Features of the biosynthesis of immunoglobulin G peculiar to the growth process

It is established that with partial hepatectomy, Shvets leukosis and hepatoma RS-1 the biosynthesis intensity of rat blood serum proteins producing aggregates in the acid medium is considerably higher than that of other serum proteins, the incorporation of the radioactive precursor into immunoglobulin G peculiar to intensive normal and malignant growth being particularly intensive. During liver regeneration as well as in malignant growth specific radioactivity of immunoglobulin G peculiar to the growth processes is three and five times, respectively, as high as this value for blood serum soluble proteins and proteins of alpha-globulin fractions.     

Effect of various respiration substrates and growth marker proteins on oxygen consumption in rabbit heart mitochondria

Oxygen uptake by myocardium mitochondria of healthy, carcinomatous and new-born rabbits in the presence of different substrates was studied under the effect of immunoglobulin G and beta-globulin peculiar to the normal and malignant growth. It is stated that the growth marker proteins representing a subfraction of immunoglobulin G of tumour patients blood serum and beta-globulin of new-born rabbits blood serum in the presence of pyruvate, alpha-ketoglutarate and glutamate inhibit the oxygen uptake by healthy rabbits heart mitochondria. Studies conducted on mitochondria of new-born and carcinomatous rabbits show that the action of the proteins under study depends on the respiration substrate. In the presence of succinate the proteins under study activate the oxygen uptake and pyruvate, alpha-ketoglutarate and glutamate inhibit this process. A conclusion is drawn that the effect of proteins peculiar to the growth process on the biological oxidation depends both on the substrate and structural state of mitochondria.     

The carbohydrate component of immunoglobulin G peculiar to malignant growth

The total amount of carbohydrates and some carbohydrate components was studied in total preparations of immunoglobulins of blood serum of healthy people and cancer patients as well as in immunoglobulin G subfraction peculiar to cancer and in the fraction isolated from immunoglobulin G of healthy people blood serum corresponding to the place of column elution. An insignificant increase is established in the content of carbohydrates in the protein peculiar to cancer as compared to their amount in immunoglobulin G of blood serum from healthy people (1.93 and 1.46 g per 100 g of protein, respectively). A conclusion is drawn that the content of the studied substances in the protein peculiar to cancer cannot determine the peculiarities of its physicochemical, immunological and biological properties.     

Iron-sulfur centers and free radicals of the electron transfer chains of mitochondria in chemical and hormonal carcinogenesis

One of the possible reasons for peculiar features of the tumour cell energetics is discussed. Regular variations in the iron-sulphur protein content in the mitochondrial electron transport chains were shown in models of chemical and hormonal carcinogenesis. A decrease in the content of these proteins in the tumoural tissue is found at early stages of malignant growth against a background of higher concentration of free radicals. Irreversible nature of an iron-sulphur protein decrease was observed in the liver and adrenals of tumour-bearing animals both under development of hormonal genesis tumours and at various stages of chemical carcinogenesis. Coming from the results obtained it is suggested that disturbances in the state of iron-sulphur proteins affect the oxidation phosphorylation efficiency and that modification of compensatory mechanism results in the glycolysis activation, which is a characteristic feature of the tumour cell energetics.     

EPR study of blood of anemic patients with urological cancer

Changes in Fe³⁺-transferrin (Fe-Tf) and Cu²⁺-ceruloplasmin (Cu-Cp) concentrations in venous blood sampled from anemic patients with urinary bladder and kidney cancer of stages I–IV were assessed using EPR spectroscopy. In malignancy-associated anemia, the paramagnetic Fe³⁺ concentration in Tf proved to be below the norm, while in anemic non-oncology patients the Tf iron saturation was normal. Moreover, in patients with malignancy-associated anemia the Cu-Cp on average was nearly twice higher than in healthy volunteers (p < 0.01). Thus, simultaneous EPR measurement of protein-borne paramagnetic centers (such as Fe-Tf and Cu-Cp) in the blood of anemic patients can be used as an express biomarker for urological cancer even at early stages of malignant growth.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Incidence of monoclonal gammopathies in blood donors]

A routine screening of monoclonal gammopathies (M.G.) was performed in the serum from 36, 015 blood donors by cellulose acetate electrophoresis. The incidence of M.G. was estimated to 0.14 per cent. About 86 per cent of cases can be classified as asymptomatic M.G. and 14 per cent as malignant M.G. (myeloma or Waldenstr?m macroglobulinemia). In asymptomatic forms, heavy chain classes are only IgG or IgM with a large predominance of IgG (86,4%). It is suggested that donors in whom M.G. have been detected should not be allowed to give blood. A yearly clinical, hematological and an immunoglobulin check-up is recommended to these patients in order to defect the first sign of a malignant process.     

Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.     

Properdin and the protein composition of lymph and blood following resuscitation]

It was shown in experiments on dogs that the terminal blood letting with the subsequent reanimation by intrabone autoblood force pumping caused regular redistribution of proteins between blood, lymph, and tissues. Properdine and alpha-globulin retention in interstitium not eliminable by reanimation measures, and also stress secretion of gamma-globulins from the lymph nodes was noted.     

Angiogenesis and vascular growth factor receptor expression in malignant melanoma.

