A new method for fish chromosome preparation

A new method for the preparation of fish chromosomes from abdominal cavity fluid has been developed. Cells were collected from fish abdominal cavity fluid after an in vivo PHA treatment, and cultured for a short time in medium with colchicine. After 30 min hypotonic treatment for marine fish and 35 min for freshwater fish, slides were prepared by the conventional air-drying method. The advantages ofthe method are: (1) it is technically simple; (2) it produces a reasonably high mitotic index; (3) chromosome spreading is good (4) there is very little cell breakage. Using this method, the chromosomes ofrainbow trout (2n=62); cod (2n=4546) and plaice (2n=46,47 and 48) were investigated.     

A new method for the screening of solid-phase combinatorial libraries for affinity chromatography

A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography.     

Alpha-glutamyl oligopeptides: preparation by the solid-phase method

The synthesis of the oligomeric peptides (Glu)_n–Phe(NO₂)–Phe (up to n = 4) by the solid-phase method is reported. Fractionation by ion-exchange column chromatography of the crude materials cleaved from the resin and subsequent amino acid analysis revealed that the desired peptides were obtained, although contaminated by several by-products whose number depends on the length of peptide chain and on the experimental conditions.     

A rapid method for the preparation of amino acid resin esters for Merrifield solid-phase peptide synthesis

An improved method for the preparation of Merrifield resin esters is presented. This method is rapid, is free of racemization, and is not complicated by a quaternization side reaction. Chloromethylated resin beads, t-butoxycarbonyl amino acid, and potassium t-butoxide are heated at 80 °C in dimethylsulfoxide for one-half hour to yield resin esters of suitable substitution for solid-phase petide synthesis. All twenty of the BOC protected common amino acids were esterified to the resin by this method. Resin substitution values lie in the range of 0.13 meq/g (BOC-Glu (NH₂)) to 0.66 meq/g (BOC/Pro), with most of the amino acids yielding 0.3–0.4 meq/g (on a resin containing 0.8 meq Cl/g).     

A new method for preparation of undecalcified bone sections

A new method for preparation of sections of undecalcified bone is described. Samples of ovine bone were embedded in methylmethacrylate and thick-sectioned with a cutoff machine or commercial band saw. Composite slides were prepared by gluing white acrylic to glass using cyanoacrylate glue. Bone sections were glued to the composite slide and then surface polished by grinding or ultramilling. The polished surface of the section was then etched and stained. The techniques described in this paper reduce the time spent grinding or milling sections and improve resolution of surface-stained features of undecalcified bone sections.     

A new method for the preparation of metaphase chromosomes for flow analysis

A new method for the preparation of metaphase chromosomes for flow analysis has been evaluated. It has been shown that this method, which involves detergent lysis of metaphase cells and polyamines to stabilize the DNA, yields lower coefficients of variation and background levels in the DNA histograms than is currently obtained by hexylene glycol based methods. A conventional flow cytometer (FACS-II) has been used to resolve the human karyotype into about 14 peaks after ethidium bromide staining and excitation with a relatively low level of illumination (0.4 W at 488 nm). Flow karyotypes have also been obtained from suspension cell lines, in particular from the mouse cell line, Friend 707/B10. The only disadvantage of this method is that the chromosomes are highly condensed and therefore banding studies on sorted chromosomes may not be possible.     

A new method for the preparation of mammalian spermatogonial chromosomes.

After the seminiferous tubules were minced and washed with phosphate-buffered saline to remove sperms, spermatids and spermatocytes (Hoo and Bowles, 1971), they were repeatedly treated with 0.1% trypsin solution to liberate spermatogonia. It was concluded that the spermatogonial chromosomes can be analysed much more easily and accurately by this procedure than by previous ones.     

