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Retroplacental blood samples were received after vaginal deliveries from 22 healthy primi- and multiparae aged between 20 and 30 years. Simultaneously venous blood samples were taken immediately after delivery. Pregnancy specific beta 1-glycoprotein (SP1), pregnancy associated alpha 2-glycoprotein (PZ), immunoglobulins G, M, A, secretory immunoglobulin A (S-IgA), alpha 2-macroglobulin (AMG) and unconjugated estriol were estimated quantitatively in venous as well as in retroplacental blood samples. The concentrations of SP1 in retroplacental blood were significantly lower compared with concentrations in venous blood, whereas estriol concentrations were significantly higher in retroplacental blood. All other factors differed not significantly. Only the IgM concentrations correlated in venous and retroplacental blood samples. The percentages of estimated proteins related to total protein content showed similar proportions in venous as well as in retroplacental blood. The relative protein content of SP1 was significantly lower in retroplacental blood than venous blood samples. The percentage of PZ was significantly higher in retroplacental blood. Among other things these increased concentrations of estriol and PZ in retroplacental blood be able to prevent the rejection of the trophoblast by the maternal endometrium.
The serological and physicochemical properties of the following three forms of human anti-gamma-globulin factors were compared: (a) rheumatoid factors; (b) Milgrom type anti-gamma-globulin factors; and (c) factors directed against an antigen in human gammaG-globulin that is hidden in the intact molecule and revealed by enzymatic digestion at low pH. The property common to these factors is ability to interact with human gammaG-globulin; they are distinguishable because they react with different antigenic groups on this molecule. In all of five sera, the Milgrom type anti-gamma-globulin factors were gammaM-globulins. They reacted with various human gammaG-globulin antibodies but failed to interact with gammaM-globulin type antibodies in agglutination and absorption experiments. When isolated from other anti-gamma-globulin factors, they agglutinated red cells coated with intact anti-Rh antibodies, but failed to react with cells cells coated with pepsin-digested anti-Rh antibody. These observations indicate that the agglutinator reacts with the crystallizable, inert fragment of gammaG-globulin. Anti-gamma-globulin activity directed against an antigen in human gammaG-globulin revealed by pepsin digestion was demonstrated in gammaG-, gammaA-, and gammaM-globulins. This anti-gamma-globulin factor could be absorbed by antigen-antibody precipitates containing human antibody, which shows that the hidden antigen in human gammaG-globulin is revealed not only by enzymatic digestion at low pH, but also when gammaG-globulin is present as antibody in an antigen-antibody precipitate. Rheumatoid factors and Milgrom type anti-gamma-globulin factors were also absorbed by antigen-antibody precipitates containing human antibody. The results indicate that the three distinct forms of antigamma-globulin factors may all be produced as a result of antigenic stimulation by autologous antigen-antibody complexes.
Cold-insoluble globulin (CI globulin) was purified from human plasma and identified on the basis of its sedimentation coefficient, electrophoretic mobility, and concentration in normal plasma. CI globulin was distinguished from antihemophilic factor (AHF) by amino acid analysis, position of elution from 4% agarose, and electrophoretic migration in polyacrylamide gels in the presence of sodium dodecyl sulfate without prior reduction. CI globulin and AHF could not be distinguished by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction and probably have very similar subunit molecular weights. CI globulin apparently consists of two polypeptide chains, each of molecular weight 2.0 x 10(5), held together by disulfide bonds. CI globulin was a substrate for activated fibrin-stabilizing factor (FSF, blood coagulation factor XIII). FSF catalyzed the incorporation of a fluorescent primary amine, N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulfonamide, into CI globulin and also catalyzed the cross-linking of CI globulin into multimers, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction. In the presence of fibrin, cross-linking of CI globulin by FSF occurred without the formation of CI globulin multimers. Instead, polypeptides with apparent molecular weights of 2.6 x 10(5) and 3.0 x 10(5) were seen. The formation of these polypeptides coincided with the loss of the alpha chain of fibrin and CI globulin. The polypeptides were not seen when fibrin alone was cross-linked. The formation of the polypeptides was greater in fine clots than in coarse clots, and greater in clots incubated at 0 degrees than in clots incubated at 37 degrees. In clots made from purified fibrinogen, CI globulin, and FSF, the concentration of CI globulin in the clot liquor was greater if either FSF or calcium ion was omitted and cross-linking did not take place. These observations suggest that CI globulin is enzymically cross-linked to one of the chains of fibrin, most likely the alpha chain, and is thus covalently incorporated into the fibrin clot. CI globulin is very similar to a protein in the plasma membrane of fibroblasts. The cross-linking of CI globulin to itself and to fibrin may typify reactions also involving the fibroblast membrane protein.
