Radioimmunological quantitation of urinary trypsin inhibitor

Using monospecific antibody to human urinary trypsin inhibitor, we developed a highly specific and sensitive radioimmunoassay (RIA) for measuring human urinary trypsin inhibitor. No cross-reactivity of the antibody with protein standard serum, which contained albumin, alpha 1-antitrypsin, haptoglobin, alpha 2-macroglobulin, transferrin, IgG and IgA, was observed. The sensitivity of the system was 10 ng of trypsin inhibitor per assay tube, and 5-10 microliters of urine was sufficient to determine the concentration of trypsin inhibitor in urine. The amounts excreted in the urine of 10 healthy men and 10 healthy women were 4.83 +/- 2.46 (mean +/- SD) and 3.86 +/- 1.35 mg/day, respectively. The correlation between estimates by RIA and those by enzymic assay was r = 0.96 (p less than 0.005). The method proposed here can be used to determine the concentration of urinary trypsin inhibitor in a small amount of biological fluids and cells.     

Isolation and purification of rat acute-phase alpha 2-macroglobulin

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.     

Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary cultures

The regulation of the three major acute-phase proteins alpha 2-macroglobulin, cysteine proteinase inhibitor and alpha 1-antitrypsin by recombinant human interleukin-1 beta, recombinant human interleukin-6 and recombinant human tumor necrosis factor alpha was studied in rat hepatocyte primary cultures. Synthesis and secretion of the acute-phase proteins was measured after labeling with [35S]methionine and immunoprecipitation. Incubation of hepatocytes with interleukin-6 led to dose-dependent and time-dependent changes in the synthesis of the three major acute-phase proteins and albumin, similar to those occurring in vivo during experimental inflammation. alpha 2-Macroglobulin and cysteine proteinase inhibitor synthesis was induced 54-fold and 8-fold, respectively, 24 h after the addition of 100 units/ml interleukin-6. At the same time synthesis of the negative acute-phase protein albumin was reduced to 30% of controls. Half-maximal effects were achieved with 4 units interleukin-6/ml. Interleukin-1 beta had only a partial effect on the regulation of the four patients studied: only a twofold stimulation of alpha 2-macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor alpha did not alter the synthesis of acute-phase proteins. The stimulation of alpha 2-macroglobulin and cysteine proteinase inhibitor synthesis by interleukin-6 was inhibited by interleukin-1 beta in a dose-dependent manner. In pulse-chase experiments the effect of interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha on the secretion of acute-phase proteins was examined. Interleukin-6 markedly accelerated the secretion of total proteins and alpha 2-macroglobulin, whereas the secretion of cysteine proteinase inhibitor, alpha 1-antitrypsin and albumin was not affected. The inhibition of N-glycosylation by tunicamycin abolished the effect of interleukin-6 on the secretion of alpha 2-macroglobulin, indicating a possible role of interleukin-6 on N-glycosylation.     

A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P < 0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.     

The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

Long term production of acute-phase proteins by adult rat hepatocytes co-cultured with another liver cell type in serum-free medium

Three acute-phase proteins, haptoglobin, alpha 2-macroglobulin and hemopexin, as well as albumin, have been measured daily in the hydrocortisone-supplemented serum-free medium of pure and mixed cultures of adult rat hepatocytes for 5 and 20 days respectively. Whereas plasma protein production rapidly declined in pure culture, it remained relatively stable when hepatocytes were co-cultured with rat liver epithelial cells. In the latter cultures, an early stimulation of albumin and alpha 2-macroglobulin secretion was observed. In addition, four other plasma proteins, fibrinogen, alpha 1-acute-phase protein, alpha 1-acid glycoprotein and alpha 1-antitrypsin were shown by immunodiffusion to still be produced by day 20 of co-culture. These results suggest that hepatocyte co-cultures represent a suitable model for studying the mechanism which controls synthesis of plasma proteins, including acute-phase proteins by liver cells.     

Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide.

The radioimmunological (RIA) determination of prostaglandin (PG) E2 and of PGF2alpha in urine of humans and rats is described in detail. After extraction and chromatography PGE2 was determined by using a PGE specific antibody or by using either PGB or PGF2alpha specific antibodies after the respective conversion procedures. The three different RIA procedures were compared to each other. PGF2alpha was determined by a specific antibody to PGF2alpha. Basal excretion of PGE2 and of PGF2alpha in healthy women on free diet was 9.3 ng/hour+/-0.98 and 18.3 ng/hour +/- 2.5 respectively. Furosemide increased the excretion of PGE2 and of PGF2alpha in humans significantly, while PG-excretion rates decreased on indomethacin. In rat urine PGE2 and PGF2alpha increased markedly from 46.2 pg/min +/- 9.3 and 27+/- 3.4 to 253.8 +/- 43.3 and 108 +/- 12.6 pg/min (per one kidney) in the anesthetized-laparotomized animal. This increase was abolished after giving two different PG synthetase inhibitors.     

