Macroporous Polymer Monoliths for Affinity Chromatography and Solid-Phase Enzyme Processing.

Nowadays, monolithic stationary phases, because of their special morphology and enormous permeability, are widely used for the development and realization of fast dynamic and static processes based on the mass transition between liquid and solid phases. These are liquid chromatography, solid-phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including the bioaffinity mode, represents unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In the edited chapter, the examples of the application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by the combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents the original and practically valuable method that can be used in biotechnology. In addition, macroporous monoliths were adapted to the immobilization of such different enzymes as polynucleotide phosphorylase, ribonuclease A, α-chymotrypsin, chitinolytic biocatalysts, ß-xylosidase, and ß-xylanase. The possibility of use of immobilized enzyme reactors based on monoliths for different purposes is demonstrated.

[Tumor-like amyloidosis of the upper respiratory tract]. / Opukholevidnyi amiloidoz verkhnikh dykhatel'nykh putei.

The paper describes 11 cases of local tumor-like amyloidosis (LTA) of the upper respiratory tract, among which laryngeal amyloidosis was most common. The clinical diagnosis of suspected local amyloidosis was made in only two cases. The diagnosis of local amyloidosis was established at a morphological examination of a distant neoplasm, by using special Congo red staining followed by polarizing microscopy. Attention is drawn to the localization and sequence of amyloid deposition and morphological changes related to the age of patients and the duration of the disease. The paper discusses the nature of local amyloidosis as stromal vascular proteinosis with the deposition of AL amyloid (immunoglobulin light chain amyloid) that are formed apparently by local immunocytes of the mucosa-associated lymphoid tissue (MALT) system. It emphasizes the need for the clinical monitoring of patients with LTA to rule out systemic amyloidosis.

Affinity chromatography of proteins on monolithic columns.

At present, monolithic stationary phases, because of their morphology, are widely used for development and realization of fast dynamic and static processes based on mass transition between liquid and solid phases. These are liquid chromatography, solid phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including bioaffinity mode, represents a unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In this work, the examples of application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator from E. coli cell lysate and Chinese Hamster Ovary cell culture supernatant, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents an original and practically valuable method that can be used in biotechnology.

Polymer monoliths as efficient solid phases for enzymatic polynucleotide degradation followed by fast HPLC analysis.

Two ribonuclease A bioreactors based on lab-made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free-radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to the solid surface via direct covalent binding, while in the second bioreactor the flexible-chain synthetic polymer was used as an intermediate spacer. The effect of temperature, substrate flow rate, and loaded sample volume on the biocatalytic efficiency of the immobilized enzyme was examined. The kinetic parameters of the enzymatic degradation of synthetic polycytidylic acid were calculated and compared to those found for hydrolysis with soluble ribonuclease A. The monitoring of substrate splitting was carried out by means of fast anion-exchange HPLC on an ultra-short monolithic column (disk) using off- and on-line analytical approaches.

[Choice of surgery scope in occlusive obstruction of the colon]. / Vybor ob''ema operativnogo vmeshatel'stva pri obturatsionnoi neprokhodimosti obodochnoi kishki.

From 1980 five hundred and seventy-three patients underwent surgery for occlusive obstruction of the colon (OOC). Radical surgeries (left-sided hemicolonectomy, Hartman's surgery, subtotal colonectomy) were performed in 440 (77%) patients, 133 (23%) patients underwent palliative surgeries. One hundred and sixty-one patients of radically operated underwent one-stage surgeries (93 right-sided hemicolonectomies and 68 subtotal colonectomies). Postoperative lethality after radical surgeries was 16.5%. Postoperative lethality, time of hospital stay, rate of postoperative complications after Haptman's surgery and subtotal colintcyomy don't differ, but patients after subtotal colonectomy don't require reconstruction surgery.

In vitro comparison of complementary interactions between synthetic linear/branched oligo/poly-L-lysines and tissue plasminogen activator by means of high-performance monolithic-disk affinity chromatography.

The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-L-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.

[Toxicity of mushrooms Paxillus involutus and Paxillus atrotomentosus]. / K voprosu o toksichnosti gribov vida svinushka tonkaia i tolstaia.

Thirty-eight patients with mushroom (Paxillus involutus and Paxillus atrotomentosus) poisoning were treated. Slight poisoning (acute gastroenteritis) was diagnosed in 17 patients, medium-severe in 13, severe in 6, and extremely severe in 2 patients. Changes in the LPO-AOD system correlated with the severity of hepatorenal involvement. The treatment included hepatotropic therapy; patients with acute renal failure were treated by hemodialysis. Paxillus mushrooms induced functional evacuatory disorders in the small intestine. Eleven patients with adhesions in the abdominal cavity developed ileus. Two patients died: a man aged 26 years after eating fried (not boiled) mushrooms and a woman aged 76 years with ileus with symptoms of multiple organ dysfunction. The rest patients were discharged from hospital in satisfactory condition. Clinical course of poisoning with Paxillus mushrooms is discussed.

