Non-reactive Fraction Research Articles

Microheterogeneity of α1-antichymotrypsin in the management of giant-cell arteritis and polymyalgia rheumatica

1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the glycan microheterogeneity of alpha 1-antichymotrypsin in sera from patients with giant-cell arteritis and/or polymyalgia rheumatica, and its variation in the serum of several of these patients during induction of disease remission by prednisone therapy and possible further recurrence of giant-cell arteritis and/or polymyalgia rheumatica. 2. In the serum of patients with active disease we observed increased proportions of concanavalin A nonreactive and concanavalin A weakly reactive fractions. The results were expressed as the ratio of concanavalin A non-reactive fraction plus concanavalin A weakly reactive fraction to concanavalin A reactive fraction, called R alpha 1-ACT. An R alpha 1-ACT higher than 1.8 (upper normal value) was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 2/22 sera from patients with inactive disease (specificity 91%). 3. The erythrocyte sedimentation rate and the serum C-reactive protein level, common markers of biological inflammation, are usually elevated in active giant-cell arteritis and/or polymyalgia rheumatica. The two parameters are commonly used to guide the therapy during the course of the disease, but they have no specificity. An erythrocyte sedimentation rate of greater than 30 mm/h was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 5/22 sera from patients with inactive disease (specificity 77%).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on alpha-fetoprotein produced by renal cell carcinoma

A 58-year-old man was found to have a Stage IV primary renal cell carcinoma (RCC) invading the colon and producing alpha-fetoprotein (AFP). The preoperative serum AFP level of 418 ng/ml had decreased to the normal level of 0.9 ng/ml 3 weeks after the radical operation. As the high serum AFP level in RCC without metastasis to the liver is a rare condition, histopathologic studies were performed on surgical specimens. Histologically, the tumor was clear cell adenocarcinoma and AFP was detected in tumor cells immunohistochemically. A Concanavalin A (Con A) sepharose affinity chromatogram was performed to detect the component of AFP in this case. The Con A nonreactive fraction rate was as high as 47%, which indicated that the AFP of this RCC was of fetal type.

The microheterogeneity of bovine serum .ALPHA.1-acid glycoprotein.

The microheterogeneity in bovine serum alpha 1-acid glycoprotein (alpha 1AGP) was examined by crossed affino-immunoelectrophoresis with concanavalin A(Con A). In healthy cows, serum alpha 1AGP revealed 2 fractions which were named fraction 1 and 2 according to the degree of binding affinity to Con A. The mean +/- SD of serum alpha 1AGP concentration was 0.31 +/- 0.09 g/l and the relative amounts of fraction 1 and 2 were approximately 67% and 33% respectively on this method. In bovine leukemia cases, 5 out of 8 had a high serum alpha 1AGP concentration of more than 0.7 g/l. On the electrophoretic pattern, the other 2 fractions (Con A weakly and non-reactive fractions, named fraction 3 and 4) were observed in 5 cases and their relative amounts of 4 fractions were 17.8-28.5%, 35.5-46.0%, 17.9-24.4% and 12.0-17.2%, respectively. In bovie leukemia virus infected cows with no clinical symptoms, however, serum alpha 1AGP concentration and the electrophoretic patterns were almost the same as those in healthy cows.

Alpha-fetoprotein and human chorionic gonadotropin-producing lung cancer

A 73-year-old man had primary lung cancer that produced both alphafetoprotein (AFP) and human chorionic gonadotropin (HCG). The preoperative serum AFP level of 1039 ng/ml decreased to the normal range 8 weeks after surgery. The preoperative serum HCG level of 11 mIU/ml, which temporarily decreased to the normal range after operation, soon increased thereafter. The serum HCG level decreased, however, to the normal range after postoperative mediastinal radiation therapy. During relapse, only the serum HCG level increased gradually to 26,000 mIU/ml 7 weeks before his death. The lung cancer was classified histologically as poorly differentiated adenocarcinoma. Immunohistochemically, AFP was detected in the mononuclear tumor cells of the primary tumor in the lung, and HCG was found in the giant cells of the subcarinal metastatic lymph node. The concanavalin A non-reactive fraction rate for AFP was 81.3%, and appeared to differ from those of hepatocellular carcinoma and yolk sac tumor.

Affinity chromatographic separation of alpha-fetoprotein variants: development of a mini-column procedure, and application to cancer patients.