Sequence analysis of the putative regulatory region of rat alpha 2-macroglobulin gene

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase reactant, concentration of which in serum increases more than 100-fold in the course of inflammation. Glucocorticoid and some protein factors such as interleukin 1 (IL-1) have been known to be involved in the regulation of this plasma protein synthesis. To understand the regulatory mechanism of alpha 2M production at the molecular level, we isolated genomic DNA clones of rat alpha 2M gene and characterized the promoter region of the gene by comparing the nucleotide sequence with those of other acute-phase reactant genes. Several possible regulatory signals were identified. Particularly, a sequence (T/A)T(C/G)TGGGA(A/T) was found about at 170 bp upstream from a putative capping site, which was also found in the 5'flanking region of various acute-phase reactant genes.     

Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC–RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE–RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC–RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE–RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.     

Relationship between production of acute-phase proteins and strength of inflammatory stimulation in rats

The relationship between intensity of inflammatory stimulation and production of α(2)-macroglobulin (α2M) and α(1)-acid glycoprotein (AAG) in rats was investigated. Sprague-Dawley rats were injected with turpentine oil at doses of 0.05, 0.2 or 0.4 mL/rat. Serum levels of α2M, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured by enzyme-linked immunosorbent assay, and AAG was measured by single radial immunodiffusion. Peak serum levels of α2M and AAG in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. However, no significant differences were observed for peak serum levels of these acute-phase proteins between 0.2 and 0.4 mL/rat. Furthermore, peak serum levels of IL-6 and CINC-1 in rats injected at 0.05 mL/rat were significantly lower than those at 0.2 or 0.4 mL/rat. Thus, the production of these acute-phase proteins has upper limits, even under increased strength of inflammatory stimulation in rats injected with turpentine oil.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Development of time-resolved immunofluorometric assays for rat follicle-stimulating hormone and luteinizing hormone and application on sera of cycling rats

The aim of this study was to develop time-resolved immunofluorometric assays (TR-IFMA) for measuring rat (r)FSH and rLH. The advantages of these IFMAs are higher sensitivity due to lower background values, higher specificity as only intact molecules of FSH and LH can be measured, and a very long shelf life of the nonradioactive biotin antigens compared with radiolabeled iodine antigens. For rFSH, IFMAs are lacking, while for rLH, if present, the resources for antibodies are scarce or the mouse monoclonal antibodies (mMAbs) against LHalpha are inactive with FSH. Thus specific antibodies need to be obtained. With the final TR-IFMAs, rFSH and rLH levels were assessed during the estrous cycle and compared with those obtained with the more classical RIAs and fluoroimmunoassays (FIAs). Two IFMAs for rFSH were developed with mMAbs against the recombinant human (rec h)FSHbeta subunit (FSH56A) attached to the wall and two different rabbit polyclonal antibodies (PAbs) against the alpha subunit of rec hFSH (R93-2705) or recombinant rat (rec r)LH (R95-2715) conjugated with biotin as signal antibody. With both IFMAs, rFSH holo-molecules can be measured. Rat FSH standards could be assessed between 0.02 and 10 ng/ml with a detection limit of 0.05 and 0.24 ng/ml in buffer and serum, respectively. These detection limits in four IFMAs were 8- to 16-fold lower than those in RIAs and FIAs. This detection level allowed the measurement of FSH levels in serum of hypophysectomized (HYPEX) rats at 0.18 ng/ml. In serum of cycling rats, the FSH levels of the IFMA were 2-fold lower than those of the FIA, while in ovariectomized (OVX) rats the IFMA levels were comparable. A peak level of FSH was found during proestrus of Day 2 and gestation with both RIA and FIA, but with IFMAs at gestation only. An IFMA for rLH was set up with mMAb (hCG77A) reacting with rLHbeta as capture and rabbit PAb to rec rLHalpha (R95-2712) as signal antibody. Rat LH standard could be assessed between 0.001 and 10 ng/ml with a detection limit of 0.012 and 0.1 ng/ml in buffer and serum, respectively, which was 8-fold lower than that in RIA/FIA. In serum of HYPEX rats, LH was undetectable (< 0.04 ng/ml), whereas a high background level of 2.5 ng/ml was measured in the FIA. In serum of cycling rats, only a very low LH level of 0.14 ng/ml was measured, which strongly deviated from the level of 3.46 ng/ml with an FIA. The load of LH in serum of OVX rats was 2.91 ng/ml, which was 12-fold lower than that for the FIA. The peak level of LH was detected on proestrus Day 2 with RIA, FIA, and IFMA. In conclusion, two IFMAs for rFSH and one for rLH have been developed with high sensitivity and specificity for intact gonadotropins. The LH pattern during the estrous cycle was comparable between IFMA, RIA, and FIA, although the overall level in the IFMA was much lower, as were HYPEX levels. The FSH pattern differed only on proestrus Day 2 in the IFMA from that of RIA/FIA, showing a peak level with RIA/FIA and a basal level with the IFMA. This implies that in RIA/FIA measurements, proteins other than intact FSH and LH interfere with the analysis at proestrus Day 2 for FSH and in HYPEX, cycling, and OVX rats for LH.     