A new method for surgical treatment of the short bowel syndrome.

This study was aimed to investigate the electrophysiological and morphological characteristics resulting from the structural and functional transformation of gastric tissue transplanted to the small intestine. Twelve adult mongrel dogs were studied up to 3 years. Gastric transplants preserved its main microstructure and minimal compensatory-adaptive processes developed in the mucosa and muscle layers of the graft. A significant influence on the electrical activity of the small intestine was observed, with a 10% reduction of the slow wave frequency (SWF) in the proximal and distal jejunum adjacent to the graft after meals. The SWF of the gastric graft itself, however, corresponded to the frequency of the native stomach, did not depend and was not associated with adjacent intestinal areas. In summary, the stomach graft transplanted to the small intestine keeps the properties of gastric tissue, there are functional adaptations to conditions of digestion in the small intestine and the graft has minimal effects on intestinal motility.

[Broadening of the organ specificity of the action of polynucleotide interferon inducers]. / Rasshirenie organospetsifichnosti deistviia polinukleotidnykh induktorov interferona.

Interferon titers in the blood and brain of mice and their protection from the herpes virus were compared after the animal exposure to poly(G).poly(C) duplex, both native and modified with cis-diammine dichloroplatinum (II). It was shown that the duplex platination especially at the level of the poly(G) strand resulted in sharp rising of the interferon titers in the extracts of the animal brain and rearrangement of the types of interferon induced in the brain to predominance of gamma-interferon. The interferonogenesis indices correlated with the duplex protective activity against the herpes virus. It was concluded that the platinum binding could increase the membrane specificity of the duplex and stimulate its penetration through the hematoencephalic barrier. Possible structural changes in the duplex under the action of platinum (II) resulting in the observed effect are discussed.

Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography.

A new affinity method for the direct quantitative analysis of monospecific anti-peptide immunoglobulins (antibodies) and, simultaneously, their semi-preparative isolation from blood serum of the immunized animals has been developed. Immunoaffinity discs based on macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were used as the supporting stationary phase. The specifically prepared synthetic peptides with biological activity imitating that of the immunoglobulin binding sites of various proteins were used as the selective ligands instead of native proteins. These ligands were immobilized by a single-step reaction that involves epoxy groups located on the pore surface of the porous polymer disc with amine groups of the peptide molecules. A spacer between biospecific ligands and the linking site was not required to achieve good separation. These novel immunosorbents characterized by large binding capacity are well suited for high throughput screening. Dissociation constants of the peptide-antibody complexes calculated from the experimental adsorption isotherms confirm the excellent selectivity of the proposed separation method. The discs were used in a single step enrichment of antibodies both from precipitated blood fraction and crude blood serum of immunized animals. The quantitative data of the immunoaffinity disc chromatography were compared to those obtained by an enzyme-linked immunosorbent assay. Gel electrophoresis was also used to demonstrate the high degree of purity of the final product. In contrast to typical techniques that involve proteins, this immunoaffinity approach allows for the first time direct determination of concentration of specific antibodies using the immunosorbent prepared from the short peptide molecules immobilized on the internal surface of reactive porous polymer discs.

[Morphofunctional disorders in the small intestine in acute occlusive ileus]. / Morfofunktsional'nye narusheniia v tonkoi kishke pri ostroi obturatsionnoi neprokhodimosti.

Activity of liminal and membrane enzymes and morphologic state of small intestinal mucosa were studied in experimental acute occlusive ileus in rats. It was established that degenerative alterations of intestinal mucosa epithelium above the site of obstruction are more pronounced than in the distal part. The activity of membrane enzymes (invertase) remained unaltered during 72 hours after the obturation, however, serious morphologic damage of the intestinal mucosa epithelium and sharp suppression of the liminal digestion were observed.

[Antiviral activity of poly(A)-poly(U) duplexes modified with cis-diammine-dichloroplatinum (II)]. / Protivovirusnaia aktivnost' dupleksov poli(A)-poli(U), modifitsirovannykh tsis-diammin-dikhloplatinoi(II).

Polyribonucleotide duplex poly(A).poly(U) was modified with cis-diammine dichloroplatinum (II) (cis-DDP). It was shown that the antiinfluenza protective activity of the modified duplex in mice increased with the degree of modification (rb) rising up to 0.2. The effect was different from that for poly(I).poly(C) and poly(G).poly(C). The interferon titers in the murine brain increased in parallel with increasing of the antiviral activity. It was assumed that the structural specificity of the poly(A).poly(U) duplex was responsible for the phenomenon and that cis-DDP interaction with N(7) atoms of the adenine heterocycles blocked the "abnormal" Hoogsteen pairing of adenines with uracils. As a result the antiviral activity increased because of lowering the quantity of the intramolecular defects and increasing the length of the regular double-stranded regions.

[Biological activity of the poly(G).poly(C) complex, modified by divalent platinum compounds]. / Biologicheskaia aktivnost' kompleksa poli(G).poli(C), modifitsirovannogo soedinenimi dvukhvalentnoi platiny.

