[Mutation analysis of the phenylalanine hydroxylase gene of phenylketonuria patients of Kemerovskaya Oblast' and Saha Republic].

Phenylketonuria (PKU) associated mutations in phenylalanine hydroxylase (PAH) gene were identified by direct DNA sequencing in 46 PKU patients and members of their families from Kemerovskaya Region and Saha Republic. Mutations found included both widespread known mutations (R158Q, R252W, R261Q, P281L, IVS10-11G>A, R408W, IVS12+1G>A) and several rare mutations (IVS2+5G>A, R155H, Y168H, W187R, E221_D222>Efs, A342T, Y386C, IVS11+1G>C). We observed the increase in diversity of PKU-associated alleles in the populations studied, probably due to their complex mixed ethnic structure.

[Identification of mutation of the phenylalanine hydroxylase gene using an automated DNA sequencer]. / Identifikatsiia mutatsii fenilalaningidroksilazy s ispol'zovaniem avtomaticheskogo sekvenatora DNK.

Mutations were studied in phenylalanine hydroxylase gene of phenylketonuria patients from Kemerovo oblast and Altaiskii krai (15 and 2 families, respectively). The following mutations were identified in exons of this gene: R408W, R261Q, R243Q, Y414C, Y386C, P281L, Y168H, R68S (lead to amino acid substitutions), R243X (leads to stop codon formation), and three splice site mutations (IVS12nt 1g-->a, IVS2nt-13t-->g, IVS7nt 1g-->a).

Review: immunomodulatory activity of pregnancy-associated plasma protein-A.

This mini-review highlights the growing number of indications for the immunological importance of pregnancy-associated plasma protein-A (PAPP-A), which is a remote member of the alpha-macroglobulin plasma protein family. PAPP-A can bind a variety of cytokines and specifically cleave a binding protein for insulin-like growth factors, thereby serving as a modulator of cytokine activity. Important immune functions, such as lymphocyte proliferation response to alloantigens and lectins and expression of HLA-DR molecules are predominantly suppressed in vitro by PAPP-A. It is likely that the immunoregulatory properties of PAPP-A are very similar to that of alpha 2-macroglobulin. The experimental data allows us to suppose that PAPP-A serves to prevent the recognition of the fetus by the maternal immune system and to suppress locally the host's immune response to the tumour.

The effects of alpha 2-antiplasmin complex and alpha 2-antiplasmin on the secretion of IgG and IgM by cultured human mononuclear cells.

We tested alpha 2-antiplasmin (AP) and its complex with plasmin (PL) for their capacity to modulate spontaneous secretion of immunoglobulins (Ig) G and M by mononuclear cells (MNC). MNC were obtained from peripheral blood of 10 donors (5 males and 5 females) and cultured for 7 days in the presence of various concentrations of AP and AP-PL complex. The 18 hr incubation of MNC with AP-PL complex resulted in a dose-dependent increase in IgG and IgM secretion but the stimulatory effect was less significant than under conditions of incubation of MNC with the complex during a 7 day culture period. The AP-PL complex-induced stimulation occurred only in the cultures in which the dose of the complex was either close to or higher than the mean value of the complex levels in plasma of healthy donors. Elevation of IgG secretion in AP-PL complex-treated cultures from females was slightly higher than in those from males. No significant change in IgG and IgM secretion was observed in the presence of native AP.

[Reactions between protease inhibitors in blood plasma and collagen]. / Izuchenie reaktsii mezhdu ingibitorami proteinaz plazmy krovi i kollagenom.

The interaction of three macroglobulins and three serpines, as well as inter-alpha-trypsin inhibitor (ITI) with alpha 1- and alpha 2-chains of collagen I which were immobilized on nitrocellulose. All seven proteinase inhibitors were shown to have a certain affinity for collagen. The binding of alpha 2-macroglobulin and gestation-associated protein A with collagen chains is largely determined by the conformational state of these macrophages. alpha 2-Antiplasmin, proteinase alpha 1-inhibitor, antithrombin III and ITI with collagen yield complexes that are resistant to urea, sodium dodecylsulfate, and Trilon B.

[Concentration of plasmin complexes with alpha-2-macroglobulin and alpha-2-antiplasmin in blood plasma and serum]. / Kontsentratsiia kompleksov plazmina s alfa-2-makroglobulinom i alfa-2-antiplazminom v plazme i syvorotke krovi.

A methodological approach has been developed for measuring the concentrations of proteinase complexes with their inhibitors in various biological fluids. This approach is based on the use of polyclonal antibodies to the complex complements and their detection by enzyme immunoassay. Appreciable individual differences in the levels of alpha 2-macroglobulin and plasmin in donor blood plasma samples were revealed. Complexes of this inhibitor with plasmin are responsible for about 3% of the total alpha 2-macroglobulin pool in blood plasma and sera.

