Development of a radiometric metabolic viability testing method for human and porcine skin

A radiometric viability assay based upon the conversion of [¹⁴C]glucose into ¹⁴CO₂ by the viable cells on the dermal side of whole skin has been developed. The assay proved to be sensitive, reproducible, and practical, and was based upon the use of a microbiological growth detection system commonly used in many hospitals and laboratories. Relatively small samples of skin (0.25–1.00 g) were used in the test, and it was found that microbiological contamination did not interfere with the assay under normal conditions. The linear proportionality of the assay with both time and amount of skin assayed precluded the difficulties of nonlinear proportionality in other systems, allowing direct comparisons to be made between skin samples of different sizes and different incubation times. The assay could also detect ¹⁴CO₂ released from many radiolabeled substrates, including glucose, aspartate, glutamate, ornithine, orotic acid, and glycerol. Thus, the method could be used to test a number of cellular functions necessary for viability, including glycolysis, the functioning of the citric acid cycle and the pentose phosphate pathway, sugar and amino acid metabolism, pyrimidine biosynthesis, and cryopreservative agent metabolism. Since any of these tests could be performed in 4 hr, a viability assay based upon glycolysis alone, or in combination with any of the other tested substrates, could be carried out after allograft skin procurement before a decision needed to be made on skin cryopreservation.     

A test for endogenous interference in superoxide dismutase assays

Endogenous interfering substances can be detected by applying the techniques of parallel-line analysis of variance to the assay of superoxide dismutase (SOD) activities in crude tissue extracts. The technique also allows expression of specific activities in terms of units of activity as defined by commercially available standards as opposed to definition by the specific assay conditions utilized. This analysis may be broadly applied to many of the indirect SOD assays currently in use. In the current investigation, SOD was assayed by its ability to inhibit the rate of reduction of acetylated ferricytochrome c by superoxide anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) generated via the xanthine-xanthine oxidase reaction. The parallel-line analysis was effective in detecting the presence of interference by exogenous ascorbate in the reaction system, and by unidentified endogenous reactants within extracts of whole blood and ocular choroidal tissues of the rainbow trout, Salmo gairdneri.     

Chemical quantitation of hemoglobin glycosylation: fluorometric detection of formaldehyde released upon periodate oxidation of glycoglobin

A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ?-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia.This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.     

Effect of acetate on glucose utilization by isolated rat thymocytes

The effects of acetate and ammonium salts on glucose metabolism, aminoisobutyric acid influx, and radioiodinated insulin binding in isolated thymocytes were studied. Acetate in the concentration range, 0.1–30 mm, was found to inhibit basal and insulin-stimulated CO₂ production whereas ammonium chloride at concentrations greater than 0.3 mm was slightly stimulatory. Ammonium salts inhibited glucose incorporation into glycogen and aminoisobutyric acid influx only at high concentration (30 mm). Neither acetate nor ammonium salts had significant effects on glucose incorporation into glycogen or aminoisobutyric acid influx at lower concentrations. No effect on insulin binding was observed. The observation that very low concentrations of acetate can perturb these biological assay systems suggests that other biological functions may be affected by trace amounts of buffer salts carried over from protein isolation steps.     

An approach to the identification of adenosine''s inhibitory site on adenylate cyclase

The separate and combined effects of molybdate and dithiothreitol on the stability of human uterine 9 S estrogen receptor were studied. Maximal, short-term, protection of the 9 S estrogen receptor was achieved by the joint inclusion of both stabilizing agents in cytosol buffers. This molybdate—dithiothreitol-mediated stability was dependent on reducing agent concentration inferring sulphydryl involvement in 9 S receptor protection by molybdate. The study also showed that molybdate—dithiothreitol could not prevent the gradual decay of the 9 S estrogen receptor to the 4 S form in cytosols stored at 4°C over prolonged periods.     

Pregnancy termination by prostaglandin F2α stimulates maternal behavior in the rat

Treatment with PGF_2α resulted in the termination of pregnancy in 16- and 19-day pregnant rats, but not in 10- or 13-day pregnant rats. Rats that aborted displayed a rapid onset of maternal behavior when tested with foster pups. Aborted rats also displayed sexual receptivity and ovulation: these phenomena resemble the sequence of events following hysterectomy on the same days of pregnancy. Both can be related to the events surrounding normal parturition in the rat. The results are interpreted as due to a pregnancy-induced deactivation of the factor in the uterus that prevents estrogen from stimulating maternal behavior in nonpregnant females. In the absence of this factor, the PGF_2α-induced rise in estrogen secretion facilitates maternal behavior and sexual behavior and induces ovulation.     

Kinetic studies of the applicability of the common site concept to the 3β-hydroxysteroid dehydrogenase: 5-ene-3-ketosteroid isomerase activities of human placental microsomes

Pregnenolone (3β-hydroxy-5-pregnen-20-one) and DHA (3β-hydroxy-5-androsten-17-one), substrates for 3β-hy-droxysteroid dehydrogenase (3β-HSD), with K_M values of 15–40 nM, were ineffective inhibitors of 5-ene-3-ketosteroid isomerase (isomerase), with K_I values >40 μM in each case. Progesterone and androstenedione (4-androstene-3, 17-dione), 3β-HSD inhibitors with K_I values of 5.0 μM and 0.8 μM respectively, were also relatively ineffective inhibitors of isomerase, with K_I values of 30 μM and 16.5 μM respectively. Exposure of microsomes to hydrogen peroxide, which significantly increases the K_M for pregnenolone as a 3β-HSD substrate, had no effect on the K_M for the isomerase substrate 5-pregnene-3, 20-dione.It is concluded that the data do not support the common site concept with regard to the conversion of pregnenolone to progesterone by human placental microsomes.