Interrelationships between the virulence and the presence of M-protein in hemolytic streptococcus group A]

Experiments on mice demonstrated that of hemolytic streptococci A M types 2, 3, 12, 22, 46, and 49 characteristic is the absence of parallelism between their virulence for mice and the presence of M+-colonies in the microbial population. There was also a greater virulence for mice of M-variants. The established changes in serological properties of the cultures passaged in vitro were brought to the acquisition of polyagglutinability by the passage cultures, and to the loss of type-specific protection by the immune sera against these cultures. A possibility of acquisition by passage cultures of a factor determining their high virulence for mice, nonidentical to M-protein, is discussed.     

Stimulation of the Growth of Excised Cultured Roots of Soya Bean by Abscisic Acid

Excised root cultures of soya bean cultured at 30 °C inWhite's medium (1943) supplemented with 0•1 per cent yeastextract have been serially sub-cultured over 13 culture passagesof 7 days although a decline in the linear growth and lateralformation begins in the third passage and the roots are devoidof laterals from the 8th passage onwards. Applications of abscisicacid within the concentration range 0•01–0•001mg l^–1 and during the first two culture passages enhancedthe linear growth of the main axis, the number of emergent lateralsand the total length of laterals. This effect has been shownfor two cultivars, with roots derived from seed in both itsfirst and second year of storage and under different conditionsof culture and culture pH.     

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.     

A clonal analysis of anthocyanin accumulation by cell cultures of wild carrot

The accumulation of anthocyanin by clones and subclones from a cell suspension culture of wild carrot (Daucus carota L.) has been measured under standard conditions. Clones which accumulate low amounts of anthocyanin were shown, by recloning after maintenance by serial passage, to have become heterogenous and to contain cells with increased accumulation of anthocyanin. There appears to be a maximum amount of anthocyanin that clones can accumulate. Clones which accumulate the maximum amount of anthocyanin were shown by recloning after maintenance by serial passaging, to have become heterogenous and to contain many cells which accumulate less than the maximum possible amount of anthocyanin. When clones which accumulate the maximum amount of anthocyanin are maintained by serial passage, the decline in anthocyanin accumulation is different in different media. The results indicate that the changes in the ability of cells to accumulate anthocyanin involve no qualitative change in the genetic information of the cells, i.e., the changes are not the consequence of mutations.     

Carbon Isotope Fractionation during Anaerobic Degradation of Methyl tert-Butyl Ether under Sulfate-Reducing and Methanogenic Conditions

Methyl tert-butyl ether (MTBE), an octane enhancer and a fuel oxygenate in reformulated gasoline, has received increasing public attention after it was detected as a major contaminant of water resources. Although several techniques have been developed to remediate MTBE-contaminated sites, the fate of MTBE is mainly dependent upon natural degradation processes. Compound-specific stable isotope analysis has been proposed as a tool to distinguish the loss of MTBE due to biodegradation from other physical processes. Although MTBE is highly recalcitrant, anaerobic degradation has been demonstrated under different anoxic conditions and may be an important process. To accurately assess in situ MTBE degradation through carbon isotope analysis, carbon isotope fractionation during MTBE degradation by different cultures under different electron-accepting conditions needs to be investigated. In this study, carbon isotope fractionation during MTBE degradation under sulfate-reducing and methanogenic conditions was studied in anaerobic cultures enriched from two different sediments. Significant enrichment of ¹³C in residual MTBE during anaerobic biotransformation was observed under both sulfate-reducing and methanogenic conditions. The isotopic enrichment factors () estimated for each enrichment were almost identical (−13.4 to −14.6; r² = 0.89 to 0.99). A value of −14.4 ± 0.7 was obtained from regression analysis (r² = 0.97, n = 55, 95% confidence interval), when all data from our MTBE-transforming anaerobic cultures were combined. The similar magnitude of carbon isotope fractionation in all enrichments regardless of culture or electron-accepting condition suggests that the terminal electron-accepting process may not significantly affect carbon isotope fractionation during anaerobic MTBE degradation.     

Is beta-galactosidase staining a marker of senescence in vitro and in vivo?

Cytochemically detectable beta-galactosidase (beta-gal) at pH 6.0 has been reported to increase during the replicative senescence of fibroblast cultures and has been used widely as a marker of cellular senescence in vivo and in vitro. In this study, we have characterized changes in senescence-associated (SA) beta-gal staining in early and late passage cultures, cultures established from donors of different ages, virally immortalized cells, and tissue slices obtained from donors of different ages. The effects of different culture conditions were also examined. While we confirm the previous report that SA beta-gal staining increased in low-density cultures of proliferatively senescent cells, we were unable to demonstrate that it is a specific marker for aging in vitro. Cultures established from donors of different ages stained for SA beta-gal activity as a function of in vitro replicative age, not donor age. We also failed to observe any differences in SA beta-gal staining in skin cells in situ as a marker of aging in vivo. The level of cytochemically detectable SA beta-gal was elevated in confluent nontransformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density, and in low-density, young, normal cultures oxidatively challenged by treatment with H2O2. Although we clearly demonstrate that SA beta-gal staining in cells is increased under a variety of different conditions, the interpretation of increased staining remains unclear, as does the question of whether the same mechanisms are responsible for the increased SA beta-gal staining observed in senescent cells and changes observed in cells under other conditions.     