Both synthetic and catabolic processes determine the serum gamma-globulin level. The rate of gamma-globulin synthesis appears to be the primary factor determining the amount of serum gamma-globulin. Increase of gamma-globulin synthesis (as may occur following immunization or development of plasma cell tumor) elevates the serum gamma-globulin level. This, in turn, accelerates the fractional rate of gamma-globulin catabolism. The change in catabolic rate reduces the dimensions of the serum change from that which would occur if synthesis alone determined the serum gamma-globulin level. The present studies indicate the existence of a homeostatic mechanism controlling the rate of gamma-globulin catabolism. The mechanisms of gamma-globulin catabolism are specific and selective. Marked serum increase of other immunoglobulin components (beta(2A)-globulins and gamma(1)-macroglobulins) do not accelerate gamma-globulin catabolism. Similarly, serum albumin increases do not influence gamma-globulin catabolism. The site determining gamma-globulin catabolism is restricted to a part of the gamma-globulin molecule; i.e., on the F piece obtained by papain digestion and, by inference, on the H chains obtained by reduction and alkylation of gamma-globulin molecules.
A procedure is presented for the purification of alpha2u globulin, a protein synthesized in the liver, secreted into the serum, and excreted in the urine of male rats. The steady-state levels of alpha2u globulin in the serum and liver cytosol fraction of adult male rats have been determined using a radial immunodiffusion assay. A cell-free protein synthesizing system, easily prepared from commercial wheat germ, has been used to identify and quantitate the mRNA coding for alpha2u globulin. Poly(A)-containing RNA isolated from male rat liver directs the synthesis in the wheat germ translational system of a protein which is precipitated by rabbit anti-alpha2u globulin and migrates with authentic alpha2u globulin on sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-containing RNA from the livers of female rats or from the kidneys of male rats, tissues which synthesize no alpha2u globulin, do not direct the synthesis of alpha2u globulin in the wheat germ system. Thyroidectomized male rats had no detectable alpha2u globulin in their sera or liver cytosols, and the livers from these thyroidectomized males were found to contain no translatable mRNA for alpha2u globulin, as measured in the wheat germ system. Administration of L-thyroxine or triiodo-L-thyronine to hyroidectomized males resulted in the synthesis of alpha2u globulin, as measured by increased levels of this protein in sera and liver cytosols. This increase in alpha2u globulin synthesis following thyroid hormone treatment was accompanied by a parallel increase in the functional level of the hepatic mRNA coding for this protein. Treatment of thyroidectomized males with a variety of androgens failed to stimulate alpha2u globulin synthesis, and no alpha2u globulin mRNA could be detected in the livers from these androgen-treated thyroidectomized males. These findings indicate that thyroid hormones influence alpha2u globulin synthesis in male rat liver by acting pretranslationally, possibly by modulating gene transcription, and rule out the possibility of an indirect androgen-mediated effect of thyroid hormones in modulating alpha2u globulin biosynthesis.
