Cyclic adenosine 3',5'monophosphate/protein kinase A and mitogen-activated protein kinase 3/1 pathways are involved in adenylate cyclase-activating polypeptide 1-induced common alpha-glycoprotein subunit gene (Cga) expression in mouse pituitary gonadotroph LbetaT2 cells

Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.     

Comparison of properties of thiol proteinase inhibitors from rat serum and liver

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.     

Rat plasma 25-hydroxyvitamin D3 binding protein: An inhibitor of the 25-hydroxyvitamin D3-1 α-hydroxylase

An antibody was prepared from serum of rabbits injected with a pure inhibitor protein obtained from rat serum for chick renal 25-hydroxyvitamin D₃-1α-hydroxylase. The antibody was separated from the endogenous inhibitor in rabbit serum. The antibody shows a single precipitin line with the rat serum antigen and with crude calf serum. Furthermore, the antibody removes the 4.0 S 25-hydroxyvitamin D₃ binding protein from rat serum. The removal of the 25-hydroxyvitamin D₃ binding protein from rat serum with antibody brings about a proportionate removal of inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase. The pure inhibitor binds 25-hydroxyvitamin D₃, as demonstrated by sucrose density gradient sedimentation, and shows specificity of binding identical to the serum transport globulin for 25-hydroxyvitamin D₃. Thus, the previously reported inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase in rat preparations is the serum 25-hydroxyvitamin D₃ transport protein or some derivative thereof. The antibody added to rat renal mitochondrial preparations does increase the activity of the 1- and 24-hydroxylases slightly but not markedly.     

Comparative characteristic of serpin--alpha-1 proteinase inhibitor in human and mice serum

Serpin alpha-1-proteinase inhibitor have been studied in human subjects and in mice of different lines as acute phase reactant and during tumor development. In humans, there was no difference of serpin activity between men and women. Increased activity was noted in men with acute trauma (acute phase reaction). Comparatively to male, in female mice of different lines decreased activity of serum alpha-1-proteinase inhibitor, was shown. There was no increase of alpha-1-proteinase inhibitor activity during inflammation induced by zymosan administration in mice. alpha-1-proteinase inhibitor belongs to acute phase reactants in humans but not in mice; for mice alpha-2-macroglobulin is a more typical acute phase reactant as compared to alpha-1-proteinase inhibitor. Murine tumor development (hepatoma HA-1, lymphosarcoma LS, Lewis lung adenocarcinoma) was followed by a decreased activity of serum alpha-1-proteinase inhibitor both in successfully treated and untreated groups. According to data of literature, similar dated were obtained in humans with tumors. It was suggested that changes of expressiln of alpha-1-proteinase inhibitor by tumors and its secretion were involved in decreased activity of alpha-1-proteinase inhibitor.     

Proteolytic enzymes; further studies on protein,polypeptide, and other inhibitors of serum proteinase,leucoproteinase, trypsin,and papain

1. Serum proteinase precursor was found in plasma protein fractions I and III of Cohn. Inhibitors of serum proteinase, leucoproteinase, trypsin, and papain were found in fractions IV-1 and IV-4, and to a lesser extent in fractions V and I. 2. Pancreatic, soy bean, lima bean, and egg white inhibitors inhibited trypsin stoichiometrically. Pancreatic inhibitor had comparable inhibitory activity against serum proteinase; soy bean inhibitor had somewhat less, lima bean inhibitor even less, and egg white inhibitor very little. None of these inhibitors appreciably inhibited leucoproteinase or papain. 3. Serum and fractions IV - 1 and IV - 4 had marked inhibitory activity against trypsin and leucoproteinase, and somewhat less against serum proteinase and papain. The inhibitory activity of the plasma proteins against trypsin and leucoproteinase was due almost entirely to fractions IV - 1 and IV - 4; against serum proteinase and papain fraction V was slightly more important. The "reconstituted plasma proteins" accounted for 8 to 25 per cent of the proteinase-inhibitory activity of whole serum or plasma. 4. The proteinase-inhibitory activity of serum, plasma protein fractions, and soy bean inhibitor was heat labile, while that of pancreatic, lima bean, and egg white inhibitors was relatively heat stable. 5. Reducing and oxidizing agents, in very high concentration, inhibited serum proteinase, as well as trypsin and leucoproteinase. These proteinases were not influenced by mercurial sulfhydryl inhibitors, indicating that free sulfhydryl groups do not play an important part in their activity.     

