Isoenzymes of wheat o-diphenolase revealed by column isoelectric focusing

Summary Common wheat o-diphenolase at different purification steps has been submitted to column isoelectric focusing electrophoresis. The fraction purified with calcium phosphate gel shows multiple forms focused at pI 3.60, 4.95, 6.80, and 9.60. The enzyme activity and the purification degree of the four enzymatic components obtained have been calculated. The major fraction recovered after purification by chromatography on DEAE-cellulose does not display other multiple forms; however, it has some enzymatically inactive proteins well separated in the pH-gradient. The enzymatic protein, focused at pI 9.60, thus achieves a 1753-fold purification over the crude extract. The application of the technique allows a characterization of the wheat o-diphenolase and its recovery in highly purified form.

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Isoenzyme von o-Diphenoloxydase aus Weichweizen, identifiziert durch isoelektrisches Fokussieren über Säule

Zusammenfassung Weichweizen-o-Diphenoloxydasen verschiedener Reinigungsstufen wurden über eine Säule isoelektrisch focussiert. Das durch Calciumphosphatgel gereinigte Enzym (Fraktion C) zeigt 4 multiple Formen, focussiert bei pI 3,60; 4,65; 6,80 und 9,60. Die Aktivität und der Reinheitszustand von diesen vier Formen wurden berechnet. Die Fraktion C wurde durch Chromatographie über Cellulose weiter gereinigt. Die in größeren Mengen gewonnene Fraktion zeigte beim isoelektrischen Focussieren keine multiple Formen mehr, nur eine Trennung von enzymatisch inaktiven Proteinen beim pH-Gradienten. Das enzymatisch aktive Protein, focussiert bei pI 9,60, zeigte einen 1753 mal besseren Reinheitszustand als das Rohenzym. Die Anwendung dieser Technik erlaubt die Charakterisierung der Weichweizen-o-Diphenoloxydasen und ihre Gewinnung in einem hochgereinigten Zustand.

     

Un particolare metodo per la determinazione di autovalori di matrici tridiagonali simmetriche

In this paper we given an algorithm of low computational complexity which determines the eigenvalues of a symmetric tridiagonal matrix. The algorithm uses the technique of spectrum slicing together with methods for finding the zeros of polynomials. An application of algorithm for computing Gauss quadrature formulas is given.

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Lavoro svolto nell'ambito del Gruppo Nazionale di Informatica Matematica del C.N.R.     

Transfusion-related acute lung injury associated with an NA1-specific antigranulocyte antibody

Transfusion-related acute lung injury (TRALI) is an infrequent complication of hemotherapy. Antigranulocyte antibodies, most of them present in donor's serum, have been implicated in its pathogenesis. We describe a case of TRALI, following red blood cell transfusion, associated with an antigranulocyte antibody with NA1 specificity in the patient's serum.     

Flow-cytometric detection of the RI alpha subunit of type I cAMP-dependent protein kinase in human cells

cAMP-dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA, designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8-N3-32P-cAMP or by radioimmunolabeling techniques. Both techniques are time consuming and require a high number of cells and the use of radioactive reagents. Using the MCF-10A normal human mammary cell line infected with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. MCF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone (1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.     

Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.     

Artificial neural network identification of heterotrophic marinebacteria based on their fatty-acid composition

The traditional approach to biochemical identification of marine fresh isolates requires considerably long culture preparation times and large quantities of expensive materials and reagents, and the results are not very reliable. On the other hand, taxonomy tests based on DNA composition, although sensitive and reliable, require long execution times and high costs, A method is presented for the classification of fatty-acid profiles, extracted from marine bacteria strains, at genus level based on supervised artificial neural networks. The proposed method allows the correct identification of all patterns belonging to the training set and almost all patterns belonging to the test set. Moreover, a quantitative measure of the importance of each fatty acid for bacterial classification is also achieved. This measure allows the determination of a cluster of fatty acids to be controlled with greater care. The results show that the proposed method is reproducible and rapid, so that it can be routinely used in the marine microbiology laboratory to identify fresh isolates     

Presence of a non-NMDA glutamate receptor subtype in the sympathetic nervous system of neonatal swine

Calcium-dependent protein kinases (CDPKs) in plants are characterized by a four-domain structure including conserved sequences in the catalytic domain, and in the C-terminal calmodulin-like domain. Based on this conservation we have PCR-amplified and isolated a potato cDNA clone (StCPK1) from a library representing an early stage of tuber development. DNA sequence analysis revealed that in the catalytic domain, StCPK1 shares more homology with CDPK-related kinases than with CDPKs; however, like CDPKs, it possesses canonical EF-hands at the calmodulin-like 3' end. StCPK1 exists in a few copies in the potato genome and is abundantly expressed in the sepals of mature flowers. Floral expression of genes homologous to StCPK1 appears to be widespread in the family Solanaceae.     

The IL-6 gene expression by leukemic cells from acute lymphoblastic leukemia common and T type and modulation of IL-6 production by TNF

The tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) mRNA accumulation and the release of the cytokines TNF-alpha and IL-6 was determined in leukemic cells isolated from bone marrow biopsy from patients with acute lymphoblastic leukemia: ALL-common type (cALL), 11 patients; ALL-T type, nine patients. The non-leukemic bone marrow cells (BMMC) and peripheral blood mononuclear cells (PBMC) from healthy donors were used as a control. The mRNA was assessed by fluorescent in situ hybridization in cell suspension and analyzed with flow cytometry. The accumulation of cytokine mRNA was higher in cALL cells as compared to ALL-T and PBMC (control) and was comparable to cytokines mRNA accumulation in BMMC. The production of IL-6 by leukemic cells from both types of leukemia was significantly lower than in BMMC. The bioactive TNF was not detected in either of the leukemia groups studied. TNF-alpha protein was produced by ALL-T cells and BMMC but not by cALL type of leukemic cells. The synthesis of IL-6 was significantly enhanced by TNF-alpha in BMMC and ALL-T while the presence of TNF-alpha had no effect on IL-6 synthesis in the culture of cALL leukemic cells. It was concluded that despite IL-6 and TNF-alpha mRNA contents, leukemic cells representing early stage of B-cell development (CD10+) showed disregulation of production of these cytokines.