The discovery and development of green fluorescent protein (GFP) from the jellyfish Aequorea victoria and,more recently red fluorescent protein (dsRed) from the sea anemone Discosoma striata, have revolutionized our ability to study biological function such as protein localization, dynamics and interactions in living cells. The usefulness of GFPs derives from the finding that the fluorescent property of GFPs requires no other cofactor: The fluorophore forms spontaneously from the cyclization of the peptide backbone. Although GFPs promise high sensitivity and great versatility for biological applications, they sometimes send irresponsible signals because of the properties such as their sensitivity to pH and chloride fluctuations and photobleaching. Therefore, GFP users should be careful in interpreting the fluorescent signals from GFP variants to obtain reliable biological data. On the other hand, by using the characteristics, a wide range of application could be done. In this review, we will present 1) an overview of the physicochemical properties of GFPs, and 2) the valuable applications of GFP for biological research, including circularly permuted GFP technology and GFP-based fluorescence resonance energy transfer technique as well as their potential in a variety of biological sensors.
Flow-cytometry is used for the analysis of leukemia and lymphoma cells, and, less frequently, erythrocytes or platelets. In this article, the authors shows the complimentary roles of flow-cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria(PNH) and idiopathic thrombocytopenic purpura (ITP), which represent erythroid and megakaryocytic diseases, respectively. PNH is characterized by expansion of one or more stem cell clones with a glycosylphosphatidylinositolanchored protein(GPI-AP) mutation. Flow-cytometry analysis of peripheral blood cells and detection of GPI-AP-deficient cells is a simple and reliable method for establishing the diagnosis of PNH. Sensitivity for detection of GPI-AP-deficient cells with flow-cytometry was higher than that of the Ham test and sugar water test. The Proportion of GPI-AP-deficient cells was greater in monocytes and granulocytes than in erythrocytes. Not only CD55 and CD59 assays but also CD14, CD16, CD24 assays were suitable for detection of GPI-AP-deficient monocytes and granulocytes. Measurement of platelet-associated immunoglobulin G has been applied for the diagnosis of ITP. To prepare platelet-rich plasma, whole blood should be diluted with more than the same amount of buffer. To wash platelets, buffer with albumin and EDTA was used at a room temperature to attain higher recovery rate. Sodium citrate was used as anticoagulant since the sample anticoagulated with EDTA showed nonspecific reaction during its storage.
There are some technical issues concerning the accurate flowcytometric Immunophenotyping(It is essential to take several factors into consideration in order to conduct accurate flowcytometric immunophenotyping.). We evaluated following factors: (1) the fluorescence intensity of major fluoroscence probes used for monoclonal antibody conjugation.;(2) the compensation requirements for the third color dye, such as PerCP, PECy5, PerCP-Cy5.5 ;(3) the reactivity differences resulted from clones used and sample treatments (ie., fixation). Results demonstrated that (1) antibodies labeled with bright fluorescence should be used to detect markers expressed at low levels on cell surface when determined with two-color combination for CD4 and CD25;(2) in 4-color analysis using APC conjugated antibodies, substantial compensation adjustment was required when used a tandem dye as the third color, particularly PE-Cy5, as a result of direct excitation by a 2nd red laser; (3) antibody reactivity was dependent on clones used and methods of sample preparation represented by CCR5 antibody.
The guinea pig has been one of the valuable animal models in studying various diseases and developing counter measures. However, many immunological parameters remain unclarified. Using commercially available anti-guinea pig antibodies or usual gating, leukocyte fractionation has been difficult to differentiate by flow cytometry. We found that cross-reactive anti-porcine antibody MIL4 is effective in differentiating leukocytes into 4 distinct populations by gating with SSC. Each of the 4 populations were sorted and identified in which: fraction 1 that was MIL4negativeSSClow was ‘lymphocyte’; fraction 2 that was MIL4highSSChigh was ‘neutrophil’; fraction 3 that was MIL4intermidiateSSCintermediate was 'monocyte'; and fraction 4 that was MIL4negativeSSChigh possessed a large PAS+ inclusion body and was CD4high, FcR+, CD8low and T-cell markers. Thus, a novel MIL4/SSC parameter enabled the differentiation of peripheral blood leukocytes of guinea pigs into 4 characteristic leukocyte fractions. This may provide a tool to define the phenotypes of each leukocyte fraction of the guinea pig.
Using the in site DNA nick end labeling methods and immunohistochemistry, the incidence of apoptosis (apoptotic index, AI) and labeling index of Ki-67 (LI) were determined in the superficial spreading type (superficial type) of early gastric cancer. The relationship of the growth pattern and the heterogeneity of AI and LI in 3 different portions within the tumor (the central surface, lateral margin, and the deepest portion) was studied and compared with small-sized submucosal cancers (penetrating type). In the superficial type cancer, the AI values (median) were the lowest (0.23%) in the lateral marginal portion among the 3 portions, and were as high in the deepest portion (0.85%) as in the central surface (1.12%). In the penetrating type, no apparent differences of the AI values among the 3 portions were observed. Concerning the LI values, a similar heterogeneity pattern was observed in both types of cancer: the LIs were the lowest in the deepest portion among the 3 portions, and were as high in the lateral marginal portion as in the central surface portion. Thus, in the superficial type cancer, the proliferative activity was preserved and apoptotic cell loss was decreased at the lateral portion, whereas the proliferative activity was decreased and apoptotic cell loss was preserved at the deepest portion. These results suggested that the impaired balance of the apoptotic cell death and proliferation within the tumor in the superficial type of early gastric cancer might be related to its characteristic predominant growth in the horizontal direction compared with the growth in the vertical direction within the stomach wall.
