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PubMed2003

A simple PCR/RFLP analysis can differentiate between Candida albicans, Aspergillus niger, and Aspergillus fumigatus.

The clinical management of immunocompromised patients depends on the rapid identification of infectious agents such as fungal pathogens. The procedure described here for accomplishing this uses a sensitive polymerase chain reaction method, previously reported, combined with restriction-enzyme digestion to distinguish between Candida and Aspergillus species and to classify Aspergillus strains.

Aspergillus fumigatus↗
PubMed2006

Comparative proteomics analysis of differentially expressed phosphoproteins in adult rat ventricular myocytes subjected to diazoxide preconditioning.

Mitochondrial ATP sensitive potassium channels (mitoK(ATP) channels) are involved in the cardioprotection afforded by ischemic preconditioning (IPC) and diazoxide, a selective mitoK(ATP) channel opener. The activation of some kinases, including phoshoprotein kinase (PKC)-epsilon and mitogen-activating protein kinases (MAPK), is involved in signal conduction of preconditioning downstream from mitoK(ATP) channel opening. Diazoxide can open mitoK(ATP) channels and activate PKC-epsilon, which will phosphorylate some substrate proteins. These proteins that exhibit altered post-translational modification via phosphorylation due to diazoxide pretreatment may be the target molecules and play an important role in cellular protection after mitoK(ATP) channel opening. To analyze and identify the phosphoproteins associated with diazoxide preconditioning, phosphoprotein enrichment and comparative two-dimensional gel electrophoresis (2D-GE) were used. Cultured adult rat ventricular myocytes were pretreated in the presence and absence of 100 micronol/1l diazoxide for 10 min and enriched phosphoproteins from control myocytes and those pretreated with 100 micromol/l diazoxide were separated by 2D-GE and stained with a silver staining kit. Phosphoproteins of interest were further identified by matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF MS). Eight protein spots with different abundance were found, of which six differentially expressed proteins were identified by MALDI-TOF MS. They included 94 kDa glucose-regulated protein, calpactin I heavy chain, chaperonin containing TCP-1 zeta subunit, hypothetical protein XP_346548, ferritin light chain and ferritin light chain 2. These findings provide new clues to understanding the mechanism of ischemic preconditioning in cardiomyocytes downstream from mitoK(ATP) channel opening.

Animals↗
PubMed1997

Analysis of differential display RT-PCR products using fluorescent primers and GENESCAN software.

Differential display reverse transcription PCR (DDRT-PCR) is a procedure used to identify the induction or repression of gene expression. In most DDRT-PCR protocols, radioisotopes are incorporated during PCR and the cDNA products are detected by autoradiography. This report describes the fluorescent labeling of cDNAs and their detection on automated sequencers from PE Applied Biosystems. A fluorescent tag can be incorporated into the PCR product by using either a labeled primer or a labeled dUTP. The fluorescent signals are analyzed by GENESCAN software. Fluorescent DDRT-PCR increases throughput and obviates the handling of hazardous radioisotopes. A PCR cycling profile, expected to give improved reproducibility, is also described. Because amplified cDNAs can't be recovered from the automated sequencer gel, suggestions are given for the identification and recovery of differentially expressed cDNAs.

Animals↗
PubMed1988

Analysis of differentiation and transformation of cells by lectins.

During differentiation cells are known to change their biological behavior according to their genotype. This is thought to be accompanied by a modulation of cell surface determinants expressed on the outer cell membrane. Vice versa, cell surface molecules are suggested to mediate extracellular signals to the genome. Most of these molecules integrated in the cell membrane have been proven to be glycoconjugates. The carbohydrate moieties of these molecules can be detected by means of lectins that are characterized by their ability to react specifically with distinct terminal sugar sequences. Thus, lectins have been used as appropriate tools for studying the modulation of functionally important membrane-associated molecules during the differentiation of cells, in particular of B- and T-lymphocytes. Moreover, lectins have been proven to distinguish between differentiated cells and malignant cell clones, according to the hypothesis that transformed cells possess a glycoconjugate profile that corresponds to the stage of differentiation at which they are arrested. Since lectins, like monoclonal antibodies, make it possible to study functionally important molecules that are associated with differentiation and malignancy, they might be of value for diagnostic purposes and, moreover, for analyzing malignant transformation.

Cell Differentiation↗
PubMed2006

Analysis of differentially expressed proteins in cancerous and normal colonic tissues.