This is an affinity chromatographic procedure for separating variants of alpha-fetoprotein in 0.1 mL of serum on a disposable mini-column containing 0.9 mL of concanavalin A-Sepharose, at pH 6.4 and with a flow rate of 5 mL/h. The binding capacity of the column is 52 micrograms. Analytical recovery was 90%. Between-run CVs varied from 2.0% for samples with a high percentage of concanavalin A nonreactive fraction to 13% for samples with a low percentage. The mean proportion of nonreactive alpha-fetoprotein was 13.4% for patients with hepatocellular carcinoma, 62.2% for testicular carcinoma, and 8.9% for nonmalignant liver diseases. This suggests that the alpha-fetoprotein variants could be used to distinguish hepatocellular carcinoma from other carcinomas. Serial determination of the concanavalin A nonreactive fraction in such patients showed a relatively constant percentage. This indicates that monitoring variants in patients with hepatocellular carcinoma throughout their clinical course may not provide useful information additional to that provided by the values for total serum alpha-fetoprotein.

Studies on Lectin Affinities of Chicken Alpha-Fetoprotein During Ontogeny

Alpha-fetoprotein in the sera of chicken embryos (ch-AFP) of the 7th, 10th, 13th, 16th, and 19th day of incubation and of one-day-old chickens were examined in crossed-immuno-affino-electrophoresis (CIAE) using different lectins [concanavalin A (Con A), lentil, wheat germ lectin, and soybean lectin] as ligands. Differences in lectin affinities, suggesting different degrees or forms of glycosylation of ch-AFP during ontogeny, were demonstrated by this method. Sera of chicken embryos of the same stages were also subjected to Con A affinity chromatography and the elution patterns were evaluated qualitatively in CIAE and quantitatively in rocket Immunoelectro-phoresis. These experiments revealed a major Con A-nonreactive and a minor Con A-reactive fraction at the 7th day of incubation. In contrast, a minor Con A nonreactive fraction and a major Con A-reactive fraction were observed at later stages of embryonic life. Extracts from embryos of the 8th and 15th day of incubation did not exhibit heterogeneity when subjected to CIAE. The biological significance of these observations, as well as the biological role of AFP in general, remains to be elucidated. Examination of ch-AFP glycosylation patterns might be included into the spectrum of anlayses when certain early teratological processes in the chicken embryo are to be monitored. In addition, these molecular changes have to be considered when in vitro or in vivo experiments on the biological, especially immunological, role of ch-AFP are envisaged.

Molecular heterogeneity of human chorionic gonadotropin and its subunits in testicular cancer.

The secretion of human chorionic gonadotropin and its subunits was examined by a heterologous hCG/hCG-beta radioimmunoassay and homologous radioimmunoassays of the alpha- and beta-subunit in patients with nonseminomatous testicular germ cell tumors. Of 53 patients, 32 (60%) had elevated levels of hCG (9 IU/l-4.5 million IU/l), 12 (23%) elevated serum concentrations of hCG-beta (20-286 micrograms/l) and 8 (15%) of hCG-alpha (14-498 micrograms/l). A selective elevation of hCG-subunits without the hormone was never observed. Serum concentrations of hCG-beta were to low to interfere in the heterologous radioimmunoassay using 125I-hCG and anti-hCG-beta antiserum. Gelfiltration of patient's sera (n = 5) on Ultrogel AcA 44 column confirmed the secretion of free subunits and additionally a high molecular form of hCG-beta (70,000) in three of five serum specimens. Furthermore, the molecular heterogeneity of hCG and hCG-subunits was examined by affinity chromatography on Concanavalin A (Con A)-sepharose. There was a significant difference of Con A nonreactive hCG and hCG-alpha between patients with testicular cancer (hCG:2-82%; mean, 22%; n = 7; hCG-alpha: 11-61%; mean, 31%; n = 7) and pregnant women (hCG: 0%; n = 3; hCG-alpha: 0-13%; mean, 6%; n = 5). Con A nonreactive and reactive hCG of patients with cancer revealed similar binding in a radioreceptor gonadotropin assay. The Con A nonreactive fraction of the beta-subunit was determined to be 20-73% (mean, 50%; n = 6) in serum samples of patients with testicular cancer and did not differ from the percentage in pregnant women (29-67%; mean, 46%; n = 4). The lectin binding heterogeneity of hCG and hCG-alpha indicate structural variations in the carbohydrate chains. It is assumed that Con A nonreactive hCG and hCG-alpha are directly liberated by nonsecretory mechanism from tumor cells into the circulation. Determination of hCG released by cell damage may have clinical significance in patients with testicular cancer under chemotherapy.