The lectin binding that is related to the structure of sugar chain of cell surface is available an indicator to changes for the character of cell. Tetraploid and octaploid Meth-A cells were established from the diploid cells. To examine the lectin binding, diploid, tetraploid and octaploid Meth-A cells growing exponentially were double-stained with propidium iodide and 7 sort of FITC labeled lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), Ricinus communis agglutinin Ⅰ(RCA120), peanut agglutinin (PNA), soybean agglutinin (SBA), Ulex europeaus agglutinin Ⅰ(UEA-Ⅰ) and Dolichos bifluorous agglutinin (DBA), and measured for their fluorescence by flow cytometry. The extent of lectin binding was different by the lectins used. It was suggested that the expression of sugar chain changes by polyploidization
The standard ploidy generated by Fairfield DNA Ploidy System was described in some reports and we tried to measure three kinds of malignant tumors and obtained clear data with this system. Sample preparation from formalin-fixed, paraffin-embedded tissue specimens is fundamentally the same as the one for laser scanning cytometric DNA analysis except nuclear staining. Samples were obtained from blocks of formalin-fixed,paraffin-embedded tissues by scraping with knives for ophthalmologists. Free cells of almost naked nuclei were obtained through deparaffinization and treatment with 0.5 % trypsin solution. All samples were stained by the method of Feulgen followed by measurement for DNA Ploidy. Five hundred tumor cells and two hundred lymphocytes were picked up for measuring of DNA after selecting them from the nuclear image. DNA ploidy patterns of all cases were clearly and semi-automatically generated. In case of shortage of lymphocytes in the samples, normal lymphocytes obtained from a block of lymph node were added and picked up for measurements of a given number of them. And largest peak of lymphocytes is a position of 2n in DNA ploidy. In the case of malignant fibrous histiocytoma, different DNA ploidy patterns, diploid and aneuploid, were found in one large tumor mass, which could make us estimate the different progression in the same tumor. In cases of malignant lymphomas and esophageal squamous cell carcinomas, aneuploid peaks were recognized in their DNA ploidy patterns. The figures of relation between nuclear size and DNA content obtained at the same time showed the nuclear atypism in all cases. Instruments of Fairfield DNA Ploidy System are handy in operation and can describe nuclear image delicately, and pick up tumor cells and lymphocytes selectively.
T cells specific for minor histocompatibility (H) antigens play a crucial role in immune responses in recipients following HLA-identical stem cell transplantation. Minor H antigen-specific cytotoxic T cell clones have been generated by random cloning followed by extensive characterizations for HLA restriction; thus it was difficult to expect to obtain T cell clones restricted by HLA allele of interest efficiently. Here we report a new approach that permits both direct visualization of T cell response to minor H antigens and cloning of T cells specific for minor H antigens presented by HLA alleles of interest. Panels of Epstein-Barr virus-transformed B lymphoblastoid cell lines (LCL) transduced with each of recipient's HLA alleles were used as stimulators in an enzyme-linked immunospot assay to identify restricting HLA alleles and LCL that possess minor H antigens detectable by T cell fractions in T cell lines generated from stem cell recipients. This method allowed us to select appropriate stimulators for positive selection of T cells specific for minor H antigens presented by HLA of interest using IFN-γcatch method. We have generated four cytotoxic T cell clones restricted by HLA-A24 which is the most common allele in Japanese (~60%) by this HLA-targeted cloning method. This new method could potentially applied to isolate T cell clones that recognize any antigens in the context of specific HLA alleles of interest.
Microsatellite instability (MSI) is known to be associated with defective DNA mismatch repair in various human malignancies, and is regarded as a major factor in tumorigenesis. To establish an assay system where more precise and objective assessments are made feasible, we made use of fluorescence-labeled polymerase chain reaction (PCR) and laser scanning. Secondly, we cotntroled Taq polymerase-dependent modification of the amplified microsatellite sequences, by enzymatic modification with T4 DNA polymerase. Thirdly, we developed a dual fluorescence co-electrophoresis system, in which both samples derived from cancer and normal tissues are electrophoresed in the same lane, in order to minimize migration errors. These improvements remarkably facilitate precise and objective assessments of microsatellite instability. Using this new system, High Resolution Fluorescent Microsatellite Analysis (HRFMA), we examined MSI in various human malignancies. Intriguingly, patterns of microsatellite changes observed can be classified into two distinct subtypes; one showing relatively small changes within 6 base pairs (type A) and the other exhibiting drastic changes over 8 base pairs (type B). In gastric and colorectal cancer, Type A and Type B was observed in 20-25% and 3-10%, respectively. Although MSI has been uniformly connected to high risk for cancer, only type B microsatellite instability correlated with family history in gastric cancer. The risk for secondary malignancies and that for a familial predisposition may be independent, even in the same diseases.