A straightforward radiometric technique for measuring IMP dehydrogenase

[2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity.     

A radiometric technique for measuring l-asparagine in picomol quantities

1. HeLa cells were cultured in the presence of heterologous immunoglobulin G and guinea-pig serum together with [(32)P]phosphate. 2. Incorporation of [(32)P]phosphate was significantly stimulated by anti-HeLa immunoglobulin G and complement-sufficient serum compared with immunoglobulin G from unimmunized rabbits and complement. Within 2.5h heat-inactivated guinea-pig serum and anti-HeLa immunoglobulin G stimulated [(32)P]phosphate incorporation to the same extent as heat-inactivated complement and immunoglobulin G from unimmunized rabbits. 3. Compared with cells exposed to immunoglobulin G from unimmunized rabbits together with complement, anti-HeLa immunoglobulin G with complement increased the phospholipid content of HeLa cells twofold within 5h of incubation. 4. Exposure of HeLa cells to anti-HeLa immunoglobulin G and complement for 5-22h resulted in a twofold increase in the net accumulation of [(32)P]phosphate in sphingomyelin and phosphatidylcholine and a 50% increase in the net accumulation of [(32)P]phosphate in phosphatidylethanolamine, compared with cultures exposed to immunoglobulin G from unimmunized rabbits and complement. 5. A transient accumulation of (32)P-labelled lysophosphoglycerides in HeLa cells exposed to antibody and complement was detected, confirming previous findings (Güttler & Clausen, 1969b). 6. The stimulation of [(32)P]phosphate turnover occurred in cells filling up their cytoplasma with vacuoles. This supports the suggestion that the accumulation of phospholipid in these cells may be concerned with the synthesis and function of cytomembranes.     

A radiometric technique for the measurement of adenylosuccinate lyase

When radioactive adenylsuccinic acid (AMP-S) is metabolized to AMP and fumaric acid by the enzyme adenylsuccinate lyase (EC 4.3.2.2), a proton is released to the solvent as ³H₂O. This removal is believed to be stereospecifically identical to that catalyzed by the enzyme, l-aspartase [1–5], and therefore entails the loss of a proton from C-3 of the dicarboxylic acid moiety of the nucleotide. Advantage has been taken of this fact in the design of a facile assay for this enzyme. Adenylosuccinic acid, tritiated on C-2 and C-3 of the l-aspartase moiety, is prepared by chemical synthesis. This product is purified, lyophilized to dryness and reconstituted in a solution of unlabelled AMP-S, bringing the final concentration to 5·10^?3 M, and the final specific activity to 8.0 μCi/mol. 5-μl aliquots of this substrate are then incubated at 37°C with 5-μl aliquots of tissue extract; after an appropriate period, any tritium released to the solvent water is distilled at room temperature overnight into a 5 μl droplet of saturated aqueous KOH adherent to the lid of the sealed reaction vessel. The lid is removed and tritium thereon is measured by scintillation spectrometry. The assay, performed as prescribed, is facile, in that it permits the simultaneous estimation of the lyase activity in a large battery of samples, is not interfered with by opalescent or proteinaceous suspensions, is accurate and outstandingly sensitive.     

A stroboscopic technique for measuring Daphnia heart-beat rate

The variation of Daphnia heart-beat rate with temperature is described. The temperature can be changed and monitored continuously while the heartbeat rate is measured using stroboscopic techniques.     

A split-root technique for measuring root water potential

Water encounters various resistances in moving along a path of decreasing potential energy from the soil through the plant to the atmosphere. The reported relative magnitudes of these pathway resistances vary widely and often these results are conflicting. One reason for such inconsistency is the difficulty in measuring the potential drop across various segments of the soil-plant-atmosphere continuum. The measurement of water potentials at the soil-root interface and in the root xylem of a transpiring plant remains a challenging problem.     

A flow cytometry technique for measuring chromosome-mediated gene transfer

BACKGROUND: Using artificial chromosome expression systems (ACes), we have developed a unique and rapid screening technique to quantify delivery of foreign DNA into cells in vitro. Delivery was measured within 24 h after transfection, using flow cytometry to detect the transfer of ACes labeled with thymidine analogue. This technique can be used to optimize delivery parameters of ACes and heterologous DNA into cells and eventually tissue. METHOD: Chinese hamster ovary (CHO) cells carrying artificial chromosomes were grown in media supplemented with iododeoxyuridine (IdUrd). The 60-mb artificial chromosome was purified by flow cytometry sorting and transfected into Chinese hamster lung fibroblast cells (V79-4) or mouse connective tissue cells [LM(tk-)] using LipofectAMINE 2000trade mark, a cationic lipid, and Superfecttrade mark, a cationic dendrimer. The cells were incubated with an FITC-conjugated anti-bromodeoxyuridine (BrdUrd) antibody and analyzed by flow cytometry. IdUrd-incorporated artificial chromosome expressing green fluorescent protein (GFP) was transfected into V79-4 cells. Delivery was measured at 24 h and GFP expression was detected at 48 h. RESULTS: The delivery of intact artificial chromosomes into V79-4 and LMtk- cells was detected within 2 h and up to 48 h post-transfection. Maximum delivery rates of 20% and 14% were observed using LipofectAMINE 2000 and Superfect, respectively. Flow cytometry data correlated with microscopic observations. IdUrd incorporation resulted in less quenching after staining with Hoechst 33258 and chromomycin A3 than BrdUrd incorporation. The fluorescence intensity of the FITC-conjugated anti-BrdUrd antibody was greater with IdUrd-incorporated chromosomes than with BrdUrd-incorporated chromosomes. CONCLUSION: The results indicate that IdUrd-labeled artificial chromosomes can be detected 24 h after transfection. This efficient, sensitive, high-throughput detection technique is being used to evaluate and optimize other transfer technologies (e.g., electroporation and sonoporation), different delivery reagents, and protocols in a variety of cells in vitro. This work represents the first step in utilizing artificial chromosomes as nonviral vectors for gene therapy.     

