Sites of serum alpha-fetoprotein synthesis in the human and in the rat.

Selected tissues from human embryos of 6 to 9 weeks' gestation, from rat fetuses of 15 days' gestation, and from rats 2 days of age were incubated with (14)C-labeled amino acids. Immunoelectrophoresis of the culture fluid after incubation, using rabbit antisera against human and rat fetal serum proteins, followed by radioautography revealed that: 1) Radioactive alpha-fetoprotein was present in cultures of human liver, rat liver, and rat yolk sac, but not in cultures of human or rat brain, lung, heart, kidney, intestines, skeletal muscle, skin, or placenta; human yolk sac was not studied. 2) Radioactive transferrin was also present in rat yolk sac cultures, and the same protein was found in rat liver cultures as well. 3) Rat liver and rat placenta cultures both produced radioactive serum Ralpha(2)-globulin. Serum alpha-fetoprotein concentrations in the rat declined abruptly after birth to approximately half of the prenatal level by 2 to 3 days of age, in accord with the loss of the fetal membranes at delivery; the alpha-fetoprotein level then remained relatively constant until the rat was 6 to 8 days of age, after which synthesis of the protein was increasingly suppressed.

Development of gamma G, gamma A, gamma M, beta IC-beta IA, C 1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, alpha 1-antitrypsin, orosomucoid, beta-lipoprotein, alpha 2-macroglobulin, and prealbumin in the human conceptus.

The synthesis of gammaG, gammaA, gammaM, beta(1C)/beta(1A), C'1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, alpha(1)-antitrypsin, orosomucoid, beta-lipoprotein, alpha(2)-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in (14)C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized beta(1C)/beta(1A), C'1 esterase inhibitor, transferrin, hemopexin, alpha(1)-antitrypsin, beta-lipoprotein, alpha(2)-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of gammaM occurred as early as 10.5 wk gestation, and gammaG synthesis was found in cultures as early as 12 wk gestation; gammaA synthesis was not detected in any of the tissue cultures. With the exception of the gamma-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of gammaG and gammaM occurred primarily in the spleen, but other sites of synthesis were noted as well. Changes in the concentrations of most of these proteins and plasminogen in embryonic and fetal serum from 5.5 to 41 wk gestation, in amniotic fluid from 6.5 to 38 wk gestation, and in the sera of neonates during the 1st 3 wk postpartum are described. Although gammaA, gammaM, ceruloplasmin, or haptoglobin were not detectable in some of the embryonic and fetal sera, gammaA and ceruloplasmin were both present as early as 6.5 wk gestation, haptoglobin by 9.5 wk gestation, and gammaM by 17 wk gestation. Each of the other proteins were present in all of the sera examined.

A protein from polymorphonuclear leukocytes (LEM) which affects the rate of hepatic amino acid transport and synthesis of acute-phase globulins.

A proteinaceous secretion from phagocytizing polymorphonuclear leukocytes, termed "leukocytic endogenous mediator" (LEM), has been shown to have marked effects on hepatic amino acid transport and RNA and protein synthesis. A single injection of LEM results in a marked accumulation of labeled nonmetabolizable model amino acids in the liver of normal rats. The LEM-stimulated uptake of amino acids by liver was observed in adrenalectomized, hypophysectomized, thyroidectomized, or diabetic rats and could not be duplicated by pharmacological doses of a large variety of hormones. In addition, LEM stimulated an increased uptake of alpha-aminoisobutyric acid by isolated livers during their perfusion in vitro. LEM also stimulated an increased incorporation of orotic acid into hepatic RNA of intact rats, especially into the bound ribosomal fraction. This increased synthesis of RNA preceded an enhanced hepatic production of a number of the acute-phase plasma globulins. LEM did not stimulate the adenylate cyclase-cAMP system in liver and was not found to utilize this system as a second messenger. Thus, the effects of LEM in stimulating hepatic amino acid transport appear to be direct, without mediation by other hormones, and to be independent of cAMP. On the other hand, the ability of LEM to stimulate RNA and acute phase globulin synthesis in liver may require the presence of physiological quantities of hormones such as adrenal corticoids.

Course of serum-Ig concentrations in B12 chickens of the UM line.

The University of Munich chicken line ("UM") is isogenic in respect to the MHC and divides into normogammaglobulinemic, permanent and transient dysgammaglobulinemic individuals. Hence the immune defect is independent of the MHC. Continual analysis of the immunoglobulins until the 50th week of life revealed: one group of dysgammaglobulinemic individuals showed an initial IgG peak between the third and sixth week of life. Unusually high IgM and IgA levels occur in permanent and transient dysgammaglobulinemic individuals previous to the appearance of the IgG deficit and previous to a possible initial IgG peak. These high levels remain throughout the life of the chicken, possibly due to a missing negative feedback mechanism. Transient dysgammaglobulinemic chickens also exhibited increased IgM and IgA values after IgG normalization. Based upon our results, we postulate that the dysgammaglobulinemia defect is already preprogrammed during late embryonic development. The prevalence of a B or T-cell defect is still under discussion.