Determination of the elastase activity of the leukocyte thermostable alpha-glycoprotein from human blood plasma

The possibility of isolation of specific human granulocyte antigen--leukocyte thermostable alpha-glycoprotein (LTG) from plasma was shown. The isolation of preparations containing LTG was carried out on CNBr-activated sepharose with immobilized soluble fraction of pus. Using immunochemical analysis with standard antisera, the absence of plasma and leukocyte proteins and the presence of LTG in the preparations obtained were demonstrated. Pus may comprise a protein component capable of binding LTG. Elastase activity of the preparations obtained was established. The identity of LTG and granulocyte elastase is suggested.     

Induction of an Inhibitor of Influenza Virus Hemagglutination by Treatment of Serum with Periodate

Serum is commonly treated with potassium periodate to destroy nonspecific inhibitors of influenza virus hemagglutination. We have observed, however, that such treatment of serum without pre-existing inhibitor produced high titers of inhibitor against certain strains of influenza virus. Inhibitor was induced in the serum from several different animal species but not in hamster or mouse serum. Periodate treatment of serum albumin, fraction V, from several animals, including man, creates this inhibitor. Our data indicate that the inhibitor induced in the serum of various animal species differs in its mechanism of induction and in its resistance to receptor-destroying enzyme and trypsin. Hemagglutination by B/Singapore/3/64, B/Colorado/2/65, B/Georgia/1/65, and B/Massachusetts/3/66 strains of influenza virus is inhibited by periodate-treated human serum at dilutions as high as 1:5,120. The routine use of periodate treatment of serum in diagnostic and surveillance studies of influenza virus infections is not recommended.     

Immunochemical determination of the heterogeneity of leukocyte thermostable alpha-glycoprotein

A comparative immunochemical and physico-chemical study of leukocyte thermostable alpha-glycoprotein (LTG) has been performed in pus extract, hemolysate, leukolysate and blood plasma. Immunochemically determined LTG is heterogeneous in respect of molecular charge, sialic acid content and temperature stability. LTG in the blood plasma has a form of alpha-globulin, while in hemolysate it is a beta-globulin, with both forms present in pus. In leukolysate LTG possessing incomplete immunochemical identity with pus and plasma LTG was detected. It is suggested that LTG forms detected differ in their biological activity as well.     

Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.     

An inhibitor(s) of globin mRNA translation in rabbit serum

1. A factor found in rabbit serum inhibits globin mRNA translation in vitro. 2. Inhibition of globin mRNA translation has been demonstrated in a cell-free rabbit reticulocyte lysate. 3. The inactivation of globin mRNA translation is not attributed to either serum albumin or ribonuclease activities. 4. Dialyzing the inhibitor for 24 hr at 4 degrees C does not result in the diminution of the inhibiting activity. However, the activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin for 2 hr. 5. Ion exchange chromatography points to the inhibitor being a neutral protein, whereas, polyacrylamide gel electrophoresis reveals one major band with mol. wt 43 kDa. 6. The activity of the inhibiting material 3-fold greater in anemic serum than in normal serum. 7. These studies suggest that rabbit serum contains a protein inhibitor that may play a physiological role in regulating protein synthesis in red cells.     

Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.     

Comparative immunochemical and immunocytochemical study of 3 soluble granulocytic proteins in blood cells and serum

Using immunofluorescence, it has been shown that leukocyte thermostable alpha-glycoprotein (LTAG) and leukocyte beta-globulin (LBG) are, like lactoferrin, components of polynuclear leukocytes. These proteins were found in the perinuclear zone, cytoplasm membrane and extracellularly. Serum LTAG concentration increases in immune inflammatory diseases. In severe forms of diabetes and glomerulonephritis there is a rise in LBG concentration. The biological role of LTAG and LBG is unknown.     