The growth and metastases of many solid tumors are dependent on the recruitment of new blood vessels. Tumor angiogenesis is most likely initiated by paracrine release of growth factors that bind to their corresponding endothelial cell surface receptors. To determine whether angiogenesis and growth factor receptor expression are consistent findings in malignant melanoma, primary human melanomas were examined for mRNA expression of receptors for fibroblast growth factors (FGFR-1, FGFR-2), vascular endothelial growth factor (VEGFR-1, VEGFR-2), and the receptors Tiel and Tie2. Charts were reviewed and archival formalin-fixed, paraffin-embedded primary tumors were obtained from patients with thin (<1 mm; n = 10), intermediate (1 to 4 mm; n = 10), or thick malignant melanoma (>4 mm; n = 8). Also examined was whether melanoma cell lines could induce endothelial growth factor receptor synthesis by metabolic labeling. It was found that tumor vascularity did not correlate with clinical stage, melanoma thickness, or clinical outcome. It was also found that melanoma cell lines were not capable of directly regulating endothelial cell synthesis of growth factor receptors. However, expression of Tiel and VEGFR-2 mRNA by the tumor vasculature in select stage IA-IIB patients, and FGFR-1 mRNA expression by the tumor cells in the same clinical stages was found. The expression of these growth factor receptors did not correlate with clinical outcome. These data suggest that angiogenesis is not a prominent characteristic of primary malignant melanoma lesions and that the endothelial cell expression of Tiel and VEGFR-2 in vivo is probably not directly induced by the tumor.     

CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, and remains incurable in many cases. Developing more efficient immunotherapy strategies will require better understanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells were originally described as a unique population of T cells with the co-expression of NK cell markers. Apart from their role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells have been recently revealed as one of the key players in the immune responses against tumors. The objective of this study was to evaluate the frequency of CD3(+)/CD16(+)CD56(+) cells in the peripheral blood of 28 diffuse large B-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentages of CD3(+)/CD16(+)CD56(+) were significantly lower in patients with DLBCL compared to healthy donors (7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03), although there were no differences in absolute counts. The frequency and the absolute numbers of CD3(+)/CD16(+)CD56(+) cells were lower in advanced clinical stages than in earlier ones. The median percentage of CD3(+)/CD16(+)CD56(+) cells in patients in Ann Arbor stages 1-2 was 5.55% vs. 3.15% in stages 3-4 (p = 0.02), with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p = = 0.02). The percentage and absolute numbers of CD3(+)/CD16(+)CD56(+) cells were significantly higher in DL -BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04; 0.21 G/L vs. 0.44 G/L, p = 0.04). The percentage of CD3(+)/CD16(+)CD56(+) cells correlated adversely with serum lactate dehydrogenase (R= -445; p 〈 0.05) which might influence NKT count. These figures suggest a relationship between higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains to be explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of their suppression by the tumor cells, or their migration to affected lymph nodes or organs.     

The influence of tumor growth on natural cytotoxicity and activity of some lysosomal enzymes of human effector cells and the C3HA mouse splenocytes

In the course of malignant growth processes in patients with lung cancer, a decrease of natural cytotoxic activity of peripheral blood lymphocytes was observed. This process was accompanied by changes of activities of two lysosomal enzymes, arylsulfatase and acid phosphatase, suggesting participation of these enzymes in manifestation of effector functions of lymphocytes in cancer patients. The level of activity of granular enzyme, beta-glucuronidase, remained unchanged at all stages of disease. A study of natural killer activity of C3HA mice splenocytes after inoculation of transplantable hepatoma 22-a cells revealed a relative stability of the level of their cytotoxicity, and of the activities of lysosomal enzymes--arylsulfatase, acid phosphatase, alpha-mannosidase, acid lipase, N-acetyl-beta-D-glucosidase, and beta-galactosidase, beginning from the 3rd day after hepatoma implantation.     

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.     

The nature of serum factor precipitating autologous alpha-globulins in rabbits

The nature of serum factor, resulted from the immune response to human albumin being a precipitant of auto-alpha-globulins in rabbits, have been studied. The use of ion exchange, immunoaffinity chromatography and immunoelectrophoresis has shown, that antibodies to human albumin, carrying the binding site to alpha-globulin in antigen-binding fragment are the factor responsible for alpha-globulin precipitation. The synthesis of antibodies cross-reacting to alpha-globulin gives the evidence of the selective derangement of tolerance to one of organism self antigens during the immune response to heterologous antigen.     

The nature of the unhydrolysed fraction of alpha-globulin, the major protein component of Sesamum indicum L. hydrolysed by alpha-chymotrypsin.

Alpha-globulin, the high molecular weight protein fraction from sesame (Sesamum indicum L.) seed, was hydrolysed by alpha-chymotrypsin. The hydrolysate was resolved into two fractions, the hydrolysed part and the unhydrolysed part of alpha-globulin using gel filtration on Sepharose 6B-100. The unhydrolysed alpha-globulin residue was characterized for its sedimentation coefficient, subunit composition, fluorescence emission spectrum, secondary structure, and other biophysical properties. The results indicated a decrease in the size of the protein molecule upon hydrolysis to a very small extent. The effect of hydrolysis products on hydrolysis of native alpha-globulin as well as on a standard substrate, casein, was also investigated. The results indicated that the hydrolysis products contribute to the resistance of alpha-globulin to proteolysis by alpha-chymotrypsin to the extent of 40%.     

Inhibitory diffusible factor IDF45, a G1 phase inhibitor

An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio-Gel P150 chromatography and 2 reverse-phase FPLC. After the final step of purification, 60 ng/ml of IDF45 inhibited 50% of alpha-globulin-stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by alpha-globulin.     

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.     

Identification and properties of proteins characteristic of the Shvets rat leukosis

Blood proteins were examined in rats with the Shvets experimental leukosis positively reacting in the sedimentation test for leukosis. Application of monospecific antisera against main classes of immunoglobulins shows that this protein belongs to immunoglobulins of the class G. The sedimentation constant and molecular weight of the protein under study are determined. It is shown that immunoglobulin G of rats with leukosis activates glycolysis and inhibits respiration. The data obtained give reasons to assume not complete identity but, in any case, similarity of physicochemical properties of proteins appearing in animal and human blood channel with malignant growth.