AIM: To identify and analyze the differentially expressed proteins in normal and cancerous tissues of four patients suffering from colon cancer. METHODS: Colon tissues (normal and cancerous) were homogenized and the proteins were extracted using three protein extraction buffers. The extraction buffers were used in an orderly sequence of increasing extraction strength for proteins with hydrophobic properties. The protein extracts were separated using the SDS-PAGE method and the images were captured and analyzed using Quantity One software. The target protein bands were subjected to in-gel digestion with trypsin and finally analyzed using an ESI-ion trap mass spectrometer. RESULTS: A total of 50 differentially expressed proteins in colonic cancerous and normal tissues were identified. CONCLUSION: Many of the identified proteins have been reported to be involved in the progression of similar or other types of cancers. However, some of the identified proteins have not been reported before. In addition, a number of hypothetical proteins were also identified.

Adenocarcinoma↗
PubMed1993

[Characteristics of bladder tumors showing positive urinary cytology--respective analysis in differentiated and undifferentiated type].

To study the characters of bladder tumors which showed positive urinary cytologies, the results of urinary cytology of untreated bladder tumor patients in our hospital during 10 years between July 1980 and June 1990 were evaluated according to the degree of tumor differentiation, namely differentiated type which did not contain grade 3, and undifferentiated type which contained grade 3. Concerning the differentiated tumors, positive rate of cytology was 37.3% on the whole. Positive rates of multiple tumors or tumors around internal urethral orifice were significantly high, and those of tumors larger than 3 cm, tumors located on posterior wall, left wall or throughout the entire surface, were higher than the overall positive rates. Repeated examinations of yielded increased rates of positive cytology results. Concerning the undifferentiated tumors, the positive rate of cytology was 90.0% on the whole, was not influenced by tumor sizes or locations, and was consistently observed irrespective of the number of times of cytologic examination.

Aged↗
PubMed1976

[Fluorescence and electron-microscopic analysis of differentiation of the myocytes of the ventricles and atria of the avaian heart in ontogenesis].

The histogenesis of the ventricle and atria myocardium was studied in 240 chick embryos of from 30--34 hours up to 20 days of incubation and in 66 chickens at the age of 1--90 days. Fixators were the Bouin's Carnoy's and Newkomer's fluids and the 100% solution of neutral formalin. The paraffin sections were stained by histological and histochemical methods. RNA, DNA, glycogen and lipids were detected. The ultrafine sections of the material fixed in 1% buffer solution of osmium tetroxide (pH 7,2) and embedded in araldite were prepared for electron microscopy. The contrasting was made in solutions of acetic uranium and lead citrate. Electron microscope--UEMB-100K, accelarating voltage--75 kV. The prefunctional period of cardiogenesis is characterized by most intensive processes of specific differentiation of cardiac myocytes and by accumulation of energetic material. With the growing functional activity of the tubular heart the amount and size of lipid drops in the cytoplasm of differentiating cardiomyocytes diminished suggesting the use of lipids as the main source of energy. In the period of formation of the heart the trabecular myocardium of ventricles and the atria myocardium, seeming to perform the main functional load, are characterized by a better developed contracting apparatus and a considerable content of glycogen granules. A sharp decrease of the glycogen content at the end of the embryonic period is likely to be due to growing hypoxia, the appearance of lung respiration and transition to the other type of energy metabolism.

Animals↗
PubMed1975

[Analysis and differentiation of cytergic immune reactions: tuberculine type and cutaneous basophilic hypersensitivity (author's transl)].

The classic delayed tuberculine reaction represents a T-cell mediated immune reaction, detectable already before the formation of antibodies in the course of proteine sensitization. Myocobacteria stimulate and enhance this reaction. The skin test reaction needs the whole antigen for induction. The reaction starts 6 to 8 hours later, shows maximal development 24 to 28 (-72) hours later and is characterized by inflammation, induration, sometimes with hemorrhagies, necroses and ulceration, a macrophage infiltration is typical in the histological picture. During the reaction between antigen and sensitized T-cells mediators are set free which act by the way of unspecific stimulation on T-, B-lymphocytes and macrophages, otherwise they may inhibit macrophage migration. The delayed reaction is active in many infectious diseases and in the rejection of transplants and tumours. The cutaneous basophilic hypersensitivity (Jones-Mote) is a T-cell mediated immune reaction, detectable even before the classic delayed reaction after sensitization with tiny up to large doses of proteines. The skin reaction needs the whole antigen for induction, is visible 2 to 4 hours later in form of an inflammation with slight swelling and reaches maximal development 12 to 24 hours later without hemorrhagies or necroses. The infiltration consists mainly of basophilic leucocytes. There exists a weaker avidity of the T-cells of the cutaneous basophilic hypersensitivity compared to the T-cells of the delayed reaction or to the amount of antibodies. Therefore this reaction is easily suppressed by other immune reactions. In contactdermatitis and vaccine virus infections of the guinea pig the cutaneous basophilic hypersensitivity consists for weeks and months because under these circumstances other immune reactions do not compete. The differentiation of the two reactions shall develop to a routine laboratory procedure due to their therapeutic consequences in the immune diagnostic field.

