Theory for electrostatic interaction chromatography of proteins.

A simple theoretical framework for the effect of the eluting salt concentration on the retention factor of proteins in ion-exchange chromatography under conditions of linear elution is described. It is based on the solution of the linearized Poisson-Boltzmann equation for two oppositely charged planar surfaces in contact with a salt solution. The theory predicts a linear relation between the logarithmic retention factor and the reciprocal square root of the ionic strength of the eluent in the salt concentration range used in linear elution chromatography. A large body of retention data obtained in ion-exchange chromatography of proteins over a wide range of experimental conditions was plotted as ln k' vs 1/square root of I, where k' and I are the retention factor and ionic strength, respectively. The plots are linear or nearly so, as predicted for a moderate salt concentration range by the theory. From the slope of such plots the characteristic charges of the proteins were estimated by using only fundamental physicochemical constants. The chromatographically measured protein charges compare well to those obtained from titrimetric experiments at the same pH, although certain deviations are noted. The theoretical approach presented here offers a more realistic treatment of the ion-exchange chromatography of proteins than the stoichiometric displacement model and can serve as a convenient framework for the analysis of retention data.     

Sol derived gold–palladium bimetallic nanoparticles on TiO2: structure and catalytic activity in CO oxidation

Bimetallic AuPd catalysts were prepared by deposition of bimetallic aqueous sols formed in different ways: (i) co-reduction of the precursor Au and Pd ions by Na-citrate/tannic acid mixture, (ii) reduction of Au(III) ions onto preformed Pd sol, and (iii) reduction of Pd(II) ions onto a preformed Au sol. The Au/TiO₂ and Pd/TiO₂ samples as references were prepared from their respective sols. The structure of the samples was characterized by XRF, XRD, XPS, TEM and CO chemisorption both in the as-prepared state and after calcination and reduction. The catalytic activities of the calcined/reduced catalysts in the CO oxidation were compared. The presence of bimetallic crystalline phases was evidenced in all three samples both in the as prepared and calcined/reduced states, however, various extents of Pd surface enrichment were determined. The catalytic activity of the bimetallic samples regardless of the preparation method, is about the same as that of the mixture of the monometallic samples. No significant synergism is suggested in the present bimetallic samples.     

Preparation and characterization of hydroxyapatite from eggshell

Hydroxyapatite (HAp) was successfully produced by using recycled eggshell. The observed phases of the synthesised materials were dependent on the mechanochemical activation method (ball milling and attrition milling). The structures of the HAp were characterized by X-ray diffraction, scanning electron microscopy and infrared spectroscopy. Attrition milling proved to be more efficient than ball milling, as resulted nanosize, homogenous HAp even after milling.     

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.     

Conditionally pathogenic mycobacteria in the environment and their effect on living organisms

Since 1968 when bovine tuberculosis was eliminated in the Czech Republic, the epidemiological situation of bovine tuberculosis has been stabilized. At present the incidence of the disease in men and animals caused by conditionally pathogenic mycobacteria is worldwide increasing. In human population, especially people with impaired immunity are affected. In farm animals infections caused by conditionally pathogenic mycobacteria may often result in complications in intravital and postmortal diagnosis of bovine and avian tuberculosis. Those infections are then often incorrectly diagnosed which could have a great negative impact on health, economy and breeding. Therefore the objective of the study was to summarize data from literature and our own experience concerning the occurrence of atypical mycobacteria in environment. The study is divided into 5 summarizing chapters, supplemented with 13 Tables.     

Phage restriction and the presence of small plasmids in Salmonella enteritidis

Sequence variation within the variable region of the 16S rRNA at position 440 to 480 allowed the synthesis of specific PCR primers for the identification of groups within the species Photorhabdus luminescens, symbionts of entomopathogenic nematodes of the genus Heterorhabditis. For the second PCR primer the highly conserved region at 755 to 795 was used. The P. luminescens type strain specific primer could not recognize any other P. luminescens strain. The primer TEMPERATUS based on the sequence of strain DSM12190 (isolated from North West European H. megidis strain HSH2) identified all P. luminescens associated with H. megidis from North West Europe and two isolates from closely the related nematode strains from Ireland. The primer TROPICUS based on strain DSM12191 (isolated from the nematode type strain H. indica strain LN2) identified P. luminescens of tropical origin isolated from H. indica. Symbionts of H. bacteriophora could not yet be separated into well described groups with the primers used. A comparison of sequence data resulted in the identification of additional groups. The non-symbiotic P. luminescens isolates are distinct in the variable region. The group HELIOTHIDIS contains 15 P. luminescens associated with H. bacteriophora from North East America. The MARELATUS group contains symbionts of the nematode H. marelatus from the West Coast of the US. The data together with the specific symbiotic association of P. luminescens strains with different nematode species support the division of the taxon P. luminescens into different species.     

Galanin immunoreactive neurons in the medulla oblongata of rats

The distribution of galanin-immunoreactive (-ir) neurons in the medulla oblongata was mapped with light microscopic immunohistochemistry. No immunopositive perikarya were seen in untreated rats. Two days after colchicine treatment, galanin immuno-positive neurons were localized in the following areas: 1) raphe nuclei (magnus, pallidus and obscurus); 2) in various parts of the reticular formation, mainly in the territory of the catecholaminergic groups and in the peritrigeminal subdivision of the lateral reticular nucleus; 3) vagal nuclei (nucleus of the solitary tract, nucleus ambiguous); 4) two cell groups at the ventral surface of the rostro-caudal middle portion of the medulla oblongata (they do not correspond to any known demarkated anatomical nuclei, but related to the chemosensitive medullary area); 5) in the gelatinous part of the spinal trigeminal nucleus. The wide distribution of galanin neurons in the medulla support data that had been reported on the role of this peptide in various viscerosensory and autonomic mechanisms. In addition to these, galanin seems to be an important factor in the restoration of lesioned neurons (nerve growth factor-like activity). An increased galanin mRNA expression can be seen in dorsal vagal or hypoglossal motor neurons after intracranial transections of vagal or hypoglossal nerves, respectively. Transections of the olivocerebellar tract induced galanin gene expression in neurons of the contralateral inferior olive. After brainstem hemisection, galanin immunopositivity was seen in cells of the nucleus of the solitary tract due to the transection of ascending projections of this primary autonomic center in the medulla oblongata.