Fetuin, an inhibitor of lymphocyte transformation. The interaction of fetuin with phytomitogens and a possible role for fetuin in fetal development.

Fetuin, the bovine alpha-fetoprotein, contains glycopeptide sequences similar to those found on red cells. As a result, it is capable of strong physical interaction with the phytohemagglutinin isomitogens (H-PHAP) which possess two or more R (red cell binding) subunits as part of their tetrameric structures. Fetuin shows little or no interaction with L-PHAP, a phytohemagglutinin made up of four L subunits which also lack red cell affinity. Despite these differences fetuin is able to inhibit both H- and L-PHAP-induced lymphocyte transformation and is also capable of inhibiting the mitogenic effects of pokeweed mitogen, concanavalin A, antithymocyte antiserum, and the one-way mixed lymphocyte culture. In the case of L-PHAP, the inhibitory effect of fetuin is proportional to the intensity of the mitogenic stimulus. The inhibitory effects of fetuin upon lymphocyte transformation may result from perturbation or "blindfolding" of the cell membrane in a manner analogous to other immunosuppressive serum alpha-globulins. Alpha-Fetoproteins may play an immunoregulatory role during fetal development.

Phytohemagglutinin mitogenic proteins. Structural evidence for a family of isomitogenic proteins.

The phytohemagglutinin (PHAP) glycoproteins derived from Phaseolus vulgaris consist of five isomitogens that are tetrameric structures made up of two different glycoprotein subunits. Although identical in size (mol wt = 34,000), the subunits differ in their isoelectric points and amino acid sequences for six of the first seven amino-terminal residues, but are identical in primary structure from the 8th through the 24th amino acid residue. The isomitogen containing four L subunits (L-PHAP) is a potent leukoagglutinin and mitogen that lacks hemagglutinating properties. The isomitogen made up of four R subunits (4R H-PHAP) is a potent hemagglutinin. The hybrid isomitogens consisting of varying proportions of the two subunits (3L-1R, 2L-2R, 1L-3R) are capable of causing mixed erythrocyte-lymphocyte agglutination. These studies provide a structural basis for explaining the differences in biological activities of the various PHAP isomitogens.

Serum lipoproteins modulate oxygenated sterol insertion into human red cell membranes.

The insertion of oxygenated sterol compounds into human red blood cell membranes as well as the consequent transformation of the red cells to an echinocyte shape and the expansion of the membranes are impeded by the presence of serum lipoproteins in the incubation medium. All density classes of human serum lipoproteins bind oxygenated sterol compounds, and lipoproteins can act as acceptors of oxygenated sterols previously inserted into red cells. Since oxygenated sterols have been reported to be atherogenic, the modulating and possibly protective effects of serum lipoproteins on oxygenated sterol-induced derangement of cell membrane structure and function may provide a useful model for further study.

Cytochalasin B is a potent mitogen for chronic lymphocytic leukemia cells in vitro.

It is widely accepted that the neoplastic B cells from patients with chronic lymphocytic leukemia (CLL) respond poorly to common mitogens. The fungal metabolite cytochalasin B (0.5 micrograms/ml) is a weak mitogen for normal lymphocytes. However, when peripheral blood lymphocytes from 19 patients with CLL of B cell origin (B-CLL) were cultured with 0.5 micrograms cytochalasin B/ml, significant new DNA synthesis ( [14C]thymidine incorporation) occurred in 18. Stimulation indices with cytochalasin B varied widely (range = 1.9-28.2, mean +/- SD = 10.6 +/- 7.5; delta cpm range = 1,157-153,818; n = 26) but in 11 cases exceeded those seen with concanavalin A (Con A), phytohemagglutinin, or pokeweed mitogen. In all 11, the mitogenic response to cytochalasin B exceeded that to pokeweed mitogen, which is believed to be a T cell-dependent B cell mitogen. In three cases, the responses to cytochalasin B were 8.6, 3.5, and 2.3 times greater than those to Con A. As with other mitogens, the DNA synthetic response to cytochalasin B was time and dose dependent. Peak thymidine incorporation occurred at 72-88 h and declined thereafter. Significant mitogenic effects were observed with 0.1-5 micrograms cytochalasin B/ml with a peak at 0.5-2 micrograms/ml. Stimulated DNA synthesis was abolished by 1 mM hydroxyurea. Cells from two patients with B-CLL were separated by rosetting with sheep erythrocytes (E). Depletion of E-rosette-positive cells from the CLL cell population abolished the response to Con A but did not affect the response to cytochalasin B. Cytochalasin B is a potent mitogen for B-CLL cells and may be useful in cytogenetic studies of this often indolent neoplasm.

Heterogeneity of human alpha-fetoprotein as revealed by isoelectric focusing in urea-containing gels.

The heterogeneity of human alpha-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human alpha-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major alpha-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of alpha-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain alpha-fetoprotein charge isomers in various alpha-fetoprotein isolates may be important in considering certain biological functions of this protein.

Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.

Purification and characterization of a glycopeptide derived from Phaseolus vulgaris leukoagglutinating phytohemagglutinin.