BACKGROUND: Several factors may influence the concentration of serum globulins in healthy cows and thereby affect clinical interpretation; however, few studies have addressed sources of variation in globulin values. OBJECTIVES: The purpose of this study was to compare colorimetry-based total serum globulin values with electrophoretically-determined serum globulin fractions and with IgG concentration, and to evaluate diurnal and long-term physiological variation and the effects of lactation and venipuncture site on serum globulin concentrations in Holstein dairy cattle. METHODS: Serum total globulin and albumin concentrations were analyzed by colorimetry and electrophoresis in 86 lactating cows; IgG concentrations were determined by radial immunodiffusion in 41 dry and 34 lactating cows. Serum globulins were analyzed hourly for 24 hours in 8 lactating cows and weekly for 15 weeks in 6 additional cows. Globulin concentrations were compared in samples obtained from jugular and coccygeal venipuncture sites in 4 cows. Results were analyzed using parametric statistical tests. RESULTS: Colorimetry-based total serum globulin concentrations correlated well with gamma-globulin fractions (r2 = 0.87) and IgG concentrations (r2 = 0.91). Diurnal variation of total serum globulins concentration was significant (P =.01); however, globulins did not vary significantly over a 15-week period. Mean serum globulins concentration in samples obtained from the jugular vein was 2.35 g/L higher than that in samples obtained by coccygeal venipuncture (P <.0001). CONCLUSIONS: The colorimetric method used widely in routine laboratory analyses remains a useful test for globulins determination in dairy cattle. However, time of sampling and venipuncture site should be considered in the interpretation of serum globulins on serial or interindividual specimens.
Studies in the adult male hypothyroid rat, a known GH-deficient animal, have shown hepatic alpha 2U-globulin mRNA to be dependent on thyroid hormones. To study the effects of GH on alpha 2U-globulin synthesis in the absence of thyroid hormones, adult male rats were rendered hypothyroid before hormone treatment. The relative effects of bovine GH or T3 were studied by RIA of alpha 2U-globulin in hepatic cytosol in rats 6 weeks after thyroid ablation. alpha 2U-Globulin levels in vehicle-treated controls were 1.3 +/- 0.7 micrograms (+/- SD) alpha 2U-globulin/mg protein. After 2 days, GH (200 micrograms/100 g X day) resulted in an increase to 5.7 +/- 1.0 micrograms alpha 2U-globulin/mg (P less than or equal to 0.05), and T3 50 micrograms/100 g X day) resulted in an increase to 11.5 +/- 3.6 micrograms/mg (P less than or equal to 0.01). After 7 days, GH resulted in an increase to 12.4 +/- 4.6 micrograms/mg (P less than or equal to 0.01), and T3 resulted in an increase to 28.7 +/- 8.7 micrograms/mg (P less than or equal to 0.01). After 4 months of thyroid ablation, baseline hepatic alpha 2U-globulin levels fell to 4.8 ng alpha 2U-globulin/mg protein. Hepatic alpha 2U-globulin was determined 4 and 8 h after the injection of GH (200 micrograms/100 g). In these animals with markedly diminished hepatic alpha 2U-globulin levels, significant (P less than or equal to 0.01) increases occurred 4 h (25.4 ng/mg) and 8 h (57.2 ng/mg) after GH injection. The effects of treatment with bovine GH (200 micrograms/100 g X day) for 3 days on hepatic alpha 2U-globulin synthesis in liver slices and alpha 2U-globulin poly (A)+ RNA levels were measured in rats 10 weeks after thyroid ablation. GH significantly (P less than 0.05) increased alpha 2U-globulin synthesis as a percentage of total protein synthesis (from 0.01% to 0.035%) and alpha 2U-globulin mRNA as a percentage of total mRNA (from 0.03% to 0.24%). The results show that GH rapidly and specifically stimulates hepatic alpha 2U-globulin and its mRNA activity in thyroid hormone-deficient rats.