Interaction of human pregnancy-associated plasma protein-A with serine proteinases.

Human pregnancy-associated plasma protein A (PAPP-A) inhibited significantly the proteolytic activity of bovine trypsin and human plasmin. Trypsin or plasmin treatment of PAPP-A resulted in the generation of a major 85 kDa component and the rapid cleavage of internal thiol esters. The results indicated that both of these serine proteinases bound in a 1:1 stoichiometry to PAPP-A. The PAPP-A-bound enzymes were found to be enzymatically active towards small synthetic substrates and inaccessible to inactivation by soybean trypsin inhibitor and alpha 1-proteinase inhibitor. The mechanism of proteinase inhibition was likely to be entrapment, as described for alpha 2-macroglobulin.

[The effect of plasmin and its complexes with alpha 2-antiplasmin and alpha 2-macroglobulin on secretion of proteinase and plasminogen inhibitors from peripheral mononuclear cells]. / Vliianie plazmina i ego kompleksov s alpha 2-antiplazminom i alpha 2-makroglobulinom na sekretsiiu ingibitorov proteinaz i plazminogena perifericheskimi mononuklearnymi kletkami.

Secretion of proteinase inhibitors alpha2-macroglobulin, alpha1-proteinase inhibitor, inter-alpha-trypsin inhibitor and plasminogen was distinctly altered in mononuclear cell from peripheric blood of volunteers if these cells were preincubated simultaneously with alpha2-macroglobulin and alpha2-antiplasmin modified by plasmin. The complex plasmin-alpha2-macroglobulin was shown to stimulate most effectively the alpha2-macroglobulin and alpha1-proteinase inhibitor secretion. Besides, plasmin-alpha2-macroglobulin complex inhibited the inter-alpha-trypsin inhibitor secretion, whereas the complex plasmin-alpha2-antiplasmin--stimulated. Both these complexes augmented the rate of plasminogen synthesis in the mononuclear cells.

[A comparative study of pregnancy-associated protein A and its analogs in animals]. / Sravnitel'noe izuchenie assotsiirovannogo s beremennost'iu proteina A i ego analogov u zhivotnykh.

Glycoproteins similar in composition, structure, and functions were isolated from the blood serum of pregnant women, cows, dogs, and rats. They consist of four identical subunits, which form dimers due to covalent bonds, while dimers form tetramers due to hydrogen bonds. All these proteins are non-specific inhibitors of proteases and bind these enzymes in the same way as alpha-macroglobulins do, by capture in a "trap". Furthermore, they are capable of binding and transporting various cytokines. The results obtained indicate that these proteins belong to the family of macroglobulins.

Changes in tissue distribution of rat alpha 1-macroglobulin and pregnancy-associated alpha 1-glycoprotein after inflammatory injury.

Antiserum against rat alpha 1-macroglobulin (alpha 1MG) was produced in rabbits. Antiserum against rat pregnancy-associated alpha 1-glycoprotein (PAG) was obtained by immunization with a partly purified PAG preparation and absorption of the serum with male rat serum. Acute inflammation was produced in non-pregnant female rats by a single intramuscular injection of turpentine. The concentrations of both macroglobulins in the serum and in tissue extracts were measured by rocket immunoelectrophoresis at various times up to 7 days after injury. Inflammation produced in the rats resulted in moderately elevated serum levels of these proteins soon after injury. At first, alpha 1MG levels in a number of tissues (heart, lung, kidney, spleen, pancreas, uterus and ovary) were depressed markedly; they then stabilized. The elevated serum concentrations of alpha 1MG remained unchanged during inflammation. The store of PAG in the tissues was rapidly depleted and its serum level decreased to a normal value 7 days after injury. Our findings indicate that alpha 1MG plays a more important role in maintenance of the proteinase inhibitory potential in the rat than does PAG.

[A comparative study of the physicochemical and antigenic properties of human and animal albumins]. / Sravnitel'noe izuchenie fiziko-khimicheskikh i antigennykh svoistv al'buminov cheloveka i zhivotnykh.

Studies have been made on physico-chemical and antigenic properties of albumins from man, mammals, birds, insects, amphibians, worms, fishes and crustaceans. It was found that all the animals contain structurally identical proteins with similar physico-chemical properties. At the same time, the level of antigenic identity of the proteins decreases in the following order: type, genus, family, species.

[The effect of plasmin-modified proteinase inhibitors on macroglobulin production by human blood mononuclear cells in culture]. / Vliianie modifitsirovannykh plazminom ingibitorov proteinaz na produktsiiu makroglobulinov mononuklearnymi kletkami krovi cheloveka v kul'ture.

