Separation of urine proteins on the anion-exchange resin mono QTM

Proteins excreted in urine due to renal failure were separated on Mono Q^TM, a new strong anion exchanger designed for fast high-resolution protein separations. The separation procedure was divided into two steps. The first step involved removal of low-molecular- weight substances by rapid desalting on a Sephadex G-25 Superfine column. In the second step, the total protein fraction (3–6 ml) was loaded onto the Mono Q column with the aid of a superloop. The proteins were adsorbed onto the top of the ion-exchanger column and gradually displaced by a combined pH and salt gradient in 40 min. The choice of ion exchanger and initial operating conditions were based on data obtained from electrophoretic titration curve experiments. Identification of separated proteins was achieved by fused rocket electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively.     

Cholinesterase Levels in Blood Plasma and Erythrocytes from Calves,Normal Delivering Cows and Cows Suffering from Parturient Paresis

Plasma cholinesterase (pChE) levels and erythrocyte acetylcholinesterase (eAChE) levels were studied in 6 cows before, during and after parturition (Group I), their calves (Group II), 38 cows suffering from parturient paresis (Group III) and 14 newly delivered non-paretic cows (Group IV). The mean of the pChE level in Group I was 1.5 μkat/1 ± 0.20 before parturition and decreased significantly (P ≦ 0.05) to 1.2 ukat/1 ± 0.16 after parturition. The eAChE level was before parturition ≅ 140 ukat/1 and decreased to ≅ 130 μkat/1 4–5 weeks after parturition. At birth the pChE level was 12.8 ukat/1 ± 5.9 in Group II. After 4 weeks the level had decreased to 2.3 ukat/1 ±0.3. In the bull calves the pChE level started to increase when they were 6 weeks old and reached a level of 5.7 μkat/1 ± 0.6 before slaughter at 6 months of age. The heifers did not show this increase. They had a level of around 2 μkat/1 throughout the investigation. The eAChE level at birth was 119 μkat/1 and increased slowly to a level of 145 μkat/1 at 6 months. No differences between the sexes were found. The cows suffering from parturient paresis had a pChE level of 1.80 μkat/1 ± 0.30 before treatment with calcium (Ca). The level decreased significantly (P ≦ 0.001) after Ca-infusion to a level of 1.67 ukat/1 ±0.29. Group IV had a pChE level of 1.65 μkat/1 ± 0.42 at parturition. Two to 4 months later the cows that had recovered from milk fever had a level of 1.61 μkat/1 ± 0.31 and the control cows 1.66 ukat/1 ± 0.48. No differences between the groups were found for the eAChE level. The findings show that parturition influences the pChE level in cows and that sex influences the pChE level in calves between 6 weeks to at least 6 months of age. Furthermore the elevated pChE level found in the cows suffering from parturient paresis before Ca infusion may be a further sign of a disturbance in the cholinergic system with a special preference to the neuromuscular junctions.     

Selected ion monitoring for rapid differentiation of mycobacteria isolated from domestic animals

Gas chromatography/mass spectrometry adapted for selected ion monitoring was used to detect C₃₂ mycocerosic acid in short-term incubated cultures of procineand canine strains of mycobacteria. The method can be employed for rapid differentiation of Mycobacterium tuberculosis from M. avium-intracellulare.     

Visualization of freeze-dried and shadowed myosin molecules immobilized on electron microscopic films

The mica replication technique first described by Hall [5] has produced myosin molecules which were heterogeneous in appearance in terms of shadowing, decoration, contrast and background. Therefore, an alternative technique for the visualization of myosin molecules was developed: Myosin molecules are sprayed directly onto glow discharged or silicium-monoxide coated carbon filmed grids, omitting glycerol. After washing several times with distilled water, rapid freezing, and freeze-drying, the immobilized myosin molecules are visualized by shadow-casting at low temperature and at varying angles. After backing with carbon the "in situ" shadowed molecules are observed in the electron microscope. This technique has several advantages over the standard method in that it yields more reproducible results. It is potentially useful for investigating interactions of myosin binding proteins with myosin and for visualizing unshadowed myosin in the STEM.     

The role of N-acyl groups in the inhibitory activity of LH-RH analogues

Inhibitory analogues of luteinizing hormone-releasing hormone (LH-RH) were prepared with formyl-D-Trp¹, acetyl-D-Trp¹, valeryl-D-Trp¹, tartaryl-D-Trp¹, diacetyl-tartaryl-D-Trp¹, acetyl-Gly¹, and acetyl-Sar¹ successively replacing the position one in the analogue [D-Trp¹, D-p-Cl-Phe², D-Trp³, D-Phe⁶, D-Ala¹⁰]-LH-RH. The formyl-D-Trp¹ and acetyl-D-Trp¹ analogues yielded 100% blockade of ovulation at the 10 μg dose; the others were less potent and inhibited ovulation at the 50 μg dose. The inhibitory potency seems to correlate with the polarity of the acyl group.     

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ? = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ? determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures T_d which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.     

Neural crest cell behavior in white and dark embryos of Ambystoma mexicanum: Epidermal inhibition of pigment cell migration in the white axolotl

Melanophores in larvae of the white (dd) strain of the Mexican axolotl (Ambystoma mexicanum) are confined to the dorsal midline of the trunk and dorsal posterior part of the head, whereas those in dark larvae (D_-) are distributed over the flank as well. Our results show that this phenotype of white larvae is the result of the failure of the melanophores or their neural crest precursor cells to migrate laterally due to an inhibition of or a failure in the support of their migration in the subepidermal space by the overlying epidermis. Correlated light and scanning electron microscopy of dissected larvae showed melanophores occupying the subepidermal space on the flank of dark larvae, whereas these cells were restricted to the dorsal midline of white larvae. Grafting experiments in which patches of epidermis, the underlying mesoderm, or both, were exchanged between dark and white embryos suggested that white epidermis alone can prevent the integration of pigment cells on the flank of dark larvae and, conversely, that grafts of dark epidermis alone can support their migration on the flank of white larvae. Mesoderm, when grafted alone, could not be shown to have similar effects.