A glycopeptide obtained from tryptic digestion of citraconylated leukoagglutinating (L-subunit) phytohemagglutinin was isolated and purified. The composition of its ten amino acid and seven hexose residues, when compared to a previous analysis of the NH2-terminal amino acid sequence, indicated that the glycopeptide was derived from residues 11 through 20, with the oligosaccharide unit linked N-glycosidically to asparagine at the twelfth position. Structural studies involving a combination of alpha-mannosidase digestion, periodate oxidation, and permethylation, in conjunction with computerized mass-spectrometric and masschromatographic techniques, suggested a branched sequence of five mannose and two glucosamine residues similar to that found in many mammalian glycoproteins.

Oxygenated cholesterols synergistically immobilize acyl chains and enhance protein helical structure in human erythrocyte membranes.

Fourier transform infrared spectroscopy revealed that insertion of 20 alpha-hydroxycholesterol into human erythrocyte membranes (10% of total membrane sterol) immobilized the lipid acyl chains to a degree equivalent to enriching total membrane cholesterol by 50% (Rooney, M.W., Lange, Y. and Kauffman, J.W. (1984) J. Biol. Chem. 259, 8281-8285). Raman spectroscopy showed that the amount of acyl chain rotamers was not significantly altered by the presence of 20 alpha-hydroxycholesterol, indicating that acyl chain immobilization was limited to an inhibition of lateral motion. The presence of 20 alpha-hydroxycholesterol may synergistically enhance the acyl-chain-immobilizing behavior of membrane cholesterol. In addition, protein helical structure was not altered by 20 alpha-hydroxycholesterol. The insertion of 7 alpha-hydroxycholesterol into erythrocyte membranes resulted in an increase in protein helical structure which was comparable to that observed for erythrocyte membranes enriched with pure cholesterol by 50%. However, both acyl chain mobility and conformation were unchanged. These results suggest a synergistic behavior between oxysterols and cholesterol in modifying erythrocyte membrane packing.

The effects of cholesterol oxidation products in sickle and normal red blood cell membranes.

The oxysterol content in normal and sickle red blood cell (RBC) membranes was assessed using thin-layer chromatography and capillary gas chromatography/mass spectrometry. Several more oxysterols were present in sickle RBCs compared to normal RBCs. Sickle RBC membranes had a higher concentration of 5 alpha,6 alpha-epoxycholesterol, 5 alpha-cholestane-3 beta,5,6 beta-triol, 7-ketocholesterol and 19-hydroxycholesterol than normal RBC membranes. The increased oxysterols in sickle RBC may be an effect of the increased oxidative stress which occurs in sickle RBC membranes. Physical characteristics of normal and sickle RBC membrane ghosts with and without inserted oxysterols were examined by Fourier transform infrared spectroscopy. The data are consistent with a greater sterol content in sickle cells compared to normal RBC membranes, and a possible oxysterol-cholesterol synergism.

Human alpha-fetoprotein in the epidermal cells of Bowen's disease.

Human alpha-fetoprotein (HAFP) was demonstrated in clusters of atypical cells of an extensive Bowen's disease involving the pubic area. Frozen sections were studied by indirect immunofluorescence using monospecific rabbit anti-HAFP antiserum or purified rabbit anti-HAFP antibody followed by staining with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit gamma-globulin and by a direct technique using FITC-labeled rabbit anti-HAFP Fab2 fragments. Sections of fetal liver served as positive controls and sections of adult human skin and liver were used as negative controls. Preabsorption of the anti-HAFP antibodies with purified HAFP blocked the fluorescence. This finding adds Bowen's disease to the list of premalignant disorders which possess HAFP as an oncofetal marker of their malignant potential. HAFP might possibly enhance malignant behavior through interference with cellular immunosurveillance mechanisms.

The influence of oxygenated sterol compounds on dipalmitoylphosphatidylcholine bilayer structure and packing.

Fourier Transform Infra-red and Raman Spectroscopies indicate that 7 alpha-hydroxycholesterol and 7-ketocholesterol have a diminished capacity to condense (increase the packing order of) fluid-state dipalmitoylphosphatidylcholine (DPPC) acyl chains when compared with the effects of cholesterol and the other oxidized sterols studied. DPPC head groups were also more ordered by 7-ketocholesterol over the temperature range 10 degrees - 70 degrees C. Primary effects of these sterols appear to be associated with the hydrophillic regions of the DPPC bilayer, although packing arrangements with acyl chains are also involved. Phosphate and acyl chain ester groups were observed to possess a packing order which was invariant which indicates that these may be the target groups in the interaction with 7-ketocholesterol. A surprising observation was the synergistic amplification of the effects of 7-ketocholesterol by the presence of cholesterol in the DPPC bilayer.

Inhibition of cytolytic T lymphocyte activity by oxysterols.

The objective of this study was to investigate the effects of oxysterols (OS), namely 5 alpha-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10(-5) M 5 alpha-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h 51Cr release. At a concentration of 5 x 10(-6) M, 5 alpha-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.

The clinical significance of human alpha-fetoprotein.