The renal accumulation of alpha2u-globulin has been implicated in the tumorigenicity of many nongenotoxic chemicals to the kidney of the male rat. Several chemicals inducing renal tumors in the male rat were shown to bind to alpha2u-globulin. This binding impairs the renal degradation of alpha2u-globulin, resulting in lysosomal overload, cell death, increased cell proliferation, and, presumably, renal tumor formation. To support the role of alpha2u-globulin accumulation in the renal toxicity of a chemical, a demonstration of the accumulation of this protein in the kidney of the male rat is one prerequisite. Monoclonal antibodies to alpha2u-globulin are available for quantifying alpha2u-globulin content; however, the procedure is time-consuming and complicated. We developed a method for the quantitation of alpha2u-globulin in renal cytosol using capillary electrophoresis. Renal cytosol fractions were analyzed by capillary electrophoresis as protein-SDS complexes. Using alpha2u-globulin purified from urine of male rats, the limit of detection was 10 microg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV </= 4%) and peak areas corresponding to alpha2u-globulin (CV </= 3%) after normalization to the internal standard was observed. Significant increases in renal alpha2u-globulin content (up to 85% of total protein content) compared to controls (approx 15%) were observed in kidney cytosol of rats treated with alpha2u-globulin nephropathy-inducing agents such as trimethylpentane or the alkylphosphonates dimethyl methylphosphonate and diethyl ethylphosphonate, but not in kidney cytosol of male rats treated with tris-(2-chloroethyl)phosphate or the nephrotoxic agent hexachlorobutadiene. A good correlation of the alpha2u-globulin contents determined by capillary electrophoresis and immunoblotting with an alpha2u-globulin-specific antibody (r2 = 0.997) was obtained. Capillary electrophoresis provides a simple, rapid, and highly reproducible quantitation of alpha2u-globulin accumulation for renal tumorigens and may assist in the risk assessment process for these chemicals.
A procedure is presented for the preparation of a (3)H-labeled complementary DNA (cDNA) specific for the mRNA coding for alpha(2u)-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to alpha(2u)-globulin, which binds to the nascent alpha(2u)-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% alpha(2u)-globulin mRNA. A labeled cDNA is made to this alpha(2u)-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-alpha(2u)-globulin sequences, this cDNA preparation is hybridized to an RNA concentration x incubation time (R(0)t) of 1000 mol of ribonucleotide per liter x sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no alpha(2u)-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for alpha(2u)-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of alpha(2u)-globulin, the level of functional alpha(2u)-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of alpha(2u)-globulin mRNA sequences as measured by hybridization to the alpha(2u)-globulin cDNA. Thus, the hormonal control of hepatic alpha(2u)-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of alpha(2u)-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.
Gamma globulin and antibody obtained from inbred C(3)H mice are split by papain and cysteine into fragments roughly one-third the size of the original Molecule (S(20,w) = 3.5S). The papain digests were characterized by starch gel electrophoresis and immunological methods. The highly heterogeneous fragments could be divided into two groups with distinct antigenic determinants (S and F), which were separated by DEAE ion-exchange cellulose chromatography. Approximately two-thirds of the fragments had S antigenic groupings and one-third had F antigenic groupings. These data are consistent with the view that mouse gamma globulin is split by papain and cysteine into three major fragments, two of which are of the S type and one of the F type. Antibody activity of the original molecule was present in the S fragments. Although the S fragments did not precipitate the antigen (hemocyanin) they were shown to bind antigen specifically in the manner of univalent antibodies. The S fragments of normal gamma-globulin were very heterogeneous with a broad spectrum of electrophoretic mobilities. Comparison of S fragments from slow and fast migrating globulins showed that the mobilities of the original gamma-globulin samples were largely reflected in the mobilities of their S fragments. Additional observations indicated that the F fragments also may help to determine the electrophoretic mobility of intact gamma-globulin molecules. S fragments of differing electrophoretic mobility were shown to have the same antigenic determinants, indicating that the structural differences responsible for the electrophoretic mobility differences were not involved in the antigenic groupings identified with rabbit antisera. The F fragments of normal gamma-globulin migrated more rapidly than the S fragments, were less heterogeneous, and showed several bands on starch gel electrophoresis. The F fragments differed antigenically from the S fragments, and had no antibody activity. Two groups of F fragments (F and F') were detected with some antisera. The gamma-myeloma protein (5563) formed in a C(3)H plasma cell tumor and similarly fragmented by treatment with papain and cysteine, produced much more discrete S and F components than were found in the normal gamma-globulin digest. The electrophoretic properties of the myeloma protein fragments were within the range observed for normal gamma-globulin fragments. Although the gamma-myeloma protein shares antigenic determinants with normal gamma-globulins it lacks some of the antigenic groupings present in the gamma-globulin preparation. Both S and F fragments from the myeloma protein share antigenic determinants with the corresponding fragments from normal gamma-globulin. In addition, both S and F fragments of normal gamma-globulin possess antigenic groupings not present in fragments of the gamma-myeloma protein, accounting for the antigenic deficiency observed on comparison of the gamma-myeloma protein with normal gamma-globulins.