Evaluation of purified recombinant spike fragments for assessment of the presence of serum neutralizing antibodies against a variant strain of porcine epidemic diarrhea virus

Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.     

Long-term stability of root cultures of tomato transformed with Agrobacterium rhizogenes R1601

Tomato explants were transformed with Agrobacter rhizogenesR1601 and root clones used to initiate longterm cultures inliquid and on solid media. The liquid cultures were maintainedfor 50 passages over 25 months in the presence and absence ofkanamycin. During this time the clones retained their high growthrates and antibiotic resistance phenotypes. The nptll gene wasdetected by PCR and dot blot hybridization in DNA from clonesat the 21st passage, and NPT-II enzyme activity was detectedin cell extracts prepared at the 45th passage. The solid cultureswere main tained for 12 passages over 12 months, either withcontinual selection, or with alternation during the last sixpassages between selective and non-selective media. The nptllgene was detected by PCR in DNA from cultures at the 5th passageand NPT-II enzyme activity was detected in cell extracts ofvigorously growing clones from the 12th passage. Passages inthe absence of selection had no discernible effect on the stabilityof the transformed state. The results indicate that the Ri-transformedstate can be maintained in tomato root clones during long periodsof culture. Key words: Agrobacterium rhizogenes, long-term cell culture, root clones, tomato, transformation     

Identification of tumor antigens as potential target antigens for immunotherapy by serological expression cloning

The presence of tumor infiltrating T cells has been shown to be associated with a favorable prognosis in different tumor types. Several strategies have been developed to identify relevant tumor antigens which can be used for active immunotherapy strategies. The SEREX technique (serological analysis of cDNA expression libraries) identifies tumor antigens based on a spontaneous humoral immune response in cancer patients. This technique is not limited to tumor types that can be grown in cell culture or depends on established T cell clones recognizing the autologous tumor. Several steps of analysis are mandatory to evaluate SEREX-defined antigens before they become new target antigens for active immunotherapy: expression analysis; serological analysis with sera from tumor patients and normal individuals; identification of potential peptide epitopes for CD8 T cells and evaluation in T cell assays. This article summarizes our approach of antigen identification and evaluation giving the example of the recently cloned breast cancer antigen NY-BR-1.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Diagnostic possibilities of serology in diseases caused by members of the genus Candida berkhout

Up to now, the diagnosis of candidiases has been an intriguing problem chiefly because the detection of Candida in the sick organism is no conclusive evidence of its etiological involvement. The endeavour to use serological methods in the diagnosis has not been successful and their use has so far practically been limited to taxonomic studies. In an effort to find a serological approach to the diagnosis, we found the agarimmunodiffusion assay of Ouchterlony to be the most promising method and used it in our own modification. The first stage of the work was to select and prepare the most suitable type of antigen for this reaction (described in the present paper). Sixteen antigen types classifiable into four groups were prepared: simple water-extracted antigens, alcohol-extracted antigens, antigens obtained on disrupting cells by repeated freezing and thawing, and antigens prepared by boiling cultures. Cultures for antigen preparation were grown on Salvin's medium or on liquid Sabourad's medium and the antigens obtained were either used in their native form or dialysed. Serological tests with hyperimmune rabbit sera prepared by our own schedule were done repeatedly. By far the best were simple water-extracted antigens, nondialysed, from cultures grown on Sabouraud's medium. They reacted the most sensitively, gave high antibody detection rates and assessable precipitin reactions and showed high species specificity. Tests with positive human sera fully confirmed these findings; in fact, species specificity was even somewhat higher here.     

Determination of tularemia antibodies by an immunoenzyme method on a solid-phase carrier (ELISA)

ELISA "sandwich" techniques have been developed and the optimum assay conditions for detecting specific antibodies in human serum samples have been determined. The possibility of using these techniques for the determination of the level of antibodies to tularemia antigens in the sera of persons immunized with live tularemia vaccine has been shown. Statistically significant differences in the level of antibodies to tularemia antigen in the sera of immunized and nonimmunized persons have been established. The comparative study of five serological methods - ELISA, the agglutination test, the passive hemagglutination test, the immunofluorescence test and the defined antigen substrate sera ( DASS ) techniques - has revealed the advantage of ELISA, whose sensitivity has proved to be considerably higher than that of all other methods used in our work.     

