Agarose gel serum protein electrophoresis in cats with and without lymphoma and preliminary results of tandem mass fingerprinting analysis.

BACKGROUND: Serum electrophoretic profiles in cats are poorly characterized with respect to the proteins that comprise the globulin fractions, and interpretation of the electrophoretograms is routinely done in the absence of information about identity of the proteins found within each fraction. OBJECTIVES: The aims of this study were to compare protein fractions separated by serum protein electrophoresis (SPE) in healthy cats and in cats with lymphoma and to confirm some component proteins in the major fractions following SPE using tandem mass fingerprinting analysis (TMFA). METHODS: Total protein concentration was measured and agarose gel SPE performed on serum from 14 healthy cats and 14 cats with lymphoma. The absolute protein concentration within each fraction was compared between the 2 groups. Bands corresponding to the SPE fractions were excised from the gels of 2 control cats and 1 cat with lymphoma and analyzed by liquid chromatography coupled to mass spectrometry. Results were compared with sequences in the National Center for Biotechnology Information protein database. RESULTS: Median albumin concentrations were significantly decreased and median ß-globulin concentrations were significantly increased in cats with lymphoma. Narrow electrophoretic spikes were present in the ß/γ-globulin fraction in 3 cats with lymphoma. Following TMFA, multiple proteins were identified in each fraction, and their mobility agreed with results from previous studies generated using alternative techniques. Inter-α (globulin) inhibitor 4 was identified in feline serum for the first time. CONCLUSIONS: Cats with lymphoma had lower albumin and higher ß-globulin concentrations than did healthy cats. Despite limitations of one-dimensional agarose gel SPE, TMFA provided preliminary data to confirm the protein components of the various fractions.

Human papilloma virus in skin, mouth and uterine cervix in female renal transplant recipients with or without a history of cutaneous squamous cell carcinoma.

Some human papillomaviruses are thought to be associated with skin cancer. In this pilot study, 21 female renal transplant carriers, 10 with a history of skin squamous cell carcinoma and 11 without, together with 9 age-matched healthy women were investigated for human papillomavirus DNA in sun-exposed (forehead) and less sun-exposed (buttock) skin, mouth and uterine cervix. Paraffin-embedded tumours from 9 of the patients with a history of squamous cell carcinoma were analysed. Healthy skin from both the healthy and the immunosuppressed individuals harboured a wide variety of papillomaviruses. In the healthy individuals, samples from less sun-exposed skin showed a lower prevalence of human papillomavirus DNA than corresponding samples from the immunosuppressed patients (4/9 and 7/9, respectively). Among the immunosuppressed patients, human papillomavirus DNA was found as frequently in buttock samples (17/21) as in forehead samples (17/20). There was no increased prevalence of human papillomavirus in the cervix or mouth samples from the immunosuppressed patients.

Immunoprecipitation against exogenous antigens in the zone of alpha-1, alpha-2 and beta-globulins--a new immune phenomenon?

According to data by H. Zhabilov serum fractions in the zone of alpha 1, alpha 2 and beta-globulins from healthy people form a characteristic precipitation curve in terms of structure and density with the TNP (thymus nuclear protein) and CNP (carcinoma nuclear protein) as antigens when tested in two-dimensional agarose-gel electrophoresis. The observed immune phenomenon has not so far, to the best of our knowledge, been reported in the scientific literature and has profound implications for fundamental and applied immunology. The object of the present study was to confirm Zhabilov's data and study the ontogenetic aspect of the problem. We used 45 sera of children from different age groups, of healthy adult controls, HIV-infected and cancer patients. The sera were tested against the following exoantigens: TNP (thymus nuclear protein), CNP (carcinoma nuclear protein-Viral Genetics Inc, LA), as well as against ST (staphylococcal toxin), SL (streptolysin) and LPS (lipopolysaccharide) from Gram-negative bacteria (National Center for Parasitic and Infectious Diseases, Sofia, Bulgaria). Two-dimensional electrophoresis was used modified after the protocol of UCLA chemical labs. The results of our study show that immediately following birth the precipitation curve is barely discernible reaching the normal shape following 1 month-5 years of age. The precipitation curve from HIV-infected sera and those of cancer patients is similar to that of newborns. The results from our unpublished observations with sera from other biologic species tested against the same antigens show similar results: no curve was observed in fish whereas the reaction was positive with frogs, birds, rabbits and other mammals. This confirmation of the basic biologic law of ontogenesis being a repetition of phylogenesis gives us reason to consider those fractions as participating in the immune maturation of organisms for acquired immune response. There exists a possibility that they might be part of the already known immunomediators or a stage in the transition from non-specific to specific immunoglobulins which have not lost their importance in immunogenesis. The lack of previous studies in this respect and the observation and confirmation of this reaction in two different labs underscore the importance of this new immune phenomenon.

Enhanced engraftment of HTLV-I-infected human T cells in severe combined immunodeficiency mice by anti-asialo GM-1 antibody treatment.

