Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines

This study describes the first preparation and spectroscopic characterization of naturally occurring phospholipids separated according to degree of unsaturation. Phosphatidylserines (PS) have been prepared from bovine brain and shown to be pure by extensive thin layer chromatographic analysis as well as by infrared spectroscopy and fatty acid analysis. The PS has been separated according to degree of unsaturation and prepared using AgNO3-impregnated silica gel H thin-layer chromatography. Fatty acid analysis of the two principal PS subfractions indicates that they are enriched in the molecular species 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine. The identity of the two PS subfractions was further verified by rechromatographing on several thin layer systems and by infrared spectroscopy. With the use of a 100 MHz Fourier transform nuclear magnetic resonance (NMR) spectrometer, the spectra of bovine whole brain, white matter, gray matter, monoenoic, and hexaenoic PS were obtained. Distinct proton resonances were assigned to double bond protons, protons adjacent to a double bond, and protons between two double bonds, using fatty acid methyl ester standards. The various PS preparations gave different intensities of the various proton resonances which correlated with differences in fatty acid composition. The method provides a convenient, non-destructive spectroscopic method for distinguishing monoenoic and polyunsaturated species of intact phospholipids. Electron spin resonance studies of nitroxide-labelled cholestane in sonicated PS vesicles showed greater probe motion as the unsaturation of the acyl chains was increased. The hexaenoic PS vesicles were more fluid than monoenoic PS vesicles at all temperatures in the range 10-55 degrees C. These results suggest that neuronal membranes are more fluid than myelin membranes as neuronal membranes contain more hexaenoic phospholipids.     

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca²⁺ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca²⁺ concentration outside the vesicles (Ca₀²⁺), substrate depletion, ADP accumulation, nonspecific membrane deterioration or the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca₀²⁺ at the time that calcium content was maximal. A causal relationship between high Ca₀²⁺ and spontaneous calcium release was suggested by the finding that elevation of Ca₀²⁺ from less than 1 μM to 3–5 μM increased the rate and extent of calcium release.The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

Antigen-specific helper activity in serum of mice primed with sheep red cells. I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10⁸ SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10⁷ SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10⁷ SRBC. A system using normal mice injected with 10⁵ SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.     

A requirement for three cell types for in vitro generation of specific secondary cell-mediated cytotoxic response against SV40-induced tumor-associated antigens in mice

The present study demonstrates that a collaborative interaction among three cell types, namely, two distinct subsets of T cells and macrophages is needed for in vitro generation of specific secondary cell-mediated cytotoxicity against syngeneic SV40-transformed cells in mice. These data suggest a central role of three cell types in the generation of efficient antitumor immune response in syngeneic tumor systems.     

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.