Characterization of Escherichia coli donor-specific RNA-containing bacteriophages

The biological properties of 9 clones of Ri bacteriophages isolated from sewage water in 1981 were studied. On the basis of the activity of Ri phages with respect to E. coli donor-specific strains K12, the type of negative colonies, the ultrastructure of the virion and its sizes, adsorption on the pili of host cells, the latent period, the amount of harvest obtained from one infected cell, the clones under study were classified with small spherical RNA-bacteriophages. The neutralization of Ri phages with antiphage sera to standard phages f2 and fr made it possible to classify them with the first serological group and to divide them into 3 subgroups.     

Clonal selection in cultured human fibroblasts: role of protein synthetic errors

Protein synthetic error frequency, determined in cell-free extracts as delta leu/delta phe incorporation following poly(U) stimulation, has been found to decrease progressively in several strains of human diploid fibroblasts during their limited replicative lifespan. To explore the basis of this phenomenon, we followed a mass (uncloned) culture of one normal strain at 13 stages of its replicative lifespan. We found a progressive tenfold decline in error frequency that was inversely correlated with passage level (r = -.93, p less than .001). This could not be ascribed to the slow rates of replication associated with fibroblast senescence because slowing of growth by serum deprivation did not change error frequency. Additionally, terminal mass cultures maintained for 16 wk at saturation density to minimize cell selection did not change error frequency over this time. Error frequencies in 12 individual clones purified from the parental culture did not decline on repeated passage, either remaining constant or, in two clones, rising abruptly three- to five-fold after initial assays. Error frequencies of clones showed a weak inverse correlation with growth vigor but not with the maximum doubling number. We conclude that selective pressures favor more vigorously dividing clones with low protein synthetic error frequencies leading to their predominance in mass cultures.     

利用猪血清短期培养人体外周血淋巴细胞的研究

用猪血清代替小牛血清作人体外周血淋巴细胞短期培养,进行染色体分析及淋巴细胞转化的研究。在94例姐妹染色单体差别染色的培养中,培养液分别加入小牛血清与猎血清,细胞有丝分裂指数分别为3.91％和3.78％;21例常规法培养中,分裂指数分别为9.10％和9.21％;5例淋巴细胞转化试验,小牛血清和猪血清培养的转化率分别为69.52％和69.59％;16例阉割后公、母猪血清加入培养基中,细胞分裂指数(SCD法)分别为2.96％和3.14％。以上各项对照试验,均无显著性差异(P>0.05)本研究证实,猪血清可用于人体外周血淋巴细胞短期培养。     

Identification of autoantibodies associated with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinuclear antibodies. We performed serological analysis of cDNA expression library (SEREX) to identify autoantibodies associated with SLE. The screening of three different cDNA expression libraries with pooled sera of patients with SLE yielded 11 independent clones that reacted with pooled sera of patients with SLE. In this screening, autoantibodies to poly(ADP-ribose) polymerase (PARP), U1snRNP, and galectin-3 were prevalent in the sera of patients with SLE (26/68, 25/68, 12/63, respectively). The frequency of autoantibody to PARP was significantly higher in SLE than that of healthy donors (0/76) (38.2% vs 0%, p<0.00001). The autoantibody to PARP was infrequently detected in the serum of patients with RA (1/50). However, autoantibody to PARP was not found in the sera of patients with other rheumatic diseases including Sjogren's syndrome (0/19), systemic sclerosis (0/18), and polymyositis/myositis (0/37). The frequency of autoantibody to human galectin-3 (12/63) was significantly higher in SLE than that of healthy donors (0/56) (19% vs 0%, p=0.0006). Autoantibody to galectin-3 was not found in the sera of patients with rheumatoid arthritis (0/50), Sjogren's syndrome (0/18), and systemic sclerosis (0/19). Interestingly, autoantibody to galectin-3 was also prevalent in the sera of patients with polymyositis/dermatomyositis (16/37, 43.2%). Further functional characterization of these autoantibodies would be necessary to determine their value as diagnostic markers or to define clinical subsets of patients with SLE. Statistical analysis revealed that the presence of autoantibody to PARP was inversely related with pleurisy, and the presence of autoantibody to galectin-3 related with renal disease.     

Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from an infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.     

The Primulales: Serological contributions to the problem of their systematic position

The distribution of serological determinants in seed proteins from different species of Primulaceae, as well as from Ardisia crenulata (Myrsinaceae), Erica tetralix (Ericaceae), Manilkara zapota (Sapotaceae, Ebenales). Actinidia chinensis (Actinidiaceae) and Armeria maritima (Plumbaginaceae) has been determined by the use of corresponding rabbit immune sera. Serological reactions were carried out using a gel diffusion micro-method in calcium alginate gel, and a special pre-absorption technique was used in order to more closely specify the distribution of serological characteristics in the taxa under examination. While no systematic similarities were found between Primulales and Plumbaginales, a serological correspondence between Primulales and Ericales is very clear. It also occurs between these two taxa and species of the Theales. The Sapotaceae (as representatives of the Ebenales) showed some serological similarities with the Theales.