The kinetics of DNAse inhibition by specific antibodies.

The inhibition of beef pancreatic deoxyribonuclease I activity with specific antibodies is studied using the hypochromic shift of DNA at 260 nm. The kinetics of this reaction reveals two reaction phases. The first phase, which ends within seconds, is reversible and follows second order kinetics. It leads quickly to the formation of big complexes. The second phase, which reaches completion within minutes before precipitation is detectable, is essentially irreversible and follows zero order kinetics. A model explaining these results is suggested.

Clinical value of 14C-phenylacetic oil as a fat absorption test.

The value of [1-14C] phenylacetic oil as a clinical test for malassimilation was evaluated in 20 normal volunteers, 20 patients with various non-gastrointestinal illnesses, 10 patients with malabsorption, and 7 patients with maldigestion. All subjects were given a test meal composed of 0.5 ml of 14C-phenylacetic oil (0.5 muCi) in 30 g of cheese and 40 g of bread. The test was simultaneously run with D-xylose. Urine was collected over a 5 h period and assayed for 14C and D-xylose. Mean urinary recovery of 14C label in volunteers was 65 +/- 15% of th: administered dose. All 10 patients with malabsorption had abnormal D-xylose excretion and excreted less than 35% of the 14C-phenylacetate. 6 of 7 patients with maldigestion excreted less than 15%, while in the 7th patient excretion of 14C label was borderline (35%). 4 of the 7 patients with maldigestion had abnormal D-xylose excretion, probably as a result of bacterial overgrowth. The procedure described is a cost-effective, simple, rapid, and safe test for fat absorption which may be conveniently run with a D-xylose excretion test.

Purification and biochemical characterization of human lymphocyte mitogenic factor (LMF).

Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70 degrees C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the post-albumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 +/- 0.05 and the minor peak (MMF) had a pI OF 7.27 +/- 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000.

Phenylacetic oil: a possible simple test for fat absorption.

The preparation of a phenyl[1-14C]acetic acid oil is described. The absorption pattern of the phenylacetate in this oil is similar to that of oleic acid in triolein when examined in rats under conditions that may interfere with digestion and/or absorption. The safety of this compound, which is a normal metabolite, was established by the absence of the 14C label in various tissues five days after its administration to rats. When tested in 18 normal human volunteers 66.6 +/- 16.0 (S.D.) percent of the phenylacetate was recovered in a five hour urine collection. In two patients with malabsorption, less than 30% of the label was recovered in the urine over the same period. The half-life of the phenylacetate was determined to be 1-1.5 h. Our preliminary results indicate that phenylacetic oil may serve as a useful simple clinical test for fat absorption.

Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl.

1. [(14)C]Acetoin was enzymically synthesized from [(14)C]pyruvate with a pyruvate decarboxylase preparation. Its optical activity was [alpha](20) (d)-78 degrees . 2. Large amounts (1000-fold higher than physiological concentrations) of acetoin were incubated with rat liver mince. Acetoin disappeared but very little (14)CO(2) was evolved. A compound accumulated, which was purified and identified as butane-2,3-diol. Chromatography on borate-impregnated paper indicated the presence of both the erythro and threo forms. 3. Liver extracts capable of interconverting biacetyl, acetoin and butane-2,3-diol were obtained. These interconversions were catalysed by two different enzymes: acetoin dehydrogenase (EC 1.1.1.5) and butane-2,3-diol dehydrogenase (EC 1.1.1.4), previously identified in bacteria. Both required NAD(+) or NADP(+) as cofactors and were different from alcohol dehydrogenase. The equilibrium in both cases favoured the more reduced compound. 4. The activity of butane-2,3-diol dehydrogenase was decreased by dialysis against EDTA: the addition of Co(2+), Cu(2+), Zn(2+) and other bivalent metal ions restored activity. 5. Biacetyl reductase was resolved into multiple forms by CM-Sephadex chromatography and electrophoresis.

The precursors of the xylene ring in riboflavine.

1. The nature of the precursors of the xylene ring in riboflavine was reinvestigated with growing as well as resting cells of Eremothecium ashbyii. 2. The incorporation of acetoin into riboflavine was very low; further, [2-(14)C]pyruvate and [1-(14)C]acetate were equally effective as precursors of lumichrome, and pyruvate was much more active as a precursor of acetoin. These results exclude acetoin as a direct precursor of riboflavine. 3. Addition of unlabelled glucose decreased the incorporation of [(14)C]acetate into riboflavine more than it decreased the conversion of acetate into carbon dioxide, indicating that acetate is not a direct riboflavine precursor. 4. The incorporation of various sugars and dilution experiments suggest that a derivative of the intermediates of the pentose phosphate cycle is the precursor of the xylene ring in riboflavine.