A radiometric microassay for cellulase activity

The method described measures the release of ¹⁴C products from [U-¹⁴C]cellulose. The assay is simple and utilizes very small incubation volumes. Activity is evident within the first 30 min of reaction. This method should be particularly suited to activity surveys of culture filtrates from fermentations as well as the analysis of fractions from enzyme purification studies.     

A new automated technique for measuring respiration in soil samples

An automated 36 place valve to provide continuous soil respiration measurements was constructed. The valve is fully computer controlled and can sample and purge the soil atmosphere as frequently as every 75 minutes. The concentrations, automatically measured by the valve, are essentially identical to those measured manually by gas chromatography in the concentration range of 0.1 to 1% CO₂, and are kept in this range by adjusting the mass of soil and the sampling frequency. Data are transferred automatically to a computer spreadsheet program for data handling and plotting on either a rate or cumulative basis. The system has proved reliable over many thousands of analyses and has made detailed analysis of microbial activity on a continuous basis possible.     

A technique for measuring xylem hydraulic conductance under high negative pressures

A technique for measuring hydraulic conductances of excised xylem segments exposed to high negative pressures is described. A centrifugal force is used to generate negative pressures (P) in the sample and to create a positive hydrostatic pressure difference (ΔP) between its two ends. ΔP forces water through the sample at a flow rate (F) determined optically during centrifugation. The sample hydraulic conductance k is derived from F and ΔP. The sample vulnerability curve is given by the dependence of k on P. Results for Cedrus atlantica Manetti and Laurus nobilis L. shoots are given. The technique is appropriate for the analysis of xylem refilling under negative pressure.     

A rapid radiometric assay for dihydrofolate reductase

A rapid radiochemical procedure for the measurement of dihydrofolate reductase activity is described. The method employs separation of the radiolabeled substrate from the products of the reaction by precipitation with acetic acid and zinc sulfate. This method permits rapid processing of samples and climinates time-consumlng column chromatography techniques. Good agreement of results is obtained when the radioactive method is compared to the spectrophotometric assay method.     

A sensitive, radiometric assay for lysophosphatidylcholine

To facilitate investigation of the metabolism of lysophosphatidylcholine and choline lysoplasmalogen in small quantities of tissue, a method for the quantification of these phospholipid species that is capable of accurate and reproducible analysis in samples which contain less than 1 nmol of total choline lysophospholipid was developed. The procedure employs chloroform and methanol extraction of phospholipids from isolated tissue with subsequent separation of the choline lysophospholipid fraction by high-performance liquid chromatography. The choline lysophospholipids are then acetylated with [3H]acetic anhydride and the [3H]acetyl-lysophosphatidylcholine product is isolated by thin-layer chromatography and quantified by liquid scintillation counting. The choline lysophospholipid content in the sample is determined from a standard curve constructed from samples containing a known amount of synthetic lysophosphatidylcholine with correction for recovery based on the inclusion of [14C]lysophosphatidylcholine as an internal standard.     

A radiometric assay for HIV-1 protease

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.     

A tetrazolium technique for the histochemical localization of ATP: Creatine phosphotransferase

Summary A tetrazolium technique is presented that permits the study of ATP: Creatine phosphotransferase, or creatine kinase, in fixed skeletal muscle tissue sections, within the limits imposed by the properties of the chosen ditetrazole, nitro blue tatrazolium. There is a variation in creatine kinase activity between the muscle fibres. Those with high creatine kinase activity also have high succinate dehydrogenase activity.List of Abbreviations ADP Adenosine-5-diphosphate - ATP adenosine-5-triphosphate - CK creatine kinase - G-6-P glucose-6-phosphate - G-6-P-DH glucose-6-phosphate dehydrogenase - HK hexokinase - NADP nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PMS phenazine methosulphate - SDH succinate dehydrogenase     

An exploratory technique for measuring fertility norms

Abstract

In an attempt to remedy inherent weaknesses in traditional methods of fertility norm measurement, the evaluative dimension of the semantic differential is used to measure such norms among college women at two Southern universities. The instrument was designed to gauge the covert evaluative response elicited in people who observe behaviors relevant to norms which they have internalized. Variations in evaluations of hypothetical couples representing the parities zero through seven are associated with the race, religious preference, religiosity, and future work orientations of respondents. The significance of differences in the relative configurations of norms is determined by a special model of analysis of variance, and subgroups are compared with respect to the intensity of the norms they hold.