Populations of herpes simplex virus glycoprotein gC with and without affinity for the N-acetyl-galactosamine specific lectin of Helix pomatia.

SummaryTwo fractions of herpes simplex virus glycoprotein gC were isolated and characterized by means of immunosorbent-purification with monoclonal antibodies against gC and Helix pomatia lectin (HPA) affinity chromatography. About 25 per cent of the glycoprotein gC population demonstrated affinity for the lectin, compatible with presence of N-acetylgalactosamine as terminal sugar of the oligosaccharide. The HPA-binding populations of gC appeared as two electrophoretic bands with lower molecular weights than the non-binding gC.The gC subfraction without affinity for the HPA was subjected to treatments aiming to desialylize the carbohydrate moiety. Only 5 per cent of the initially non-reactive fraction of gC became reactive to HPA after the treatments, suggesting that masking of penultimate N-acetylgalactosamine by sialic acid was not a main reason for lack of HPA affinity. Results of treatment with alkaline Na BH4 demonstrated presence of oligosaccharide-peptide linkages sensitive to β-elimination suggesting O-glycosidic type of linkage.The subfraction of gC demonstrating affinity for HPA as well as gC devoid of HPA binding capacity both revealed affinity for Con A. Therefore N-glycosidically as well as O-glycosidically linked oligosaccharides seemed to be present on the one and same glycoprotein.On the basis of the results presented we assume that the glycosylation of HSV glycoprotein gC may lead to, at least, two populations of the glycoprotein gC, one with terminal N-acetylgalactosamine residues of oligosaccharides 0-glycosidically linked to the polypeptide and the other without affinity for HPA. However, both populations of gC contain similar proportions of oligosaccharides of the high mannose or complex types with N-glycosidic carbohydrate-peptide linkages as indicated by their affinity for Con A.

Comparison of amniotic fluid alpha-fetoprotein reactivity to Lens culinaris agglutinin and concanavalin A in crossed-affinity immunoelectrophoresis: ancillary tests in the prenatal diagnosis of severe fetal malformations.

Crossed-affinity immunoelectrophoresis of alpha-fetoprotein in amniotic fluid from 135 normal and 39 abnormal pregnancies (mainly neural-tube defects) was performed with a lectin, agglutinin from Lens culinaris. Results were compared with findings reported for another lectin, concanavalin A. Three fractions of alpha-fetoprotein were obtained by reaction with Lens culinaris agglutinin. The most weakly reactive fraction correlated strongly with the concanavalin A nonreactive fraction. With both lectins, significantly lower concentrations of these fractions were found in all samples from abnormal pregnancies than in those from normal pregnancies. In eight cases with a fetal abnormality the total alpha-fetoprotein concentrations were below the lower limit for abnormal samples. All eight samples revealed fractions weakly reactive with Lens culinaris agglutinin below the lower 0.1% limit for normal samples; seven of these samples had fractions nonreactive with concanavalin A below this limit. Although a decrease in the two described fractions in normal pregnancies was found with increasing gestational age, we find either method to be valuable as an ancillary test in the prenatal diagnosis of fetal neural-tube defects and other malformations.

Characteristics of oxidant precursor emissions from anthropogenic sources in the United States

Hydrocarbons (HC) and nitrogen oxides (NO x ) have been identified as the most important precursor pollutants for oxidant formation in the atmosphere. These pollutants are emitted both from natural and anthropogenic sources; however, these two types of sources are generally geographically separated. Anthropogenic emissions are dominant in and around urban centers, where the majority of severe oxidant problems occur. Based on data gathered by the MAP3S/RAINE emissions inventory project, anthropogenic emissions of HC in the conterminous United States were 24.8 million metric tons in 1979. HC emissions were predominantly from area sources, the transportation sector being the largest contributor with 39.8% of the total. State-by-state breakdowns are also included. Based on analyses by other emissions inventory projects, the nonreactive fraction of the emissions from the transportation sector is less than 20% by weight. The highest proportion of HC emissions occur at low altitudes (0–50 m range) in high population density areas. Anthropogenic emissions of NO x were 23.7 million metric tons in 1979; 50.8% were from point sources. The transportation sector and the electric utilities sector account for 37.1% and 30.9% of the NO x emissions, respectively. The NO 2 fraction of the emissions from the transportation and electric utilities sectors is less than 10% by weight, based on NO/NO 2 speciation data from two other emissions inventories. Highest rates of NO x emissions occur in high population density areas and are released at low altitude (0–50 m); three areas of high electric generating capacity were found to have high release altitudes.