The mt1 melatonin receptor and RORbeta receptor are co-localized in specific TSH-immunoreactive cells in the pars tuberalis of the rat pituitary.

The pars tuberalis (PT) of the pituitary represents an important target site for the time-pacing pineal hormone melatonin because it expresses a large number of mt1 receptors. Functional studies suggest that the PT mediates the seasonal effects of melatonin on prolactin (PRL) secretion. The aim of this study was the characterization of the phenotype of melatonin-responsive cells. Furthermore, we determined whether RORbeta, a retinoid orphan receptor present in the PT, was co-expressed in the same cells. We combined nonradioactive in situ hybridization (ISH) with hapten-labeled riboprobes for detection of the receptors and immunocytochemistry (ICC) for detection of alphaGSU (alpha-glycoprotein subunit), betaTSH, betaFSH, betaLH, GH, PRL, and ACTH. Expression of mt1 mRNA was found in small round cells, co-localized with alphaGSU and betaTSH. However, not all betaTSH-containing cells expressed mt1 mRNA. The distribution of mt1- and RORbeta-positive cells appeared to overlap, although more cells were labeled for RORbeta than for mt1. Gonadotrophs, as well as other pars distalis cell types, were never labeled for mt1 melatonin receptor. Therefore, this study identifies the "specific" cells of the PT as the mt1 melatonin receptor-expressing cells.     

Carbonate inhibition of leukotriene D4-dipeptidase in human serum.

Human serum contains an inhibitor of leukotriene D4 (LTD4) dipeptidase which was separated from the enzyme by ultrafiltration (Amicon, YM-10). Removal of the inhibitor resulted in a 3- to 5-fold increase in total LTD4-dipeptidase activity in the material retained by the filter. Inhibitor activity (which was assayed with a partially purified LTD4-dipeptidase) was recovered in the filtrate. Ultrafiltration of serum using YM-3, YM-1, and YC-05 membranes suggested an inhibitor molecular weight of less than 500. Elution of inhibitor activity from a Bio Gel P2 gel filtration column was identical to the elution pattern of pure carbonate. The inhibitor was heat stable (95 degrees C, 30 min), stable in 0.1 N NaOH, but rapidly inactivated by 0.1 N HCl at both 4 degrees C and 30 degrees C. Partially purified LTD4-dipeptidase was inhibited by carbonate and phosphate but not by nitrate, sulfate, or chloride. Based on these observations it was concluded that the inhibitor of LTD4-dipeptidase in human serum either was carbonate or required carbonate. The relative concentrations of LTC4, LTD4, and LTE4 appear to be important parameters in determining the duration and intensity of LT mediated reactions. The relative concentration of carbonate in serum or extracellular fluids might, therefore, be a factor in modulating localized LT mediated responses.     

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in porcine serum.

Characterization of the trypsin-, chymotrypsin- and elastase-inhibiting properties of porcine serum was carried out by gel filtration on Ultrogel, AcA 44, and agarose gel electrophoresis with subsequent processing for protease-inhibiting activity. Moreover, by allowing the fractions obtained from gel filtration to react with antibodies to porcine serum protease inhibitors, the specific inhibiting properties of these inhibitor molecules were identified. At least six protease inhibitors were identified and partially characterized in porcine serum. Two alpha 2 -macroglobulins (alpha 2 Mf and alpha 2 Ms), homologues to human alpha 2 -macroglobulin, with slightly different electrophoretic mobilities, were both found to exhibit trypsin, chymotrypsin and elastase inhibiting activity. Alpha 1 -Protease inhibitor (Mr 51 000), a homologue to human alpha 1 -protease inhibitor (alpha 1 -antitrypsin), also showed trypsin-, chymotrypsin- and elastase-inhibiting properties. Inter-alpha-trypsin inhibitor (Mr 162 000 and 129000), a porcine serum counterpart to human inter-alpha-trypsin inhibitor, showed trypsin- and chymo-trypsin-inhibiting properties. In addition, a specific trypsin inhibitor, alpha 2 -antigrypsin (Mr 58 000), and a specific elastase inhibitor, beta-elastase inhibitor, were characterized in porcine serum, and these seem to have no counterparts in human serum.     