[Biochemical study on the 1st case of analbuminemia in France]. / Etude biochimique du premier cas d'analbuminémie en France

A new case of analbuminemia was described as follows for a six month's old child of Algerian origin. The discovery of the disease was made by chance, the clinical signs were limited to small oedema. The serum albumin concentration was 64 mg/1 and its immunochemical action was identical to that of normal albumin. The system reacted by an increase of the synthesis of globulins. For the subject, the alpha1-antitrypsin, ceruleoplasmin, hatoglobin, alpha2-macroglobulin, transferrin, immunoglobulins M contents were three times higher than the standard figures. The analysis of the distribution of non esterified fatty acids ususally carried by albumin was normal. On the other hand, it was possible to show that the presence of free bilirubin independant from proteins could be detected for a concentration of 17 micronmol/l. A study of the family showed a standard repartition of albumin and globulins. The genetic origin observed in the symptoms was confirmed by the consanguinity of the parents.

[Use of molecular biology approaches in studying hereditary diseases]. / Primenenie molekuliarno-biologicheskikh podkhodov pri izuchenii nekotorykh nasledstvennykh boleznei.

The application of some molecular biological methods for studying the causes of hereditary diseases induced by quantitative changes of the normal protein synthesis is discussed. The group of hereditary anemiae in humans (alpha- and beta-thalassaemia) taken as an example, possible defects at various stages of the protein synthesis control and modern methods of the analysis of these defects are considered and promises offered by such approaches are shown.

[Biosynthesis and cellular localization of betal-g-globulin in the human placenta]. / Izuchenie biosinteza i kletochnoi lokalizatsii betal-g-globulina v platsente cheloveka

Pregnancy-specific beta1-globulin (beta1-GP) described by Tatarinov and Masyukevich, 1970) was shown by means of immunodiffusion in agar gel to be similar to specific pregnancy beta1-glycoprotein of Bohn (1971) as well as to pregnancy associated plasma protein-C of Lin et al (1974). It was found that in immature placenta (6-12 weeks of pregnancy) incorporation of 14S-amino acids into beta1-GP took place. Studies on immunofluorescence showed that beta1-GP was localized in cytoplasma of trophoblastic cells of placenta chorion. Beta1-GP production apparently begins in Langhans cells and proceeds in syncytiotrophoblastic cells, but at lower activity and secretory scale.

Structural similarity between bovine conglutinin and bovine lung surfactant protein D and demonstration of liver as a site of synthesis of conglutinin.

Conglutinin is a Ca(2+)-dependent, carbohydrate-binding, serum protein which contains an N-terminal collagen-like region and a C-terminal, C-type lectin domain. To date, conglutinin, which appears to play an important role in defence mechanisms, has been fully described, by protein sequence analysis, only in the bovine system. To allow comparison of lung surfactant protein D (SP-D) with conglutinin, within one species, a full-length cDNA clone for SP-D has been isolated from a bovine lung library. The derived amino acid sequence for bovine SP-D shows a higher (78%) level of identity to the sequence of conglutinin than to the sequence of human or rat SP-D (67 and 65% respectively). However, SP-D and conglutinin are known to have different carbohydrate-binding specificities, therefore some of the 16 residues conserved in the C-type lectin domains of all three species of SP-D, but which are not conserved in conglutinin, appear likely to be involved in determination of specificity. The use of a polymerase chain reaction (PCR)-derived DNA probe for bovine SP-D in Northern blotting studies yielded a signal from bovine liver mRNA as well as the expected signal from bovine lung mRNA. Since SP-D appears to be a lung-specific protein, it seems probable that the liver is the primary site of synthesis of conglutinin.

The cDNA cloning of conglutinin and identification of liver as a primary site of synthesis of conglutinin in members of the Bovidae.

Bovine conglutinin is a collagen-like, C-type, plasma lectin which belongs to the group of proteins called 'collectins'. Two inosine-containing oligonucleotides were synthesized, based on the published protein sequence for bovine conglutinin [Lee, Leiby, Allar, Paris, Lerch and Okarma (1991) J. Biol. Chem. 266, 2715-2723], and PCR on target DNA from a bovine liver lambda gt 11 cDNA library yielded a product of the expected size of 210 bp. Screening of the library with this cDNA fragment identified a single positive clone, with an insert of 0.9 kb, coding for bovine conglutinin from residue 70 to the C-terminus. The 5' cDNA sequence, encompassing 150 bp of the 5' non-translated sequence plus the sequence encoding the leader peptide and the N-terminal residues 1-70, was completed by the use of PCR techniques. The cDNA sequence of bovine conglutinin showed 86% identity with that of bovine lung surfactant protein D (SP-D), and the derived amino acid sequence of bovine conglutinin showed 78% identity with that of bovine SP-D, which included complete identity of the leader-peptide sequences. The amino acid sequence derived from the cDNA sequence differs from the published protein sequence at four positions. Northern-blot analysis on total RNA, purified from various tissues from cattle, sheep, humans, rats and mice, showed that a strong signal of approx. 1.8 kb is present in bovine liver RNA. A weak signal of similar size was also observed in sheep liver, but not in human, rat and mouse livers. A weak signal, also of 1.8 kb, is present in the lung RNAs of all the species tested. The signals from the lung tissues are likely to be due to the cross-hybridization of the bovine conglutinin cDNA to the SP-D mRNAs of the respective species. The finding of significant signals in only the bovine and sheep liver RNA samples is indicative that serum conglutinin may be present in significant amounts only in members of the Bovidae (the family encompassing cattle, antelopes, sheep and goats) and closely related species.