The effects of anti-asialo GM-1 antibody (AAGM) treatment on the engraftment of human T-cell leukemia virus type I (HTLV-I)-infected human T cells in severe combined immunodeficiency (SCID) mice were studied. The frequency of tumor formation in an HTLV-I-transformed human T-cell line, MT-2 cells, at the site of inoculation was significantly higher in AAGM-treated than untreated mice (P<0.05): 16/18 (89%) and 16/26 (62%), respectively. The promotive effect of AAGM treatment on tumor development was marked in the early stage (less than 3 weeks), suggesting that the immediate reaction of natural killers to the inoculated cells may be important for the prevention of tumor development. The surface phenotypes and clonality of the tumor cells were the same as the MT-2 cells inoculated. Inoculation of peripheral blood mononuclear cells (PBMC) from one of the 4 adult T-cell leukemia/lymphoma (ATL) patients resulted in the development of tumors in AAGM-treated SCID mice. However, the surface phenotypes of the cells from these tumors were a mixture of B cells and T cells, suggesting that these tumors consisted of Epstein-Barr virus-transformed B cells and HTLV-I-transformed T cells. In addition, HTLV-I was detected by polymerase chain reaction in various organs of the mice inoculated with PBMC from the ATL patient and the asymptomatic carrier examined. These results suggest that elimination of natural killer function by AAGM treatment is important, although such treatment is not always necessary for the engraftment of HTLV-I-infected cells in SCID mice.

Factors controlling the DNA-synthesis in 3T3 and EAT cells.

Beta globulins, (Cohn Fr. III), are a major source of molecules affecting the DNA-synthesis of 3T3 and EAT cells. Growth inhibitors for both cell types, chromatograph at the same position, corresponding to a mol. wt of about 50,000. A very basic, (pI 10.1), factor is isolated by gel electrofocusing, which stimulates the DNA-synthesis of 3T3 and EAT cells. Because of its extremely high cationic charge and its adsorption on gels, the estimation of the exact molecular weight and its preparative isolation, becomes very difficult. Some of the above mentioned molecules are heat-stable and express their action even after boiling for 10 min at pH 3.

Is beta t a component of HLA-A,B,C in thymus derived cells?

beta t, the 12,000 molecular weight polypeptide originally found in HTA 1, a thymus specific differentiation antigen, is a major labelled component of surface iodinated HLA-A,B,C purified by monoclonal antibody W6/32 from the T leukaemia cell line Molt 4. A correlation between the amount of beta t in HLA-A,B,C and the level of HTA 1 expression in Molt 4 and variants derived from it was established. The presence of beta t in the HLA-A,B,C complexes is not due to cross contamination with HTA 1. However, analysis of a large scale HLA-A,B,C preparation using Coomassie blue staining shows that, under conditions of surface iodination, beta t is over-represented by a factor of about 100 times relative to beta 2 microglobulin. The significance of beta t as a minor component of HLA-A,B,C in thymus derived cells remains uncertain.

Isolation and characterization of mouse beta 2-microglobulin allotypes.

Two allelic forms of mouse beta 2-microglobulin (beta 2m), the smaller polypeptide chain of the H-2 histocompatibility antigens, were purified from urine and partially characterized. The isoelectric points of the two allotypes are 7.4 (beta 2ma) and 8.1 (beta 2mb). The electrophoretic mobility of beta 2ma is decreased by reduction, whereas the mobility of beta 2mb is not. Urinary beta 2m can replace endogenous beta 2m in mouse H-2 histocompatibility antigens.

[Isolation and purification of specific beta1-G-globulin]. / Vydelenie i ochistka spetsificheskogo beta1-G-globulina.

Antigen, specific for trophoblastic tumor, was isolated from retroplacental blood by means of ion exchange chromatography on DEAE cellulose, gel filtration on Sephadex G-200, chromatography on hydroxylapatite and CM-cellulose and by preparative isofocusing using ampholines. Distinct heterogeneity of the protein and alteration in several its properties were observed as purity of the preparation was increased in course of isolation. A series of the protein preparations was obtained with 90-95% purity and with similar immunologic properties but the preparations differed in electrophoretic mobility, isoelectric points and some other patterns.

beta2-Microglobulin from normal and leukaemic guinea-pig lymphocytes.

beta2-Microglobulin has been isolated in useful quantities from the urine of strain-2 guinea-pigs after either treatment with sodium chromate or induction of the L2C leukaemia. Antibodies raised against the beta2-microglobulin were used to set up a radioimmunoassay which measured its export into culture fluid by normal and leukaemic lymphocytes. Material containing beta2-microglobulin was also obtained by digestion of the lymphocytic surfaces with papain; fractionation demonstrated both free and combined forms, with no qualitative difference between those from normal and those from leukaemic cells.

Modulation of the alternative complement pathways by beta 1 H globulin.