Clearance of acute-phase plasma proteins with no, high-mannose-, hybrid-, or complex type oligosaccharide side chains by the isolated perfused rat liver

The clearance of the rat acute-phase proteins alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein with no, high-mannose, hybrid or complex type oligosaccharide side chains was determined in the isolated perfused rat liver. The differently glycosylated forms of the three proteins were obtained from rat hepatocyte primary cultures treated with different inhibitors of glycosylation. The complex type forms of the three proteins were essentially not cleared by the liver during 2 h of perfusion. Unglycosylated alpha 2-macroglobulin and alpha 1-acid glycoprotein decreased in the perfusate by about 50% after 2 h; unglycosylated alpha 1-proteinase inhibitor was not taken up by the liver. The high-mannose type forms of the three proteins were nearly totally cleared. After 2 h of perfusion 10%, 45% and 30% of the hybrid type forms of alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, respectively, were cleared. The clearance rates of high-mannose and of hybrid type glycoproteins could be reduced to the rates of complex type glycoproteins by the addition of mannan to the perfusate. It is concluded that complex type glycosylation prevents the uptake of plasma glycoproteins by the liver.     

Progesterone levels and litter performance of sows following postpartum administration of prostaglandin F(2alpha)

A total of 101 sows was used to examine postpartum progesterone levels and litter performance following administration of 15 mg prostaglandins F(2alpha) (PGF(2alpha) n = 48) given within 12 h after farrowing. Daily blood samples and rectal temperatures were taken from all sows during the first 3 d post partum. Plasma progesterone (P(4)) concentrations were determined by radioimmunoassay (RIA). Regardless of treatment, plasma P(4) levels for all sows decreased in a similar fashion over the 3 d sampled. Mean (+/- SEM) P(4) on Day 2 (0.55 +/- 0.06 ng/ml) and Day 3 (0.38 +/- 0.04 ng/ml) were lower (P<0.01) than on Day 1 (0.98 +/- 0.08 ng/ml). Rectal temperature did not differ between PGF(2alpha) treated and nontreated sows nor was it different over the days measured. Litter characteristics, including survival rates on Day 7 and at weaning, and body weight on Days 3 and 35, were not affected by treatment. It was concluded that PGF(2alpha) administration to sows within 12 h post farrowing had no affect on the rate of luteal regression, as determined by P(4) concentration, nor on subsequent litter performance.     

Effect of cobra and viper venoms on alpha 2-macroglobulin activity in human, bovine, and goat sera

Incubation of human serum with cobra or viper venoms (10 micrograms/0.1 ml serum) caused negligible decrease in total protease inhibitory activity whereas alpha 2-macroglobulin activity was reduced by 67.0-82.0% in 16 hr. The action of venoms on MG activity was time dependent. Human alpha 2-macroglobulin activity was reduced to a much greater extent than goat or bovine factors by the venoms. While 25 micrograms venoms/0.1 ml serum caused 60-100% inhibition of human alpha 2-macroglobulin activity, the bovine factor was not affected under similar conditions. Goat alpha 2-macroglobulin was affected to the extent of 0-20%. Evidence is provided to show that venom proteases generate endogenous proteases in situ in human plasma or serum which in turn bind to alpha 2-macroglobulin. The venom-mediated action was abolished by prior dialysis of the serum or its dilution. Ethylenediaminetetraacetate at 10(-3) M concentration also blocked the reaction. While phenylmethylsulfonyl fluoride had no effect, pepstatin in the concentration range 10(-2) to 10(-3) M caused partial inhibition of the venom-mediated inhibition of alpha 2-macroglobulin activity in human serum.     

Development of a radioimmunoassay for human seminal plasma inhibin.

Using a homogeneous inhibin preparation from human seminal plasma with a molecular weight of about 19 000, a sensitive and specific radioimmunoassay (RIA) for inhibin has been developed. None of the purified hormones tested, such as LH, FSH and prolactin from different species, showed any cross-reaction in this RIA. Steroid hormones such as testosterone, dihydrotestosterone, oestradiol-17 beta and progesterone did not interfere with the assay. The antiserum had an affinity constant (Ka) of 2.379 X 10(9). The assay sensitivity was 10-15 ng per tube and the intra- and inter-assay coefficients of variation were 5-7% (n = 6) and 15% (n = 10) respectively. The recovery for inhibin added to the serum of a castrated man was 95-110%. Using this RIA, inhibin levels in various biological fluids and tissues were measured. Normo-spermic semen contained significantly higher levels of inhibin than did oligospermic semen. Human prostate contained a substantial quantity of inhibin. Monkey semen, rat serum, and bovine, ovine and porcine follicular fluids cross-reacted in the RIA, while ram testicular inhibin and bull semen did not do so. In developing (9-28 days of age) male rats, circulating inhibin levels showed an inverse relationship with serum FSH levels. In female rats of this age endogenous inhibin concentrations changed in parallel with those of serum FSH.     

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.     

The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.