Antibody Formation↗
PubMed1975

[Stereologic analysis of differentiation of immunocompetent cells during reactions of humoral type].

Previous investigations have shown that, during a primary reaction against a single injection of heterologous red blood cells in the Mouse, immunoblasts migrate from the lymph node germinal centers to the medullary cords through the paracortical area. In that study, we combined ultrastructural examination with stereological methods in order to analyze the differentiation of the immunocompetent cells during this migration. The data permit one to characterize two important events in this differentiation. Between the 12th and 24th hour after the antigen injection, when the germinal centers are being dispersed, the cytoplasmic volume of the cells increases 2.5 fold. The estimated number of ribosomes per cell increases from 350,000 to 1 X 10(6) and the volumetric and surface densities of endoplasmic reticulum increase by a factor of 10. Four days later, 120 hours after the antigen administration, the immunoblasts, now located in the medullary cords, show a spectacular increase in the volume and surface density of endoplasmic reticulum, and in the surface density of Golgi membranes. This change results in the transformation of immunoblasts into young plasma cells. Between these two developments, there is a three day period during which the immunoblasts undergo little change.

Animals↗
PubMed1993

Simultaneous PAGE, immunoblotting, and immunohistochemical analysis of differentiation associated keratins in lesions of the oral mucosa.

The expression of differentiation associated high PM Keratin polypeptides of the oral mucosa lesions were studied by immunohistochemical and immunoblotting techniques applied to adjacent sections of each biopsy specimen. The material studied included specimens of leukoplakia, verrucous carcinoma, squamous cell carcinoma, adenocarcinoma and keratoacanthoma. Little or no expression of 65-67 Kd keratins was evident in squamous cell carcinoma and adenocarcinoma. Hyperkeratotic (both benign and dysplastic) lesions such as verrucous carcinoma, leukoplakia, and keratoacanthoma, showed great variations in the intensity of 65-67 bands and a very irregular immunohistochemical staining pattern. Increased amounts of horny substance was usually accompanied by absence of, or decreased expression of 65-67 Kd keratins, thus indicating a change in the polypeptide composition of the horny layer in pathological conditions of the oral epithelium.

Adenocarcinoma↗
PubMed2002

Analysis of differentially expressed genes in hepatocellular carcinoma with hepatitis C virus by suppression subtractive hybridization.

Hepatitis C virus (HCV) infection is associated with pathogenesis of hepatocellular carcinoma (HCC). We carried out suppression subtractive hybridization to identify variable expression of genes linked to HCC with HCV infection. RNA from both tumorous (tester) and nontumorous (driver) liver tissues was isolated. The cDNA clones were subjected to MegaBACE PCR sequencing to identify those that hybridized to the subtracted library with preference. Nucleic acid sequences generated were searched against the human UniGene database. Among 576 clones screened in the tumorous liver tissue, we identified 30 genes and 28 expressed sequence tags (ESTs). Among 30 genes detected, 23 were with known functions and 7 with unknown functions. The known genes identified had diversified functions and could be divided into 10 functional categories. Twenty percent of these genes were previously known to be tumor related and those most frequently appearing were haptoglobin alpha(2FS)-beta precursor, haptoglobin related protein, and alpha-2-macroglobulin. Four out of 30 known genes (immunoglobulin lambda light chain, kappa immunoglobulin, spliceosomal protein, and X-ray repair cross-complementing protein) were related to chromosome translocation and nucleotide repair. These four genes may contribute to carcinogenesis caused by DNA-damaged agents and to the efficiency of anticancer therapy. The genes with unknown function, which were most frequently detected, were PRO2760 and PRO2955; both encode proteins that express in fetal liver. Twenty-one known and six novel genes were discovered in the nontumorous liver tissue. Apparently, these 27 genes were lost in the tumorous liver tissues. Therefore, using suppression subtractive hybridization, we have identified a number of genes associated with HCC with HCV infection. Most of these genes have not been reported in HCC. Further characterization of these differentially expressed known and unknown genes will provide useful information in understanding the genes responsible for the development of HCC.

Antigens, Neoplasm↗