Monoclonal B-cell lymphocytosis in the population of Slovenian region of Lower Carniola.

INTRODUCTION: Monoclonal B-cell lymphocytosis (MBL) is a premalignant asymptomatic condition characterized by the presence of a small population of monoclonal B lymphocytes (less than 5 × 10(9) /L) in the venous blood of otherwise healthy individuals. Incidence of MBL is higher among first-degree relatives of patients with chronic lymphocytic leukemia (CLL) and also increases with age and among men. We compared the incidence of MBL found in the same environment in both an open general population and relatives of CLL patients. METHODS: Samples were analyzed with 5-parameter flow cytometry using following monoclonal antibodies: kappa and lambda clonality, CD5, CD19, CD20. We analyzed 216 samples of patients in the Slovenian area of Lower Carniola aged 23-88 years. Fifty-four patients who came from families where CLL is present were compared by age and gender to 162 healthy individuals from the general population of the same region. RESULTS: We found 10 cases of MBL (4.6%). In 9.4% of younger (≤ 50 years) first-degree relatives of CLL patients with immunophenotypic characteristics of atypical MBL, we observed a prevailingly normal κ to λ ratio. The difference in incidence of MBL cases between relatives and the general population decreases with age. CONCLUSION: MBL in Slovenia can be coincidentally found with a similar frequency to previously studied populations of other parts of Europe. We found, however, a higher incidence of atypical MBL among first-degree relatives.

Transfer of gradient chromatographic methods for protein separation to Convective Interaction Media monolithic columns.

Convective Interaction Media (CIM) columns are monolithic columns optimized for the separation of macromolecules. Some of them operate in the axial mode while others operate in the radial mode depending on the column size. In this work we tested the approach suggested by Yamamoto [Biotechnol. Bioeng., 48 (1995) 444] for transfer of gradient methods between columns of different size. A simplified equation for transfer was derived together with a criterion for its application. Separation was evaluated for a standard protein mixture and peroxidase enzymes present in fermentation broth. Salt and pH gradients were applied. Similar resolutions were obtained for each sample on all columns which demonstrates that the proposed approach can be successfully used for method scale-up on this type of column.

Isolation and characterization of Mn(III) tartrate from Phanerochaete chrysosporium culture broth.

High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium. Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed. To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution. High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate. Oxidation of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol. In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability. The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine.

Laccase production by Phanerochaete chrysosporium--an artefact caused by Mn(III)?

AIMS: The possibility of laccase production by Phanerochaete chrysosporium was studied. METHODS AND RESULTS: A relatively high initial Mn(II) concentration (1-4 mM) in the growth medium leads to the development of reddish-brown coloration and intensive oxidation of 2.2'-azino-bis(3-etilbenz-tiazolin-6-sulfonate) (ABTS). The peak of ABTS oxidation was obtained approximately 1 day after the peak of MnP activity. CONCLUSION: ABTS oxidation was not caused by manganese peroxidase (MnP) nor by laccase but was the consequence of the action of Mn(III) which was stabilised in the growth medium. Decomposition of the complex took place after the biomass was removed from the growth medium and especially after the aeration of the culture was interrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: Mn(III) seems to be the cause of false positive laccase reactions. More reliable data on MnP activity can be obtained if the complex is decomposed by the fungus before MnP activity is measured in the medium.

The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium.

Convective Interaction Media (CIM) monolithic columns were applied for the HPLC monitoring of Phanerochaete chrysosporium lignin peroxidase (LiP) isoforms during cultivation. The influence of the agitation mode (circular, elliptic) and rate (130 and 200 rpm), as well as the initial nitrogen concentration (1.6-6 mM) in the growth medium was investigated. Identical rotation rate but different agitation modes resulted in different LiP activities and isoenzyme compositions. On the other hand, at different agitation types and rates, similar LiP activities were obtained at different isoenzyme compositions. Although LiP H2 and LiP H6/H7 were predominant isoenzymes obtained at various cultivation conditions, relative isoenzyme amounts differ considerably when initial nitrogen concentration was changed between 1.6 and 5 mM.

A method of fast separation of lignin peroxidases using convective interaction media disks.

The HPLC separation of lignin peroxidase isoenzymes using Convective Interaction Media disks containing quaternary amine and diethylaminoethyl ion-exchange active groups is proposed. In contrast to standard HPLC procedures the separation can be performed within a few minutes without considerably affecting the separation resolution. The method is reproducible and gives a linear response of integrated peak area to protein concentration for all measured isoenzymes. The separation resolution is retained unchanged by applying crude culture filtrate instead of a sample previously frozen and dialyzed. The optimized method might therefore be used for on-line monitoring of lignin peroxidase isoenzyme composition during fermentation. On the other hand, the proposed method is comparable in time to the original method of lignin peroxidase activity measurement (proposed by Tien and Kirk), providing additionally the isoenzyme composition.

Analysis of Fungal Pellets by UV-Visible Spectrum Diffuse Reflectance Spectroscopy.

The application of the UV-visible spectrum diffuse reflectance spectroscopy for the determination of intracellular pH in vivo, for determination of cytochrome content, and for the noninvasive in vivo detection of the redox state of fungal mitochondrial cytochromes in filamentous fungi is introduced. The time course of the intracellular pH values, mitochondrial cytochromes, and CO-binding pigments content and the correlations between the actual redox state of cytochrome aa(3) and saturation of growth medium with oxygen in pellets of the basidiomycete Phanerochaete chrysosporium were determined. As the test microorganism, the yeast Saccharomyces cerevisiae was used. UV-visible spectrum diffuse reflectance spectroscopy proved to be a promising method for the quick and simple analysis of light-impermeable biological structures for which the classical transmittance spectrophotometric methods are difficult to implement.