Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA

To investigate the possibility of producing charge-neutral gene delivery complexes with extended, non-particulate structures, DNA was allowed to self-assemble with a series of hydrophilic cationic polymers containing quaternary charged trimethylammonio ethylmethacrylate (TMAEM, 5, 15, 50, 100 mol%) copolymerised with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA, 95, 85, 50, 0 mol%, respectively). Copolymers were all able to bind DNA, assessed using ethidium bromide fluorescence, although copolymers with low TMAEM content did not expel ethidium bromide. Increasing TMAEM content of the copolymers changed the morphology of the complexes from extended (5-15 mol% TMAEM), through partially condensed particles (50 mol%) to discrete nanoparticles (100 mol% TMAEM). Complexes based on copolymers with low TMAEM content (5-50 mol%) showed less resistance to degradation by nucleases and lower surface charge (21.2+/-5.9-45.1+/-3.9 mV) than those formed using 100 mol% TMAEM (57.8+/-8.2 mV). They also showed significantly less association with phagocytic cells in vitro (human leucocytes, uptake decreased by up to 92.3%; murine peritoneal macrophages, uptake decreased by up to 69.6%), although in vivo their hepatic accumulation was only slightly decreased (maximum decrease 27.6%). Finding the appropriate balance of hydrophilicity and stability is key to development of effective vectors for gene delivery.     

Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds

Fucoidans and laminarans from Laminaria cichorioides, Laminaria japonica, Fucus evanescens, laminaran from Laminaria gurjanovae, other beta-D-glucans (translam, pustulan and zymosan) and lambda-carrageenan from Chondrus armatus were used to study the effect of water-soluble polysaccharides from seaweeds on the alternative pathway of complement (APC). beta-D-Glucans and fucoidans under study differed appreciably from each other by structural characteristics, and also by degree of purification. beta-D-glucans, on ability to bind complement, ranked in a line according to a degree of their purification. Highly purified beta-D-glucans under study did not reveal an ability to bind complement. The fucoidans were divided conventionally into three groups according to their action on APC. Highly sulfated alpha-L-fucan from L. cichorioides with the greatest activity toward APC and caused 50% inhibition of reaction of activation (RA) of APC in a concentration of 0.5-0.7 mg/ml. Opposite 50% of inhibition of lysis of erythrocytes by sulfated heterogeneous fucoidan from L. japonica was achieved with 20 mg/ml. All other fucoidans and lambda-carrageenan have activity at 6-10 mg/ml concentration. Decreasing the sulfate content from 36% up to 9% in sample fucoidans under study was not reflected practically in the 50% inhibition concentration. Apparently, the degree of sulfating of fucoidans did not influence their action on APC. But the positive influence of fucose in structure of polysaccharide was obvious.     

Effect of preparations of protein Yop encoded by Yersinia pestis calcium-dependence plasmid on mice

The impact of the preparations of Y. pestis secreted proteins Yop (YopH-M, YopB, YopD-N, YopE) on mice immunized with 3 s.c. injections was studied. Though these proteins failed to protect the animals from plague, they stimulated the immunobiological transformation in the immunized animals. YopB and YopD-N were found to have the highest immunobiological activity with respect to mice. The preparation of YopB induced the production of the highest titers of specific antibodies and stimulated cell-mediated immune response. The injection of YopD-N to mice led to a considerable decrease in the proliferative capacity of splenocytes in vitro in response to stimulation with nonspecific mitogen ConA, as well as to pathological changes in the kidneys.     

Reversible inhibition of Escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic intermediates in cryo-crystallographic studies

Here, we describe high-resolution X-ray structures of Escherichia coli inorganic pyrophosphatase (E-PPase) complexed with the substrate, magnesium, or manganese pyrophosphate. The structures correspond to steps in the catalytic synthesis of enzyme-bound pyrophosphate (PP(i)) in the presence of fluoride as an inhibitor of hydrolysis. The catalytic reaction intermediates were trapped applying a new method that we developed for initiating hydrolytic activity in the E-PPase crystal. X-ray structures were obtained for three consecutive states of the enzyme in the course of hydrolysis. Comparative analysis of these structures showed that the Mn2+-supported hydrolysis of the phosphoanhydride bond is followed by a fast release of the leaving phosphate from the P1 site. The electrophilic phosphate P2 is trapped in the "down" conformation. Its movement into the "up" position most likely represents the rate-limiting step of Mn2+-supported hydrolysis. We further determined the crystal structure of the Arg43Gln mutant variant of E-PPase complexed with one phosphate and four Mn ions.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Polymorphism of human blood serum cholinesterase in populations of Evenks and Yakuts in Krasnoyarsk Territory

Polymorphism for E2 locus of human serum cholinesterase was studied in populations of evenks and yakuts of Krasnoyarsky region by the method of starch gel electrophoresis. Thee frequencies of C5+ phenotype correspond to C5+ frequencies in other mongoloid populations of Siberia. The activity of cholinesterase was determined by semiquantitative technique. Three individuals with a sharply decreased cholinesterase activity have been revealed.