Production of monoclonal antibodies and development of an ELISA for solamargine

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml^–1 to 11.5 nmol ml^–1 of solamargine.     

DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation

The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation.     

The glycoprotein 71 of ecotropic Friend murine leukemia virus. Structure of the oligosaccharides linked to asparagine-12

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography-mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.     

Protein-free models for the proton-coupled reduction of haemoproteins

Reduction of the bis-pilocarpate-haemin complex at pH greater than or equal to 10 involves the simultaneous uptake of an electron by the Fe(III) ion and a proton by the pendant alkoxide group of an axial ligand. This provides a protein-free model for reactions such as the proton-coupled reduction of cytochromes which involve cooperative Coulombic interaction between two non-bonded sites.     

Maternal and neonatal disposition of pethidine in childbirth--a study using quantitative gas chromatography-mass spectrometry

An assay for pethidine using gas chromatography-mass spectrometry with single ion monitoring has been developed for its analysis in the blood of neonates whose mothers received pethidine during childbirth. Concentrations were determined from the height of the peak at m/e 218 derived from pethidine compared with that at m/e 234 derived from the internal standard lignocaine. Results from 10 mothers and their babies show the time-related passage of pethidine across the placenta. The elimination half-life of the drug from neonatal blood was 22.7h compared with 3h in the adult.     

Identification of diacylglycerol-O-(N,N,N-trimethyl)-homoserine in the halotolerant alga, Dunaliella parva

An unusual zwitterionic polar lipid component isolated from the halotolerant alga, Dunaliella parva Lerche, has been identified as 1(3),2-diacylglyceryl-3(1)-O-4′-(N,N,N-trimethyl) homoserine by means of infrared spectrometry, ¹H- and ¹³C-NMR spectrometry and field desorption mass spectrometry of the intact lipid, as well as by its TLC mobilities and staining behaviour. Mass spectrometry of this lipid indicated the presence of the following major molecular species: 16:0–18:0 (24%); 18:3–18:3 (19%); 16:0–18:2 (16%); 16:0–18:1 (8%).     

Chromatographic resolution of amino acid adducts of aliphatic halides

Numerous xenobiotics are known to be bioactivated and to covalently bind to proteins, but the resulting amino acid adducts (AAAs) are unknown. In this study the AAAs of twelve ¹⁴C-labeled aliphatic halides were examined after formation in an in vitro microsomal system. After exhaustive solvent extraction of the precipitated microsomal protein, the AAAs were isolated by Pronase digestion, followed by filtration through a 500 mol. wt. exclusion membrane. The liberated AAAs were applied to a constant flow DC-4A cation exchange column, resolved by stepwise buffer elution, collected and counted for radioactivity. Column recovery for applied radioactivity was 100 ± 4%. Generally, 1–4 different AAAs (defined by eluting radioactivity) were resolved, with each organohalogen displaying a characteristic elution profile. Methyl iodide, trichloroethylene and 1,2-dichloroethylene had a single major AAA while bromotrichloromethane, 1,2-dibromoethane, 1,1,1-trichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, chloroform and carbon tetrachloride had up to 4 AAAs or more, indicating combinations of binding site(s) and reactive intermediate(s). The single AAA formed following incubation of methyl iodide with the microsomes was identified as S-methylcysteine. Thus, this method appears capable of resolving binding sites and is the initial isolation step for identifying specific adducts to proteins.     

Differences in relative growth rate in 11 grasses correlate with differences in chemical composition as determined by pyrolysis mass spectrometry

Summary Eleven grass species varying in potential relative growth rate (RGR) were investigated for differences in chemical composition by pyrolysis mass spectrometry. The spectral data revealed correlations between RGR and the relative composition of several biopolymers. Species with a low potential RGR contained relatively more cell wall material such as lignin, hemicellulose, cellulose, polysaccharide-bound ferulic acid and hydroxyproline-rich protein, whereas species with a high potential RGR showed relatively more cytoplasmic elements such as protein (other than those incorporated in cell walls) and sterols.     

Flavonoids of Derris araripensis

Nineflavonoids: a dihydrochalcone,a flavone,four 3-methylflavonols,a flavanone, a 3-methylflavanonol and a flavan were isolated from the roots of Derris araripensis. Eight of these compounds are reported for the first time. Structures were established by spectral analysis and chemical degradation.     

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart.

Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.