The interactions of the non-specific inhibitor of hyaluronidase with hyaluronidase and proteolytic enzymes in vivo

1. Following intravenous administration of testes hyaluronidase in rabbits and dogs, there is a decrease in the level of the non-specific inhibitor of hyaluronidase in serum. 2. If a large amount of hyaluronidase is injected, the inhibitor level is reduced to zero and hyaluronidase may be present in serum for some time after the injection. The hyaluronidase activity of such samples of serum increases when the serum is incubated with papain. 3. Hyaluronidase activity is found in the livers of the injected animals in large amounts and this activity is increased considerably when the homogenate of this tissue is incubated with papain. 4. Intravenous administration of several proteases or venom produces a decrease in the serum inhibitor level. Intravenous administration of streptokinase produces such a decrease in rabbits but not in dogs. 5. There is a correlation between the depletion of the inhibitor from the serum and the occurrence of a slow, persistent depression of blood pressure upon administration of proteolytic enzymes.     

The detection of streptococcal cell-wall proteins that form complexes with human macroglobulins]

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.     

Protease inhibitors in porcine serum and their immunological relationships to human protease inhibitors.

A close molecular relationship exists between the protease inhibitors of porcine serum and those of human serum as shown by studying their immunological cross-reactivities with gel diffusion and immunoelectrophoretic methods. On studying seven different antisera to human protease inhibitors, five were found to cross-react with porcine serum, and on this bisis it was possible to identify alpha 2 -macroglobulin f, alpha 2 -macroglobulin s, alpha 1 -protease inhibitor, inter-alpha-trypsin inhibitor, antithrombin and alpha 2 -antiplasmin in porcine serum. Antisera to four of these porcine serum inhibitors (alpha 2 -macroglobulin f, alpha 2 -macroglobulin s, alpha 1 -protease inhibitor and inter-alpha-trypsin inhibitor) were produced and were shown to react immunologically with their human serum protease inhibitor counterparts.     

Structural characterization of the Mr = 55,000 antigen (ZP3) of porcine oocyte zona pellucida. Purification and characterization of alpha- and beta-glycoproteins following digestion of lactosaminoglycan with endo-beta-galactosidase

The major macromolecular component of the porcine oocyte zona pellucida is a Mr = 55,000 antigen, termed ZP3, comprised of greater than 25 charge isomers. ZP3 was purified to apparent electrophoretic homogeneity from nonreduced, sodium dodecyl sulfate-treated porcine zonae pellucidae by chromatography on Sephacryl S-400 and hydroxylapatite resins. The carbohydrate moiety of purified ZP3 was comprised of a heterogeneous population of acidic lactosaminoglycans as evidenced by the saccharide composition and size distribution of glycopeptides produced by Pronase digestion of ZP3, as well as by the sensitivity of ZP3 to digestion with Escherichia freundii endo-beta-galactosidase. Endo-beta-galactosidase-digested ZP3 was resolved by gel electrophoresis into two components, termed alpha-glycoprotein and beta-glycoprotein, with Mr values (nonreduced) of 46,000 and 42,000, respectively. Each was comprised of fewer and more neutral charge isomers than ZP3. Following purification by reverse phase high performance liquid chromatography, the alpha- and beta-glycoproteins of endo-beta-galactosidase-digested ZP3 were distinguished on the basis of amino acid and carbohydrate compositions, amino-terminal sequence analyses and peptide mapping experiments, thus suggesting differences in the primary structures of their respective polypeptide moieties. Corresponding dissimilarities in the immunoreactivities of the alpha- and beta-glycoproteins toward polyclonal antisera raised against ZP3, alpha-glycoprotein, and beta-glycoprotein were revealed by competitive binding radioimmunoassays as well as by immunoblotting experiments. Collectively, the data were interpreted to indicate that the Mr = 55,000 antigen of porcine oocyte zona pellucida is in fact comprised of overlapping families of charge isomers corresponding to two structurally and immunologically distinct lactosaminoglycan-containing glycoproteins.