Modification of poly(G).poly(C) with cys-diaminodichloroplatinum (cys-DDP) at the level of rb = 0.02 increased the in vivo antiviral and interferon-inducing activity of the complex, in contrast to the data reported for complex poly(G).poly(C). Antiinfluenza activity in this case depends on the method of modification and increases more intensively when a ready complex is treated with cys-DDP, as against treatment of poly(G) alone before the formation of a complex with poly(C). If rb is increased, the activity reduces again. Modification with trans-DDP at rb = 0.02 also leads to an increase of antiinfluenza activity of poly(G).poly(C), but mainly after pretreatment of poly(G).

[Non-occlusive dyscirculatory intestinal necrosis ]. / Neokkliuzionnyi distsirkuliatornyi nekroz kishechnika.

70 autopsy cases of NDIN are analysed. Necrosis in 85% of cases was observed against basic pathology, mainly cardiovascular diseases (45.7%) and after surgery in the thoracic and abdominal cavities. NDIN was considered as a main disease in the remaining 15% of cases, with its duration from 1 to 7 days. Morphologically NDIN was revealed in the colon in 71.2% of cases, in combination with the small intestine in 30.5%. Predominance of the small intestine was in 28.8%, ileum 64%. Primary circulatory disturbances in the capillary-venule system of the mucous membrane and submucosa were found histologically.

Biophysical studies of the modification of poly(rG) . poly(rC) by cisplatin. Relations to the biological activity of the complex.

The integrity of the double-stranded complex polyriboguanylic.polyribocytidylic acid [poly(rG).poly(rC)] modified by antitumour cis-diamminedichloroplatinum(II)(cis-DDP) was studied with the aid of differential pulse polarography and terbium fluorescence measurement. The modification was made to level corresponding to rb = 0.05 (rb is defined as the number of platinum atoms covalently bound per one nucleotide residue). Two modes of the modification of the polynucleotide complex were employed: The action of cis-DDP on poly(G) before formation of the complex with poly(C) and on the complex already formed from non-modified polynucleotides. It was shown that in the latter case modification disordered the integrity of the complex only negligibly. while in the former case the modification resulted in a noticeably more extensive disturbance of the double-stranded polynucleotide complex. Moreover, the modification of the complex (after its formation) at rb = 0.02 led to improved interferon-inducing and antiviral activity of poly(rG).poly(rC) tested on mice infected by influenza virus. It was suggested that the combined effects of interferon-inducing and antiviral activities of poly(rG).poly(rC) and antiviral activity of cis-DDP may result in an increased effect over and above what may be expected from the actions of the two modalities separately.

Modification of duplex poly(G).poly(C) by platinum (II) compounds.

The modification of the double-stranded poly(G).poly(C) complex by cis-diamminedichloroplatinum(II) was studied by two modes: the action of cis-DDP on poly(G) before formation of the duplex with poly(C) and that on the prepared duplex. It was shown that in the latter case modification disordered the integrity of the duplex only negligibly at rb less than or equal to 0.05 and led to improved interferon-inducing and antiviral activity tested on mice infected by Influenza and Herpes viruses.

[Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex]. / Otsenka razmera nepreryvnogo uchastka poli(G), neobkhodimogo dlia biologicheskoi aktivnosti kompleksa (G).poli(Ts).

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.

[Thermoactivation of poly(G).poly(C): a description of the effect and its hypothetical mechanism]. / Termoaktivatsiia poli(G).poli(Ts): opisanie éffekta i ego predpolagaemyi mekhanizm.

Heating of poly(G).poly(C) complex solutions at a temperature about 100 degrees C was shown to overcome a decrease in the antiviral and interferon-inducing activity of the preparations which were obtained at relatively high concentrations of polynucleotides from poly(G) stored in solution, or were stored frozen themselves. These unfavourable conditions contributed to stabilization of the poly(g) secondary structure and decrease in the degree of regularity of the complex molecules. The results suggest that thermal activation of such poly (G). poly(C) preparations occurred in 2 stages by melting residual free regions of poly(G) and their subsequent interaction with poly(C) with formation of a more regular complex.

[Modification of the poly G-poly C complex by incorporation of adenosine in the purine chain]. / O modifikatsii kompleksa poli(G)-poli(Ts) vkliucheniem adenozina v purinovuiu nit'.

Antiviral and interferonogenic activity of the complexes of poly(G,A) . poly(C) and poly(G) . poly(C) was studied in mice and cell cultures. Three out of 4 complexes of poly(G,A) . poly(C) had insignificant antiviral and interferonogenic activity in chick embryo cells. One of the complexes induced low levels of interferon production in mice and decreased the rate of their death from experimental forest-spring encephalitis. The activity of poly(G) . poly(C) in the above cell systems was much more pronounced. Unlike this complex, some complexes of poly(G,A) . poly(C) showed a noticeable activity in the cells of Primates. The effect of the noncomplementary base in the purine thread of poly(G) . poly(C) on its biological activity and nucleotide composition is discussed.