A study was made of the influence of plasmin (PL) complexes with alpha 2-macroglobulin (MG) and alpha 2-antiplasmin on secretion of MG, pregnancy-associated alpha 2-glycoprotein (PAG), and pregnancy-associated plasma protein A (PAPP-A) by mononuclear cells (MC), obtained from human peripheral blood. It has been shown that incubation of the MG-PL complexes with MC resulted in the increase in concentrations of all these macroglobulins (MG, PAG, and PAPP-A) in supernatants of cultured MC. We revealed the same effects of AP-PL with regard to the secretion of PZP and PAPP-A. However, this complex was found to suppress MG production by cultured MC. The effects found were dose dependent and some of them differed greatly in men and women, resp.

[The use of colloidal gold for the immunocytochemical analysis of subpopulations of immunocompetent cells]. / Ispol'zovanie kolloidnogo zolota dlia immunotsitokhimicheskogo analiza subpopuliatsii immunokompetentnykh kletok.

Immune colloid gold is suggested to be used for assessment of human peripheral blood lymphocyte subpopulations. The method is simple, highly sensitive, and compatible with immunofluorescent technique. Double label permitted detection of beta 2-macroglobulin-positive cells within every lymphocyte subpopulations.

[Study of structural features of macroglobulins complexed with plasmin]. / Izuchenie strukturnykh osobennostei kompleksov makroglobulinov s plazminom.

Activities of plasmin and trypsin contained in complexes with alpha 2-macroglobulin, related to pregnancy alpha 2-glycoprotein and with protein A were studied in presence of various inhibitors with a molecular mass of 0.2-70.0 kDa. Specific characteristics of interaction between plasmin and these macroglobulins inhibitors as well as with alpha 1-inhibitor of proteinases, antithrombin III and alpha 2-antiplasmin were studied using electrophoresis and immunoblotting.

[The interaction of staphylococci with plasmin and its inhibitors]. / Vzaimodeistvie stafilokokkov s plazminom i ego ingibitorami.

According to the data obtained by immunoblotting and other immunochemical techniques, the cell wall of Staphylococcus aureus, strain 209 P, contain proteinaceous receptors for plasminogen, plasmin and its complexes with alpha 2 glycoprotein and protein A, alpha 2 proteinase inhibitor, alpha 2 antiplasmin. Strains Cowan 1 and Wood 46 have no such receptors. All three strains have been found to contain the receptor for alpha 2 interferon, its molecular weight being equal to 50 kD.

[Hepatocyte receptors for proteinase inhibitor complexes with plasmin]. / Izuchenie retseptorov gepatotsitov dlia kompleksov ingibitorov proteinaz s plazminom.

Hepatocyte receptors have been found for such complexes as alpha 2-macroglobulin-plasmin, pregnancy-associated plasma protein A-plasmin, and pregnancy-associated alpha 2-glycoprotein-plasmin, molecular masses of these receptors being estimated. The proteinaceous component of hepatocyte membranes contains heavy chains of plasmin and a 50 kDa fragment of plasmin molecule. These chains and the fragment are able to bind the native alpha 2-antiplasmin. Using immunoblotting no hepatocyte receptors for native macroglobulins, and alpha 2-antiplasmin-plasmin, alpha 1-proteinase inhibitor were detected.

[The evolutionary problems of the family of human and animal macroglobulins]. / Problemy évoliutsii semeistva makroglobulinov cheloveka i zhivotnykh.

Studies have been made on physicochemical and immunochemical properties of macroglobulins, as well as of associated with pregnancy glycoproteins, from human subjects, mammals, birds, fishes and invertebrates. It was shown that these proteins exhibit similar composition, structure and capacity to bind proteinases inhibiting the latter. Using immunochemical methods, reactions of antigenic identity of these proteins were investigated. A hypothesis of evolutionary formation of macroglobulin family is discussed.

[The immunohistochemical detection in the human placenta of proteins related to the blood coagulation system]. / Immunogistokhimicheskoe vyiavlenie v platsente cheloveka nekotorykh belkov, sviazannykh s sistemoi svertyvaniia krovi.

Distribution in mature human placenta of plasminogen, pregnancy-associated inhibitors of proteases (pregnancy-associated protein A (PAPP-A) and alpha 2--pregnancy-associated glycoprotein, and also of not associated with pregnancy alpha 2-macroglobulin and alpha 1-antitrypsin was examined. Primary monospecific antibodies and secondary antibodies labeled with colloid gold were used. Plasminogen was detected in the fetal and maternal blood, and also on the surface of some placental villi (as thin positively stained rims). Pregnancy-associated protease inhibitors were detected in the syncytium of the villi of all histological types and also in the fetal and maternal blood. Staining for alpha 2-macroglobulin was most intensive. This antigen was detected in the maternal and fetal blood and on the surface of the villi. alpha 2-antitrypsin was detected in the fetal and maternal blood. It has been shown that both free plasminogen and its inhibitors are retained on the surface of the placental villi.