OBJECTIVE: Treatment with heparin and low-dose aspirin improves fetal survival among women with antiphospholipid syndrome. Despite treatment, however, these pregnancies are frequently complicated by preeclampsia, fetal growth restriction, and placental insufficiency, often with the result of preterm birth. Small case series suggest that intravenous immune globulin may reduce the rates of these obstetric complications, but the efficacy of this treatment remains unproven. This pilot study was undertaken to determine the feasibility of a multicenter trial of intravenous immune globulin and to assess the impact on obstetric and neonatal outcomes among women with antiphospholipid syndrome of the addition of intravenous immune globulin to a heparin and low-dose aspirin regimen. STUDY DESIGN: This multicenter, randomized, double-blind pilot study compared treatment with heparin and low-dose aspirin plus intravenous immune globulin with heparin and low-dose aspirin plus placebo in a group of women who met strict criteria for antiphospholipid syndrome. All patients had lupus anticoagulant, medium to high levels of immunoglobulin G anticardiolipin antibodies, or both. Patients with a single live intrauterine fetus at </=12 weeks' gestation were randomly assigned to receive either intravenous immune globulin (1 g/kg body weight) or an identical-appearing placebo for 2 consecutive days each month until 36 weeks' gestation in addition to a heparin and low-dose aspirin regimen. Maternal characteristics, obstetric complications, and neonatal outcomes were compared with the Student t test and the Fisher exact test as appropriate. RESULTS: Sixteen women were enrolled during a 2-year period; 7 received intravenous immune globulin and 9 were given placebo. The groups were similar with respect to age, gravidity, number of previous pregnancy losses, and gestational age at the initiation of treatment. Obstetric outcomes were excellent in both groups, with all women being delivered of live-born infants after 32 weeks' gestation. The rates of antepartum complications such as preeclampsia, fetal growth restriction, and placental insufficiency (as manifested by fetal growth restriction or fetal distress) were similar between the 2 groups. Gestational age at delivery (intravenous immune globulin group, 34.6 +/- 1.1 weeks; placebo group, 36.7 +/- 2.1 weeks) and birth weights (intravenous immune globulin group, 2249.7 +/- 186.1 g; placebo group; 2604.4 +/- 868.9 g) were similar between the 2 groups. There were fewer cases of fetal growth restriction (intravenous immune globulin group, 0%; placebo group, 33%) and neonatal intensive care unit admission (intravenous immune globulin group, 20%; placebo group, 44%) among the infants in the intravenous immune globulin group than those in the placebo group, but these differences were not significant. CONCLUSION: A multicenter treatment trial of intravenous immune globulin is feasible. In this pilot study intravenous immune globulin did not improve obstetric or neonatal outcomes beyond those achieved with a heparin and low-dose aspirin regimen. Although not statistically significant, the findings of fewer cases of fetal growth restriction and neonatal intensive care unit admissions among the intravenous immune globulin-treated pregnancies may warrant expansion of the study.