C3b inactivator accelerator (A-C3bINA) was isolated from human plasma. An antiserum produced against the purified protein gave a reaction of identity with beta 1 H, a well-documented contaminant of C3 preparations. Beta 1 H appears to be composed of a single polypeptide chain containing a significant quantity of carbohydrate, and having a sedimentation coefficient of 5.6 on analytical, and 6.4 on sucrose density gradient ultracentrifugation. Its mol wt based on SDS polyacrylamide gel electrophoresis and equilibrium sedimentation is approximately 150,000, whereas it elutes from Sephadex G200 with an apparent mol wt of 300,000, suggesting that beta 1 H is an asymmetric molecule. Beta 1 H potentiates the inactivation of C3b by C3b inactivator, binds to EAC43 to limit the formation of EAC43bB and EAC43bBP, and in contrast to C3b inactivator, it increases the rate of loss of hemolytic sites from EAC43bB and EAC43bBP. For the C3b inactivator-potentiating effect, beta 1 H and C3b inactivator must necessarily be simultaneously present. The kinetics of inactivation of C3b by C3b inactivator and beta 1 H are first order, suggesting that potentiation is not a multistep process. The mechanisms of binding to C3b and inhibition of the alternative pathway convertases C3bB and C3bBP are currently unknown.

[Immunological importance of a new protein: the beta2-microglobulin (author's transl)]. / Importanza immunologica di una nuova proteina: la beta2-microglobulina

The Authors are presenting a concise review on a protein, beta2-microglobulin, that has gained in notable importance in the immunological field during these last years. The story of this protein begins with nephrology because it has a notable importance in the diagnosis of renal tubular diseases; in fact its presence in the electrophoretic pattern of proteinuria puts in evidence renal tubular damage. Nextly immunological studies have shown the beta2-microglobulin has strict correlations with the HL-A system; probably it forms a part of this system, as shown by the new immunofluorescent methods applied to the lymphocte. A recent study has shown also the beta2-microglobulin serum levels are very high in some malignant diseases, like in acute leukemia; however this low molecular weight protein could also be the expression of malignancy because it can be a product of non mature cells in a large quantity.

Steroid-binding proteins in rabbit plasma: Separation of testosterone-binding globulin (TeBG) from corticosteroid-binding globulin (CBG), preliminary characterization of TeBG, and changes in TeBG concentration during sexual maturation.

Two distinct steroid-binding proteins are present in rabbit plasma. One of the proteins (TeBG) binds [3-H]5 alpha-dihydrotestosterone (5 alpha DHT) and [3-H]testosterone. The affinity of this binding protein for 5 alpha DHT was 3-4 times greater than for testosterone. Binding of [3-H]5 alphaDHT could be inhibited by unlabeled 5 alpha DHT, testosterone, 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), and 17 alpha-methyl- B-testosterone (skf) 7690). The relative affinity of the competitors was: 5 alpha DHT greater than 3 alpha-diol greater than testosterone greater than SKF 7690. The antiandrogens, cyproterone (1,2 alpha-methylene-6-chloro-pregn-4,6-diene-17 alpla-ol-3,20 dione), cyproterone-17-acetate, and 6 alpha-bromo-17 beta-hydroxy-17 alpha-methyl-4-oxa-5 alpha-androstan-3-ine (BOMT) were ineffective in competing for [3-H5d alpha DHT binding sites, as were 4-androstene-3, 17-dione, 17 beta-estradiol (E2), progesterone, and cortisol. The formation of the [3-H]5 alpha DHT-TeBG complex was extremely rapid; the binding reaction was essentially completed in 15 s. The complex dissociated rapidly in the presence of charcoal. The dissociation rate constant (Kdiss) was 0.157 min- minus 1 and the dissociation half-time t-1/2) was 4.5 min. In the presence of charcoal and unlabeled 5 alpha DHT the Kdiss was 0.268 min- minus 1 and the t=1/2 was 2.6 min. The sedimentation coefficient of TeBG was congruent to 4.6 S and its molecular weight, estimated by gel filtration on a calibrated Sephadex G-200 column, was congruent to 75,000. The concentration of TeBG in male rabbit plasma decreased with sexual maturation and was approximately three times higher in adult females than in adult males. The other protein (CBG) bound both [3-H]cortisol and [3-H]progesterone. Binding of these compounds could be inhibited by unlabeled cortisol and progesterone, but not by unlabeled 5 alpha DHT, testosterone, or E2. CBG had a sedimentation of congruent to 3.9 S and an apparent molecular weight of congruent to 105,000. TeBG could be separated from CBG by a 60% ammonium sulfate precipitation and by gel filtration chromatography. Both proteins are thermolabile; TeBG is inactivated at temperatures above 30 degrees C and CBG is inactivated at temperatures above 50 degrees C.

Studies on the cytophilic properties of human beta2 microglobulin.

Human beta2 microglobulin is cytophilic for mouse, rat, and to a lesser extent guinea pig lymphocytes and polymorphonuclear leukocytes but not for human, rabbit, chicken, frog, and turtle cells, nor for any mature erythrocytes tested. Mouse splenic lymphocytes bind 5- 7-fold more beta2 microblobulin than thymocytes although hydrocortisone-resistant thymocytes resemble spleen cells in this regard. The beta2 microglobulin binding by B and T cells in mouse spleen is similar. The structure responsible for beta2 microglobulin binding to the surface of cells is distinct from the Fc receptor specific for aggregated IgG.