Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.     

1H- and 13C-NMR, FTIR, UV-VIS, ESI-MS, and PM5 studies as well as emission properties of a new Schiff base of gossypol with 5-methoxytryptamine and a new hydrazone of gossypol with dansylhydrazine

A new Schiff base of gossypol with 5-methoxytryptamine (GSTR) and a new hydrazone of gossypol with dansylhydrazine (GHDH) have been synthesized and studied by Fourier transform infrared (FTIR), 1H and 13C nuclear magnetic resonance (NMR), ultraviolet-visible (UV-VIS), electrospray ionization-mass spectroscopy (ESI-MS) as well as the parametric method PM5. The spectroscopic methods have provided clear evidence that GSTR exists in chloroform solution as an enamine-enamine tautomer, whereas GHDH is present in chloroform as a N-imine-N-imine tautomer. The fluorescence spectra of both compounds indicate that their quantum yield of fluorescence is increased by one or two orders of magnitude compared to that of pure gossypol. The ESI-MS spectra of the 1:1 mixtures of GSTR or GHDH with formic acid have demonstrated that both compounds exist as protonated monomers in the gas phase, whereas GHDH can also exist in a stable protonated dimeric structure. The structures of the stable tautomers are calculated and visualized using the PM5 semiempirical method. The intra- and intermolecular hydrogen bonds within these structures are discussed.     

Nitrous acid deamination of methylated amino-oligosaccharide glycosides

G.l.c.-mass spectrometry has been used to characterize the products of N-deacetylation-nitrous acid deamination of per-O-methylated derivatives (8–11) of methyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-glucopyranoside(1), methyl (2) and benzyl (3) 2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-β-D-glucopyranosides, and methyl 2-acetamido-2-deoxy-6-O-β-D-galactopyranosyl-α-D-glucopyranoside (4). 2,5-Anhydrohexoses have been converted into alditol trideuteriomethyl ethers, alditol acetates, and aldononitriles. The importance of side reactions that lead to the formation of 2-deoxy-2-C-formylpentofuranosides is discussed.     

Novel competitive PCR methods for quantitation of T-cell receptor delta (TCRD) gene rearrangements

Since the invention of the polymerase chain reaction (PCR) several quantitative PCR-based approaches have been described. Recently, the real-time PCR method became a standard in quantitative PCR, although high costs of the necessary equipment and reagents make it unaffordable for many laboratories. In this paper we describe two novel competitive PCR techniques, which were used to determine the frequency of T-cell receptor delta gene (TCRD) rearrangements in peripheral blood leukocytes. In the reference gene competitive PCR (rgc-PCR) the rearranged TCRD gene competes with the reference gene (RAG1) for common reagents (dNTPs and Taq polymerase). The intensity ratio of amplification products, TCRD/RAG1, corresponds to the portion of cells containing a rearrangement. A series of reactions was performed, in which RAG1 primers were added to the PCR after different numbers of cycles. On the basis of the number of cycles needed to obtain equal band intensity, the frequency of cells containing a rearrangement was calculated. In the common primer competitive PCR (cpc-PCR), two gene rearrangements, Vdelta1-Jdelta1 and Vdelta2-Jdelta1, compete for the common Jdelta1 primer. The competing genes are amplified from the same genomic DNA template; therefore unlike in the method using the internal competitor, the results are not affected by the quantity or quality of the analysed sample. We showed that the rgc-PCR and cpc-PCR are reliable and give reproducible results. The methods do not require any expensive equipment or reagents, and can be used to determine the frequency of gene rearrangements.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Functional studies on sciatic nerve blood flow in respect to its vascular supply and tonic neural activity.

