does not involve complement activation or intravascular proteolytic activity, whereas the liberation of a specific peptic-like cleavage product of albumin appears to be the consequence of an acid protease.

Human monoclonal gamma G-cryoglobulins with anti-gamma-globulin activity.

Seven human gammaG-myeloma proteins which were also cryoglobulins were studied with respect to their reactivity with gammaG-globulins as well as with regard to their antigenic classification within the gammaG-heavy chain subclasses. Five of the seven cryoglobulins studied were positive in at least two of the three tests used to assay for anti-gamma-globulin activity. One protein was only weakly positive in one test system and another was negative in all test systems. The structures which were recognized by the cryoglobulins were localized to the Fc-fragment. Only primate gammaG-globulins contained these antigenic determinants and in some cases the cryoglobulin appeared to show specificity for one human heavy chain subclass over the others. Antigenic analysis revealed that four of the five cryoglobulins with definite antibody activity belonged to the gammaG3-subclass, the fifth belonged to the gammaG1-subclass. The two cryoglobulins which reacted only weakly or failed to combine with gammaG-globulins were both of the gammaG1-subclass. These findings taken together with the localization of the combining site to the Fab-fragment suggests that many of these cryoglobulins may represent antibodies to gammaG-globulin, and that the cryoprecipitate in these cases represents antigen-antibody complexes of such a nature that they precipitate only in the cold.

The characterization of soluble amyloid prepared in water.

Amyloid was extracted from the spleen of a patient with primary amyloidosis by homogenizing it at high speed with water after preliminary treatments, first to remove proteins soluble in saline, and then to remove salts. The extracts containing amyloid appeared to be clear at concentrations up to 6 mg/ml of protein. The material gave little sediment on being centrifuged up to 20,000 g for 1 hr, but the protein was sedimented at 100,000 g in 1 hr. The amyloid could be precipitated from the extracts by addition of NaCl to 0.0075 mole/liter or of CaCl(2) to 0.0025 mole/liter. The protein-bound Congo red formed a red precipitate and this property was used to estimate recovery and purity of amyloid during extraction. On electronmicroscopy the isolated amyloid proved to be morphologically pure. It existed either as single filaments measuring 60-80 A in diameter or as large aggregates of these filaments.Freshly isolated amyloid in water sedimented as a single homogeneous peak with an s degrees (20,[unk]) of about 45-50S. On standing, the solution became cloudy and more rapidly sedimenting components appeared. On electrophoresis the material migrated as a homogeneous peak towards the anode. The protein had an amino acid composition different from that of all known serum proteins. It was rich in acidic amino acids and had little cysteine and methionine and no hydroxyproline. The total content of carbohydrate was less than 2%.