Carbohydrate structure of the concanavalin A molecular variants of alpha-fetoprotein.

The structure of the carbohydrate units of alpha-fetoprotein from fetal calf serum has been studied. Glycopeptides and oligosaccharides were prepared from alpha-fetoprotein by protease treatment and hydrazinolysis, respectively, and subjected to carbohydrate and amino acid analysis. Two N-glycosidic glycans are present in each alpha-fetoprotein molecule. These were separated into concanavalin A (ConA)-reactive and nonreactive species on ConA-Sepharose. Methylation analysis, Smith degradation, sequential exoglycosidase treatments, and sizing suggested that the major, ConA-nonreactive fraction is composed of triantennary and the minor, ConA-reactive fraction, of biantennary complex-type ConA-reactive and -nonreactive fractions of intact alpha-fetoprotein, respectively, and refractionated on Con A-Sepharose. The results indicate that 75% of alpha-fetoprotein molecules contain two triantennary complex-type glycans, 20% contain one triantennary and one biantennary glycan, and 5% contain two biantennary glycans. The last two molecular variants are bound to ConA. These results explain, at least in part, the previously found heterogeneity of alpha-fetoprotein with respect to charge and molecular size, and provide a biochemical basis for the differing reactivities toward ConA of alpha-fetoprotein from the yolk sac, fetal liver, and various tumors.

Human large granular lymphocytes: spontaneous and interferon-boosted NK activity against adherent and nonadherent tumor cell lines.

Human natural killer (NK) cells against K-562 tumor cells have been morphologically identified as large granular lymphocytes (LGL). We have investigated whether LGL are also responsible for NK activity against a variety of adherent and nonadherent tumor cell lines. LGL-enriched fractions, obtained by centrifugation of nonadherent mononuclear cells on discontinuous density gradients of Percoll, contained high levels of spontaneous and interferon- (IFN) boosted NK activity against each of the target cells tested. In contrast, the LGL-depleted functions, which contain most of the typical small lymphocytes, were devoid of NK activity and did not develop appreciable activity after treatment with IFN. The ability of the different fractions to exert NK activity correlated with their capacity to form conjugates with nonadherent tumor cells. Reconstitution of the different fractions with macrophages did not increase the NK activity of already cytotoxic fractions and did not induce appreciable activity in the LGL-depleted fractions. On the other hand, mixtures of highly cytotoxic and nonreactive cell fractions did not reveal the presence of suppressor cells for NK in the nonreactive fractions. Taken together, our data demonstrate that both spontaneous and IFN-boosted NK activity is confined to the LGL-enriched fractions and suggest that LGL are the main effector cells exerting NK cytotoxicity against a variety of adherent as well as nonadherent tumor cell lines.

Alpha-fetoproteins produced by the yolk sac and the liver are glycosylated differently

Chromatography of mouse alpha-fetoprotein (AFP) on concanavalin A (con A)-Sepharose gave two main AFP variants, one nonreactive with con A and the other bound and eluted with α-methylmannoside. The nonreactive variant showed partial reactivity with Ricinus communis agglutinin I indicating that it is at least partly glycosylated. The nonreactive fraction greatly diminished after birth when the yolk sac is lost. Internally labeled AFP was almost completely nonreactive with con A when obtained from cultures of visceral yolk sac, while more than 90% of AFP from the liver bound to con A. These results show that a developmental change takes place in the glycosylation pattern of AFP as its synthesis shifts from the yolk sac to the liver.

Protein A reactivity of various mammalian immunoglobulins.

Serum samples and immunoglobulin fractions of eight mammalian species were applied to a Sepharose--protein A column. As with the human immunoglobulin subclasses IgG1, IgG2 and IgG4, all examined IgG classes and subclasses were bound to a greater or lesser extent to protein A. However, the binding of IgG1 of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reactive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.

Human colostral IgA interacting with staphylococcal protein A.

Human colostral IgA from three apparently healthy mothers were all divided into protein A reactive and protein A non-reactive fractions on a Sepharose-protein A column. The reactive fractions, giving no direct precipitation but co-precipitations, were found to interact with protein A through the Fc-region.