The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

The effect of microsomal enzyme inducing drugs on reproductive function in the ewe.

A number of drugs cause marked increases in the steroid hydroxylase activity of hepatic microsomes. Beginning 2 days after estrus, 117 mature ewes were each given 14 injections over a 27-day period of phenobarbital sodium, diphenylhydantoin, chlorcyclizine HCl or phenylbutazone. Blood samples for luteinizing hormone (LH) and progesterone determination by radioimmunoassay (RIA) were taken on day 10 of the first estrous cycle (day 18 if no heat was observed) and on days 5 and 10 of the second cycle. On day 10 of the second cycle, the ewes were given an intravenous injection of 1 ml of 6% solution of pentobarbitol sodium anesthetic per 4.5 kg body weight, and the length of anesthetic sleep time was measured. The ewes were then killed and corpora lutea and liver were weighed.In 33 ewes treated with either phenobarbitol sodium or phenylbutazone, sleep time was shortened (18 min $\text{vs}$ 29 min in untreated controls, P<.01), indicating that enzyme induction had occurred. For 41 ewes treated with either chlorcyclizine HCl or diphenylhydantoin, sleep time was lengthened to 93 min (P<.01 $\text{vs}$ controls), indicating impaired liver function. Electron micrographs of liver cells verified that enzyme induction or hepatic degeneration had occurred.     

Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

Reconstitution of the Na+K+ pump of Ehrlich ascites tumor and enhancement of efficiency by quercetin

Plasma membranes from Ehrlich ascites tumor cells were solubilized by octylglucoside in the presence of phospholipids. The Na+K+-ATPase was purified from this extract by adsorption and elution from thio-Seph-arose 4B. The enzyme (specific activity, 7 mumoles of ATP hydrolyzed min-1 mg of protein -1) was reconstituted into liposomes by the octyglucoside dilution procedure. An ATP-dependent Na+ influx with low efficiency was observed. On addition of appropriate amounts of quercetin, the Na+ flux/ATP hydrolysis ratio was increased from 0.4 to 1.4.     

Inhibition of leukocyte adhesion by the in vivo and in vitro administration of cannabinoids

Cannabinoids have been shown to affect various aspects of arachidonic acid metabolism both in vivo and in vitro. Eicosanoid metabolites of arachidonate and related octadecanoate are believed to be involved in cell adhesion processes as agonists in some instances and as antagonists in other cases. This report shows data in which cannabinoids exhibit marked inhibitory effects on the adhesion of mouse peritoneal cells to polystyrene culture dishes. The effects could be seen by in vivo administration of the drugs as well as by direct exposure of the cells in vitro. The data suggest that this inhibition of adhesion is mediated by one or more products generated by stimulation of a lipoxygenase pathway.     

A survey of cultivated heirloom tomato varieties identifies four new mutant alleles at the green-flesh locus

The process of crop domestication occurs through the selection and subsequent propagation of novel alleles that improve traits of interest. Cultivated tomato (Solanum lycopersicum), particularly heirloom varieties, exhibit a wide range of variation in fruit size, shape and color. The green-flesh mutant of tomato possesses a stay-green phenotype resulting in fruits that ripen to a red-brown color, due to the retention of chlorophyll and the simultaneous accumulation of lycopene. The recent identification of the GREEN-FLESH gene provides a molecular tool with which to investigate the origin of a subset of cultivated tomato varieties that resemble the green-flesh mutant. Sequence analysis of the GF locus from 26 varieties revealed the existence of four previously unidentified null alleles. This study illustrates the potential of cultivated tomato varieties, including heritage cultivars, heirlooms, and land races, for uncovering new alleles in genes of interest.     

Determination of products of acetohydroxy acid synthase by the colorimetric method, revisited

The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.     

Assignment of a human serum glycoprotein SP-40,40 gene (CLI) to chromosome 8.

SP-40,40 is a serum glycoprotein consisting of two different subunits (alpha and beta) assembled into a dimer by disulfide bonds. Northern blot hybridization, using total RNA from several cell lines, showed that SP-40,40 is expressed in glioblastoma and testicular tumor cells, as well as hepatoma cells. Spot blot hybridization of flow-sorted human chromosomes, using a SP-40,40 cDNA fragment as a probe, localized the gene for SP-40,40 to human chromosome 8. This gene has been given the designation CLI, for complement lysis inhibitor, by the Human Gene Nomenclature Committee.