Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

Immunoenzyme analysis of adenovirus hexons. Indication and characteristics of rabbit and mouse antibodies against hexons

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of rabbit and mouse IgG antibodies specific to adenoviral hexon. The anti-hexon antibodies were detected by capture with purified hexon coated onto polystyrene microtiter plates and visualizing them by respective anti-IgG horseradish peroxidase conjugates. In the sera from hyperimmunized rabbits and mice as well as in the mouse ascite fluids the ELISA procedure revealed primarily type-specific (epsilon) and genus-specific (alpha) antigenic determinants in hexon but not those of intermediate specificities.     

Reproduction mode of European Bitterling (Rhodeus amarus,Bloch, 1782) determined through rapid oocyte counts and size determination using digital imaging

This study focuses on assessing the reproduction mode of an important model species for evolutionary and behavioural ecology by using digital image analysis: the European bitterling Rhodeus amarus (Bloch, 1782). Specifications of mode of reproduction were determined using oocyte size distribution, seasonal dynamics in mean oocyte diameter and total number of oocytes in ovary samples gained from April to July 2007. The rapid oocyte count was enabled by using lucia image analysis software, which also provided measurement and colour estimations of oocytes. Bitterling ovaries showed features typical for indeterminate spawners, i.e. a continuous distribution of oocyte size over the reproductive season and recruitment of new pre‐vitelogenic oocytes in the second half of the reproductive season. These results are consistent with the view that the European bitterling is a batch spawning fish with indeterminate fecundity.     

Isolation of high molecular weight eukaryotic DNA with the use of potassium acetate

The simple technique for isolation of high-molecular eucaryotic DNA has been proposed. It includes cell or nuclei lysates by sodium dodecylsulfate in the presence of pronase, proteins precipitation by potassium acetate, DNA precipitation by ethanol. The DNA isolated by this technique is easily cleaved by restriction endonucleases and can be used for analysis of the unique genes by blot-hybridization. The yield of DNA is similar or somewhat higher than that in case of using the standard methods including phenol extraction or phenol-chloroform extraction.     

Carboxyacyl derivatives of cardiolipin as four-tailed hydrophobic anchors for the covalent coupling of hydrophilic proteins to liposomes.

Two carboxyacyl derivatives of cardiolipin, O-succinyl- and O-glutarylcardiolipin, were synthesized with the aim of using them as artificial membrane anchors for the immobilization of hydrophilic proteins to liposomes. Four adjacent fatty acid residues can be introduced into a protein with only one single amino group being blocked, by reacting the cardiolipin derivatives with the protein amino groups after carbodiimide activation. alpha-Chymotrypsin, used as a model protein, and modified with on average two molecules of O-succinylcardiolipin was incorporated into liposomes, which had been prepared by different methods, with very high yield. If incorporated in preformed liposomes, the carboxyacyl cardiolipin anchors were also efficient in binding proteins to liposomal surfaces. Up to 350 micrograms chymotrypsin/mumol lipid were coupled to small unilamellar vesicles, preserving reactivity of the enzyme towards specific macromolecular inhibitors. Human IgG could also be bound to anchor-containing liposomes with high protein to lipid coupling ratio as well as high coupling yield.     

Origin of bombesin-like peptides in human fetal lung

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).     

Coupling between transmembrane calcium transport and membrane potential in retinal rod discs

Changes in the transmembrane potential of bovine rod discs were studied by use of the potential-sensitive fluorescence probes diS-C3-(5) and diBA-C4-(5). The disc membrane was shown to be impermeable to potassium ions. Their concentration in the disc is as high as 2.1 +/- 0.3 mM. The permeability of the disc membrane to Ca2+ was shown to be selective. The accumulation and release of Ca2+ were found to be accompanied by the generation of inside positive and inside negative transmembrane potentials, respectively. The uptake of Ca2+ in the discs may operate against the concentration gradient of the ion. The value of the potential developed is directly proportional to the logarithm of free Ca2+ concentration in the medium (delta phi m = 11.2 +/- 1.6 mV at 4.85 microM Ca2+fr). The accumulation of Ca2+ is decreased by sodium ions and totally inhibited by monensin. This indicates that a Na-Ca exchange process participates in Ca2+ uptake of photoreceptor discs.