An introduction of laser flow meters for a continuous measurements of a tissue blood flow has opened new avenues for an accurate assessment blood flow in peripheral nerves. The aim of our study was: 1) to carry out a functional verification of anatomical sources of a sciatic nerve blood supply in the rat; 2) develop a measurement technique to facilitate standardisation of results; 3) to determine the role of nerve fibres tonic activity in the maintenance of a resting blood flow in the sciatic nerve. Based on results of the present study the following conclusions have been drawn out: 1) in order to obtain a real values of the blood flow through the sciatic nerve it is necessary to remove its muscular fascia; 2) an uninjured epineurium plays a crucial role in maintaining the resting blood flow; 3) major blood supply of sciatic nerve comes from inferior gluteal and popliteal arteries; 4) the tonic neural activity plays a role in the maintenance of the resting sciatic nerve blood flow in anaesthetised rats.     

The costs and benefits in an unusual symbiosis: experimental evidence that bitterling fish (Rhodeus sericeus) are parasites of unionid mussels in Europe

Interspecific symbiotic relationships involve a complex network of interactions, and understanding their outcome requires quantification of the costs and benefits to both partners. We experimentally investigated the costs and benefits in the relationship between European bitterling fish (Rhodeus sericeus) and freshwater mussels that are used by R. sericeus for oviposition. This relationship has hitherto been thought mutualistic, on the premise that R. sericeus use mussels as foster parents of their embryos while mussels use R. sericeus as hosts for their larvae. We demonstrate that R. sericeus is a parasite of European mussels, because it (i) avoids the cost of infection by mussel larvae and (ii) imposes a direct cost on mussels. Our experiments also indicate a potential coevolutionary arms race between bitterling fishes and their mussel hosts; the outcome of this relationship may differ between Asia, the centre of distribution of bitterling fishes, and Europe where they have recently invaded.     

Consensus sequences improve PSI-BLAST through mimicking profile-profile alignments

Sequence alignments may be the most fundamental computational resource for molecular biology. The best methods that identify sequence relatedness through profile-profile comparisons are much slower and more complex than sequence-sequence and sequence-profile comparisons such as, respectively, BLAST and PSI-BLAST. Families of related genes and gene products (proteins) can be represented by consensus sequences that list the nucleic/amino acid most frequent at each sequence position in that family. Here, we propose a novel approach for consensus-sequence-based comparisons. This approach improved searches and alignments as a standard add-on to PSI-BLAST without any changes of code. Improvements were particularly significant for more difficult tasks such as the identification of distant structural relations between proteins and their corresponding alignments. Despite the fact that the improvements were higher for more divergent relations, they were consistent even at high accuracy/low error rates for non-trivially related proteins. The improvements were very easy to achieve; no parameter used by PSI-BLAST was altered and no single line of code changed. Furthermore, the consensus sequence add-on required relatively little additional CPU time. We discuss how advanced users of PSI-BLAST can immediately benefit from using consensus sequences on their local computers. We have also made the method available through the Internet (http://www.rostlab.org/services/consensus/).     

DNA synthesis, mitosis and fusion of myocardial cells

Myocardial cells obtained from embryonic chick ventricles have been used to investigate (1) whether differentiated cells can undergo DNA synthesis and mitosis and, (2) whether heart cells when grown in culture can fuse with each other and with chick skeletal myoblasts to form heterokaryon myotubes. Electron microscopic observations have shown that myocardial cells of day 3 and day 20 chick embryos did contain myofibrils with defined sarcomeres; these cells have been observed in mitosis. Cells obtained by tryptic digestion of day 12 chick ventricles when grown in culture continued to replicate their DNA as shown by thymidine-³H radioautography with DNase controls and were observed in all stages of mitosis. Electron microscopy showed that myofibrils were present in some of the cultured cells. Bi-, tri- and tetranucleate cells were observed in the cultures. Thymidine-³H radioautography showed that these cells were formed by karyokinesis without cytokinesis and by the fusion of uninucleate cells. Since the heart cells could fuse with each other, we tested the possibility that they could fuse with skeletal myoblasts to form heterokaryon myotubes. This was accomplished by co-culturing thymidine-³H labeled ventricular cells and unlabeled skeletal myoblasts. Radioautography with DNase controls showed that some of the myotubes consisted of unlabeled skeletal muscle nuclei and labeled heart nuclei in varied proportions. The factors initiating the formation of these heterokaryons have not been elucidated.