A new simple preparation method for NaK-ATPase-rich membrane fragments

A method for the isolation of highly active membrane bound NaK-ATPase without detergents in quantity from the electric organ of the electric eel (Electrophorus electricus) is described. This method consists of the homogenation of electric organ with an isotonic solution containing sucrose, histidine, EDTA, and arginine, and of the separation of the higher active membrane fraction from the microsomal fraction by density gradient centrifugation. The enzyme has a specific activity of about 20 μmol Pi/min/mg at 37°C, and 13 μmol Pi/min/mg at 30°C. Although it is not as pure as the detergent-treated enzyme preparation based on the level of phosphorylated protein, ouabain binding, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its enzyme activity is comparable to that of the purified enzymes. This preparation is very stable and is able to change its medium by Sephadex chromatography without any loss of enzyme activity and protein content. This preparation is also expected to keep the original characteristics as well as the enzyme in the tissue.     

A new way of solid-phase microextraction fibers preparation for selected antibiotic drug determination by HPLC-MS

The polypyrrole (PPy) and polythiophene (PTh) solid phase microextraction (SPME) coatings were obtained with the use of the electropolymerisation and linear sweep voltammetry. Such fibers were modified by an ozone treatment in a gaseous phase in the concentration of 2.1 ± 0.2 × 10(-5) mol dm(-3). Both kinds of fibers were applied in the microextraction of linezolid from standard solutions to compare the extraction efficiencies displayed by these sorption phases. In these investigations a better adsorption capacity was obtained for polypyrrole fibers and hence only these kinds of fibers were utilized in the measurements from human plasma. In all measurements the concentrations of the drugs were in the range from 1 to 20 μg ml(-1) (standard solutions) and 1 to 15 μg ml(-1) (human plasma). Before the measurements, an optimization of the desorption solution experiments was performed. The correlation coefficients (R) obtained in the standard solution and human plasma were in the range from 0.8399 to 0.9970. The relative standard deviations (RSDs) were in the range of 0.1-7.6%.     

A new method for the preparation of beta-amylase from sweet potato.

A simple method for the preparation of sweet potato beta-amylase by thymol amylose adsorption is described. The method is far more efficient and gives higher recovery of the enzyme. The crystalline enzyme thus obtained is found to be homogeneous by gel chromatography, polyacrylamide gel electrophoresis.     

A new method for liposome preparation using a membrane contactor

In this article, we present a novel, scalable liposomal preparation technique suitable for the entrapment of pharmaceutical agents into liposomes. This new method is based on the ethanol-injection technique and uses a membrane contactor module, specifically designed for colloidal system preparation. In order to investigate the process, the influence of key parameters on liposome characteristics was studied. It has been established that vesicle-size distribution decreased with a decrease of the organic-phase pressure, an increase of the aqueous-phase flow rate, and a decrease of the phospholipid concentration. Additionally, special attention was paid on reproducibility and long-term stability of lipid vesicles, confirming the robustness of the membrane contactor-based technique. On the other hand, drug-loaded liposomes were prepared and filled with two hydrophobic drug models. High entrapment-efficiency values were successfully achieved for indomethacin (63%) and beclomethasone dipropionate (98%). Transmission electron microscopy images revealed nanometric quasispherical-shaped multilamellar vesicles (size ranging from 50 to 160?nm).     

Comparative studies of agarose and kieselguhr-agarose composites for the preparation and operation of immunoadsorbents.

Study was made of controlled fabrication and operation of immunoadsorbents exploiting beaded composites of agarose or kieselguhr-agarose. Materials were activated by cyanogen bromide and tresyl chloride, derivatised with human IgG antigens, and utilised in direct, one-step purifications of anti-huIgG monoclonal antibodies produced in serum-based cultures of murine hybridomas. The influence of solid phase composition, degrees of activation, concentration of immobilised antigen, capping chemistries, and mode of product desorption was studied in respect of purification performance. Maximum concentrations of immobilised huIgG could be achieved following activation of 50% available hydroxyl groups in both materials. Specific adsorption and desorption of monoclonal antibodies, expressed per mole of immobilised ligand, declined with increasing ligand concentrations. Control of activation and derivatisation of agarose solid phases enhanced the overall specification and performance of both homogeneous and composite fabricates. The large particle size of composites (150-1000 microns) restricted efficient performance in fixed bed contactors operated under non-equilibrium conditions. However, their physical nature recommended adsorptive operations with particulate feedstocks in fixed or fluidised beds, batch suspension contactors, or fast flow regimes adopted for cleaning and equilibration operations.