We describe a simple cloning procedure for alpha 2u-globulin that requires neither enrichment of mRNA for cloning nor purification of a specific probe for screening recombinant colonies. Total adult male liver poly(A)+RNA was used as template for cloning, and the subsequent recombinant colonies were screened by comparing hybridization to radioactive cDNA probes prepared from hepatic male and female mRNA, respectively. Almost all of the selected "male-specific" clones were later shown to contain alpha 2u-globulin sequences. This cloned alpha 2u-globulin cDNA has been shown to specifically hybridize to male rat liver RNA, which, when isolated and translated in vitro, codes for a 21,000-dalton protein (pro-alpha 2u-globulin) immunologically identical to alpha 2u-globulin. When translation occurs in the presence of pancreatic microsomes this in vitro synthesized pro-alpha 2u-globulin is processed to the 19,000-dalton mature form of alpha 2u-globulin. The nucleotide sequence of the alpha 2u-globulin cDNA has been determined, thus elucidating the complete amino acid sequence of alpha 2u-globulin and most of the hydrophobic "leader" sequence of pro-alpha 2u-globulin. The amino acid sequence deduced from the cDNA is in agreement with the partial sequence that we previously determined by sequential Edman degradation of the purified protein. alpha 2u-Globulin cDNA clones contain within the 3'-untranslated region one or both of the two putative polyadenylylation/transcription termination sites (A-A-T-A-A-A and A-A-T-T-A-A-A). Either of these can be used, generating alpha 2u-globulin mRNA species of two lengths. A codon usage analysis of the cDNA showed that, although all six leucine codons are used for the 14 leucine residues in mature alpha 2u-globulin, the seven leucines in the partial leader sequence reported are all encoded by the same codon, CTG. The primary amino acid sequence contains a unique Asn-Gly-Ser sequence, likely to be in beta-turn conformation, as the probable site of glycosylation for this glycoprotein.
Conventionally prepared immune serum globulin frequently produces severe side effects when administered intravenously. A modified preparation in which 4 to 5 interchain disulfide bonds have been reduced and alkylated has been made for intravenous use. However, reduction and alkylation may affect Fc-mediated functions of immunoglobulin G, particularly its ability to fix complement by the classical pathway. To determine whether reduction and alkylation alters the protective activity of immune serum globulin in vivo we compared it with two less harshly prepared globulins (pH 4 treated or ultrafiltered) in an infant rat model of Haemophilus influenzae b infection. Antibody binding to the capsular and noncapsular components of H. influenzae b and in vitro bactericidal activity were similar in the globulin preparations. Infant rats were treated with various doses of globulins adjusted to provide identical concentrations of anticapsular antibodies as measured by the Farr radioactive antigen binding assay. At high doses of anticapsular antibody (greater than 1,500 ng per pup), all preparations protected well. At marginal doses (750 ng per pup), however, rats given reduced and alkylated globulin had a significantly greater incidence of bacteremia (P less than 0.05), meningitis (P less than 0.01), and death (P less than 0.05) and a higher magnitude of bacteremia (P less than 0.02) than rats who received pH4-treated or ultrafiltered globulins. These differences were not due to differences in anticapsular antibody concentrations achieved in the serum. The 50% protective serum concentrations of anticapsular antibody in this model were 200 to 300 ng/ml for reduced and alkylated globulin and 100 to 200 ng/ml for acid-treated globulin. Absorption of the globulins with purified H. influenzae b capsule reduced in vitro bactericidal activity and rat protective activity. However, the magnitude of bacteremia was lower in rats receiving absorbed pH 4-treated globulin than in those receiving absorbed reduced and alkylated globulin (P less than 0.05). We conclude that reduced and alkylated immunoglobulin G provides significantly less protective activity against H. influenzae b infection in this model than globulins not so modified, and we suggest that the altered Fc function of the immunoglobulin G, such as the decreased ability to fix complement by the classical pathway or decreased Fc-mediated opsonization, may be responsible for this impairment.