An angle-variable three-dimensional pulsed field gel electrophoresis system

A three-dimensional pulsed field electrophoretic method based on the simultaneous application of fixed and cyclically alternating polarity fields at a right angle is described. Requiring only minimal electronic hardware it provides highly homogeneous field conditions over a large gel area and the versatility to vary the pulse vector angle. The electrophoretic parameters critical to achieve fast high resolution separation over a wide range of molecular sizes have been optimized and applied to megabase-size chromosomal DNA molecules. The empirical relationships between pulse time, field strength conditions, and resolution limits derived allow selection of coordinated experimental conditions for the separation of specific DNA size ranges.     

Purification of human C3b inactivator by monoclonal-antibody affinity chromatography

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.     

Vitellin and vitellogenin incorporation by isolated oöcytes of Locusta migratoria migratorioides (R.F.)

Vitellin and vitellogenin labelled in vitro with ¹²⁵I and in vivo with ³H were incorporated into yolk by locust oöcytes incubated in an in vitro system. This incorporation was specific and linear with the duration of incubation. Uptake of vitellin by oöcytes was 3–4 times higher than ¹²⁵I-bovine serum albumin in 2.1-mm oöcytes and 20 times higher than ¹²⁵I-bovine serum albumin in 4.0-mm long oöcytes. The uptake of the albumin was enhanced by the presence of vitellin in the incubation medium. ³H-labelled yolk protein was incorporated at higher rates than that labelled with ¹²⁵I. The addition of the juvenile hormone analogue ZR 515, caused the incorporation rates of vitellogenin to be increased. The amount of vitellin or vitellogenin taken up by the oöcytes increased with their length, and the rate of incorporation per unit surface area was highest in 3–4-mm long oöcytes. These results corroborate previously reported in vivo patterns of incorporation rates of developing oöcytes.     

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

Summary The Ca⁺⁺-mediated increase in K⁺-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca⁺⁺ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K⁺ concentration. In K⁺-free solutions the concentration of quinine to achieve 50% inhibition (K ₅₀) was 5 m, but at 5mm K⁺ the required concentration was increased 20-fold to 100 m. An increase in internal Na⁺ had the opposite effect, allowing a high potency of quinine despite the presence of external K⁺. Alterations in the internal K⁺ level, on the other hand, were without effect on theK ₅₀, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl^– by NO ₃ ^– , a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K⁺ from an external binding site, the reported K⁺-activation site for the Ca⁺⁺-mediated K⁺-permeability.     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

From compartmental analysis of radioisotope elutin measurements, fluxes of Ca²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to complete nutrient solutions containing a range of calcium concentrations ([Ca₀]) from 2 eq l^-1 to 20 meq l^-1, increasing in 10-fold steps for Ca²⁺. Except for the calcium counter-ion (usually NO ₃ ^- , sometimes Cl^- at the highest [Ca₀]), the composition of the nutrient solution was other-wise the same at all calcium concentrations. Compartmental analysis indicated that the cytoplasm had a high content of exchangeable Ca²⁺ but, in the light of evidence from animal studies, ionic activity of calcium in the cytoplasm was assumed to be no greater than 0.002 eq ml^-1. With the Ussing-Teorell flux equation as the criterion, it was concluded that at all values of [Ca₀] tested, Ca²⁺ entered the cytoplasm passively and was actively pumped back into the external solution. Entry of calcium to the vacuole from the cytoplasm was active in all cases. The conclusions regarding the character of ion transport across the plasmalemma were the same as when the whole calcium content of the cytoplasm was taken to contribute to the ionic activity. However, the electrochemical activity gradient was very much steeper than formerly estimated. Calcium was transported to the stele in proportion to the calcium content of the cytoplasm and moved in the xylem almost exclusively in the basipetal direction.     

Terpenes from pittosporaceae

The monoterpenes of the fruits of Pittosporum resiniferum and P. undulatum have been identified. The essential oil of P. resiniferum contai     

Mass spectrometric technique for the determination of N-phosphonoacetyl-l-aspartic acid in serum

N-Phosphonoacetyl-l-aspartic acid (PALA), a potent inhibitor of aspartic acid transcarbamylase, is now undergoing Phase I clinical trials. Initial experiments revealed that PALA is not metabolized to phosphonoacetic acid (PAA) in humans. Thus PALA may be quantified in serum after in vitro conversion to PAA. Serum is deproteinized with perchloric acid, lipid extracted with methylene chloride, hydrolyzed with 8 N hydrochloric acid at 100° for 3 h, and evaporated to dryness with nitrogen. The residue is silylated, and PAA is quantified by monitoring the

ions of the protonated molecular ions of trimethylsilyl derivatives of PAA and phosphonopropionic acid (internal standard) obtained in chemical ionization with methane. Limit of detection is 0.5 μM (150 ng/ml) PALA using 1 ml serum. PALA was given by continuous infusion to cancer patients at various doses. Maximum levels of PALA (50–500 μM range) were obtained at the end of infusion, followed in most cases by biexponential decay. Persistent residual PALA levels (5 μM for 48 h after infusion) correlated with increased toxicity.