Deviations from the normal of alpha-fetoprotein (AFP) concentrations in fetal serum, amniotic fluid, maternal serum and adult human serum can be explained by understanding the normal physiology and the pathophysiology of AFP synthesis and metabolism. AFP is the prototype of oncofetal markers. Emphasis is given to the usefulness of elevated serum AFP levels on the diagnosis and management of primary hepatomas and tumors of germ cell origin. The ability to detect neural tube defects early in gestation by monitoring maternal serum and amniotic fluid AFP concentrations is discussed.

Demonstration of the inhibitory effect of human alpha-fetoprotein on in vitro transformation of human lymphocytes.

We have studied the effects of human alpha-fetoprotein (HAFP), isolated from the serum and ascitic fluid of a hepatoma-bearing patient, on the in vitro transformation of human peripheral blood lymphocytes by a variety of mitogenic stimuli. At a concentration of 2.5 mg/ml, HAFP inhibited the lymphocyte response to phytohemagglutinin, concanavalin A, and rabbit anti-human thymocyte serum, but failed to inhibit the response to pokeweed mitogen. HAFP was able to inhibit the one-way mixed lymphocyte culture at concentrations of 250-500 mug/ml, but failed to inhibit at 100 mug/ml. Exposure of lymphocytes to 2.2 mg/ml of HAFP for 18 hr did not result in significant lymphocytotoxicity, and such cells washed free of HAFP were fully capable of participating in the mixed lymphocyte culture. HAFP did not inhibit lymphocyte E-rosette formation. Fetal HAFP was more effective in inhibiting human lymphocyte responses than hepatoma HAFP. These experiments support the suggestion that HAFP plays an important immunoregulatory role during fetal development, possibly through the suppression of thymus-derived lymphocyte responses to antigenic stimuli; they also suggest that there are important differences in the biological properties of hepatoma and fetal HAFP.

Mevalonic acid as an initiator of cell growth. Studies using human lymphocytes and inhibitors of endogenous mevalonate biosynthesis.

Mevalonic acid (5 x 10(-4)-1 x 10(-2) M) stimulates DNA synthesis, morphologic transformation and cell cycling in peripheral blood human lymphocytes. Other organic acid anions which serve as cholesterol and mevalonate precursors are devoid of such effects. Both ML-236B and 25-hydroxycholesterol, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, inhibit concanavalin A-induced lymphocyte transformation, but only the inhibition by ML-236B can be overcome by exogenous mevalonate. In contrast, only 25-hydroxycholesterol inhibits mevalonate-induced lymphocyte DNA synthesis. The effects of mevalonic acid on lymphocytes cannot be reproduced by isopentenyl adenine or isopentenyl adenosine. Unregulated endogenous cellular synthesis of mevalonic acid may contribute to uncontrolled growth in certain malignant cell lines.

Unimpeded growth of tumour in hosts pre-immunized with tyrosyl- or dinitrophenyl-coated tumour cells.

Techniques are described for hapten attachment to the cell membranes of mouse tumour cells. Dinitrophenylation and tyrosylation could be achieved without substantial loss of viability as measured by dye exclusion. In addition hapten coated tumour cells were capable of initiating new tumour formation in syngeneic hosts. Pre-immunization of recipient mice with hapten coated tumour cells did not increase their resistance to tumour formation upon subsequent challenge with graded doses of untreated tumour cells.

Inhibition of human lymphocyte transformation by human alpha-foetoprotein (HAFP); comparison of foetal and hepatoma HAFP and kinetic studies in vitro immunosuppression.

Five pure isolates of human alpha-foetoprotein (HAFP) from adults with tumours of the li er or stomach, as well as HAFP isolated from foetal liver, inhibit in vitro human lymphocyte transformation induced by phytomitogens, anti-human thymocyte serum, and the mixed lymphocyte culture. Foetal HAFP produces 50% inhibition at concentrations of 1-5 mug/ml. The HAFPs isolated from tumour-bearing adults are 1-3 orders of magnitude less potent (50% inhibition achieved at approximately 20, 130, 500, and 2000 mug/ml, respectively). In order to achieve maximum inhibition HAFP must be present at the time of mitogen addition; pre-exposure of lymphocytes to HAFP, followed by washing, does not result in lymphocyte suppression. The inhibiting effect of HAFP cannot be overcome by a ten-fold increase in mitogen concentration implying that HAFP does not act by simple competition with the lymphocyte membrane for the mitogen combining site. HAFP may play an immunoregulatory role during foetal development.

An absolute requirement for serum macromolecules in phytohaemagglutinin-induced human lymphocyte DNA synthesis.

We have examined the effect of different variables such as tissue culture media, with or without various supplements, lymphocyte isolation techniques, lymphocyte contamination by autologous red blood cells and platelets, and lymphocyte numbers, on the requirement for serum during phytohaemagglutinin (PHA) induced DNA synthesis in human lymphocytes. At all mitogen doses tested, we have found that dialysable constituents of serum enrich the ability of all tissue culture media to support lymphocyte DNA synthesis; however, human lymphocytes display an absolute requirement for nondialysable macromolecular constituents of serum in order to synthesize DNA.