Trypanosoma cruzi: acute infection affects expression of alpha-2-macroglobulin and A2MR/LRP receptor differently in C3H and C57BL/6 mice.

Although a complete cellular and humoral immune response is elicited in Chagas' disease, recent data suggest that other natural elements of innate immunity may also contribute to the initial host primary defense. alpha-Macroglobulins are a family of plasma proteinase inhibitors that are acute-phase reactants in Trypanosoma cruzi-infected mice and humans. Mice contain a tetrameric alpha-2-macroglobulin (MAM) and a monomeric murinoglobulin (MUG). Heterogeneity in their reactions was observed in murine T. cruzi-infected plasma A2M levels despite an overall increase. In addition, up-regulation of the A2M receptor (A2MR/LRP) was observed in peritoneal macrophages during T. cruzi infection. Here, we show that during T. cruzi infection (Y strain), the MAM and MUG hepatic mRNA levels and the corresponding plasma protein levels were up-regulated in C3H and C57BL/6 (B6) mice, but with different kinetics. On the contrary, A2MR/LRP mRNA levels increased in acutely infected C3H mice, but decreased in B6 mice, in both liver and heart. Immunocytochemistry of infected B6 heart cryosections confirmed a less intense endothelium labeling by the fluoresceinated ligand for A2MR/LRP. On the other hand, infected B6 spleen cells displayed higher F-A2M-FITC binding and MAC1 expression, confirming higher A2MR/LRP expression in macrophages. In uninfected mice, as well as after T. cruzi infection, higher A2M plasma levels were measured in C3H mice than in B6 mice. The lower tissue T. cruzi parasitism found in C3H-infected mice could reflect an inhibitory effect of A2M on parasite invasion. Our present data further contribute to clarifying aspects of the role of A2MR/LRP in a model of acute Chagas' disease in different mouse strains.

Characterization of recombinant bovine conglutinin expressed in a mammalian cell.

We describe here the successful expression of recombinant bovine conglutinin in CHO cells as well as its physical and biological characteristics. Geneticin-resistant transformants harboring bovine conglutinin cDNA in the expression vector pNOW/CMV-A were screened by Western blot analysis for secretion into media of recombinant conglutinin. A four-day amplification of the transgene with increasing concentrations of methotrexate resulted in a dose-dependent increase in the production of recombinant conglutinin to a final concentration of 18.6 microg/ml of media. Recombinant conglutinin purified from this media by affinity column chromatography on mannan-agarose had a migration pattern similar to that of native conglutinin on polyacrylamide gel electrophoresis under reducing, nonreducing, and native conditions. The recombinant conglutinin exhibited sugar binding, conglutination, hemagglutination inhibition, and neutralization of influenza A virus, activities engaged in by the native conglutinin. This is the first report describing a high level of expression of a serum cruciform collectin with the full range of biological activity.

Primary structure of bovine collectin-43 (CL-43). Comparison with conglutinin and lung surfactant protein-D.

Collectin-43 (CL-43) is a bovine serum protein that is composed of subunits of three identical chains, each of which contains a collagen region and a C-type carbohydrate recognition domain; thus, CL-43 belongs to the collectins (group III of the C-type lectins). We have derived the complete primary sequence of CL-43 using partial protein sequencing, cDNA cloning, and reverse transcription-polymerase chain reaction techniques. The primary sequence of CL-43 shows that it contains an N-terminal region of 28 residues, followed by a collagenous domain of 38 repeats of Gly-Xaa-Yaa and then a C-terminal section of 159 residues, containing a short "neck" region and the carbohydrate recognition domain with the conserved residues found in all C-type lectins. The amino acid sequence of CL-43 showed 74% identity to bovine conglutinin and 70% identity to bovine lung surfactant protein D (SP-D), but the collagen region is considerably shorter than the 57 Gly-Xaa-Yaa triplets found in conglutinin and SP-D. Northern blot analysis showed that CL-43 was only synthesized in bovine liver, with no detectable signal in a variety of other bovine tissues, including lung. No cross-hybridizing signals were detected in mRNA from sheep, human, rat, or mouse liver. Since CL-43 and conglutinin have only been detected in members of bovidae, it is probable that an ancestral gene of these two proteins was first derived from a SP-D-like gene, and that this ancestral gene duplicated during evolution.

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.

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.

Regenerative responses to exchange perfusion.

Isovolemic exchange perfusion of conscious normal and splenectomized rats to a Hct of +/- 3% with the perfluorocarbon based oxygen transport fluid, Fluosol-DA 20%, is characterized by: 1) a greater than projected depression of fibrinogen and the plasma globulins and 2) a rapid regeneration of these and certain other plasma proteins. Similar responses were observed in a study of PFC resuscitation of hemorrhagic shock in splenectomized dogs in which there was a selective depression of the platelets, the plasma globulins, IgG and fibrinogen. In the rats, the red cells and platelets required 14-21 days to return to control levels while the leukocytes returned to normal in 1-2 days. The globulins and fibrinogen exhibited a transient rebound response at 3 and 12 hours post exchange respectively with total protein levels restored to control levels at 48 hours. In the shock study, the leukocytes, which remained at control levels throughout the shock period and for 1 hour post resuscitation were 2.5x control levels at 24 hours. The platelets which were depressed to 20% of control levels following resuscitation remained depressed through the 24 hour time course of the study. Implicit in these results is the possibility that; A) thrombosis and B) immunosuppression may be caused by some component(s) of the perfusion/resuscitation fluid.

Synthesis and secretion of some plasma proteins by tissue slices of Morris hepatoma 7777 and liver from control and turpentine-injected rats.

Slices of Morris hepatoma 7777 or rat liver isolated from control or turpentine-injected rats were incubated for 2 h with 14C-leucine. Radioactivities incorporated into albumin, alpha-fetoprotein, fibrinogen, alpha 1-AP-globulin, haptoglobin and alpha 1-acid glycoprotein were determined after the proteins had been isolated from the incubation medium or tissue homogenate by immunoprecipitation with monospecific antisera. It was found that hepatoma synthesizes fibrinogen, alpha 1-AP-globulin and alpha 1-acid glycoprotein in the amounts comparable to rat liver, whereas formation of albumin and haptoglobin is reduced 5- to 10-fold. Local inflammation elicited by injection of turpentine to tissue donors increased formation of acute-phase protein in liver slices but had no effect on synthesis of these proteins in preparations of Morris hepatoma, although certain ultrastructural changes in the Golgi complex were observed not only in the liver but also in the tumour.

Electrophoretic pattern of proteins in lepromatous leprosy.

Serum total proteins and the various protein fractions were studied in fifty cases of lepromatous leprosy and in eleven cases of lepromatous leprosy with lepra reaction. The study revealed a significant increase in serum total proteins in both lepromatous leprosy and lepra reaction groups, when compared with normal healthy subjects. The percentage rise was found to be 14.5% and 22.95% for lepromatous leprosy and lepra reaction respectively. The globulin fraction showed a significant elevation, while albumin showed a decrease. Thus a reversal of A/G ratio was observed in both the disease groups. Alpha-1 and Alpha-2 globulins were found to be significantly increased in both the disease groups. Beta globulins did not reveal any significant alteration. It was interesting to note the presence of an additional globulin fraction in seventeen patients of lepromatous leprosy and two cases of lepra reaction. Gammaglobulin showed a significant rise in lepromatous leprosy (56.16%) and in lepra reaction (60.72%). The significance of the above findings are discussed in the light of available literature.

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.

Liver dysfunction in patients with hemophilia A, B, and von Willebrand's disease.

Ninety-five patients with Hemophilia A, B and von Willebrand's disease followed over a three year period were evaluated for liver disease. Half of the patients were treated episodically and half received additional prophylactic treatment. Fifty-five per cent have significant transaminitis (elevated SGOT-SGPT), 42 per cent have biochemical evidence of chronic active liver disease and two per cent have subacute and active cirrhosis. There is an annual attack rate of Hepatitis B disease of 3.5 per cent and Hepatitis B antibody titres are present in 84 per cent. This asymptomatic liver disease requires close monitoring for clinical significance.

[Intensity renewal of skin and serum soluble proteins in rats with vitamin A deficiency]. / Intensivnist' onovlennia rozchinnikh bilkiv shkiri ta sirovatkii krovi shchuriv za nedostatnosti vitaminu a.

Intensity of skin and blood serum soluble proteins renewal under conditions of vitamin A deficiency in animal organism was studied with application of the label incorporation into the electrophoretic fractions of proteins 1,3 and 24 h after administration of 14C-lysine. Metabolism of collagen proteins, amino acid composition of skin soluble proteins, content of free amino acids and nucleic acid in skin were also examined. With vitamin A deficiency the intensity of 14C-lysine incorporation into the globulin fractions of serum proteins is 2-4 times as high and into the corresponding fractions of skin proteins is 3-5 times as low as in the control. Metabolism of gamma-globulins and collagen proteins of skin under conditions of vitamin A deficiency is found to be slow. The content of DNA and RNA in skin of the avitaminous animals is reliably 17 and 23% lower, respectively. In the skin extracts obtained by means of 0.15 M NaCl the content of free amino acids with vitamin A deficiency increases. No significant changes are found in the amino acid composition of skin proteins soluble in 0.15 M NaCl, with the exception for a 20% decrease in the content of cystine together with cystein.

[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.

[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.

[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.