Variety Of Test Systems Research Articles

Immunoserology of infectious diseases

The immune response to microorganisms not only participates in the elimination of unwanted organisms from the body, but also assists in diagnosis of infectious diseases. The nonspecific immune response is the first line of defense, assisting the body until the specific immune response can be mobilized to provide protective mechanisms. The specific immune response involves humoral or cell-mediated immunity or both, dependent on the nature of the organism and its site of sequestration. A variety of test systems have been developed to identify the causative organisms of infectious diseases. Test systems used in immunoserology have classically included methods of detecting antigen-antibody reactions which range from complement fixation to immunoassay methods. Relevant test systems for detecting antigens and antibodies are described. With numerous test systems available to detect antigens and antibodies, there can be confusion regarding selection of the appropriate system for each application. Methods for detecting antibody to verify immunity differ from immunologic methods to diagnose disease. Techniques to detect soluble antigens present in active infectious states may appear similar to those used to detect antibody, but their differences should be appreciated.

Acetaldehyde‐induced mutation at the hprt Locus in Human Lymphocytes In Vitro

Acetaldehyde (Aa) induces chromosomal aberration and sister chromatid exchange in a variety of test systems, but has not previously been evaluated for its ability to induce gene mutation in mammalian cells. We have studied the mutagenic effect of Aa at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human lymphocytes in vitro by using the T-cell cloning technique and selection of mutant cell clones in medium containing thioguanine. Cells treated with 1.2-2.4 mM Aa for 24 hr or 0.2-0.6 mM Aa for 48 hr showed a dose-dependent decrease of cell survival and a 3- to 16-fold increase of the mutant frequency. The inverse relationship between cell survival and mutant frequency was linear down to a relative survival of 15%, and showed a similar slope in the 24-hr and 48-hr treatment experiments. Forty-one mutant T-cell clones derived from cultures treated with 1.2 or 2.4 mM Aa and 15 from untreated controls were expanded for DNA extraction and Southern blot analysis to study deletion mutation using a full length hprt cDNA probe, and clonal identity on the basis of T-cell receptor rearrangements. In the culture with a 16-fold increase of mutant frequency, 4 out of 10 independent mutants (40%) showed partial deletions extending beyond the 3' coding sequences of the hprt gene. Two of 22 independent mutants derived from the other treated cultures with at most a 6-fold increase of mutant frequency, and 1 of 11 independent control clones showed rearrangement of the hprt gene, none of which affected the 3'-end of the hprt gene. These results show that Aa is capable of inducing gene mutation at the hprt locus in human cells, and suggest that deletion mutation affecting the 3'-end of the gene may be a major type of Aa-induced mutation of this locus.

6 Final Report on the Safety Assessment of Morpholine

Morpholine is used in cosmetic products as a surfactant and emulsifier at concentrations up to 5%.Morpholine is metabolized in guinea pigs but was not significantly metabolized in rats, dogs, or rabbits. Dermal LD50s in rabbits ranged between 0.3 and 1.2 g/kg. The oral LD50s in rats were between 1.1 and 1.6 g/kg; in guinea pigs the oral LD50was 0.9 g/kg.In studies of acute and short-term dermal toxicity Morpholine as an undiluted and unneutralized solution or as a diluted and unneutralized solution applied daily to the skin of guinea pigs and rabbits, respectively, caused the deaths of the test animals within 2 weeks. In both cases, the skin was necrotic.Unneutralized solutions of Morpholine caused severe corneal necrosis, but upon neutralization Morpholine was not injurious to rabbit eyes.In short-term inhalation studies (in rats) with varying concentrations of Morpholine, the effects observed included irritation of the mucous membranes and an increased respiratory rate. Chronic inhalation studies of Morpholine in rats and guinea pigs reported changes in the nervous system activity and arterial and peripheral blood pressure. At high concentrations Morpholine produced swelling of the alveolar cells and atrophy of lymphoid elements in the spleen. At lower concentrations a decrease in the size of the lymph nodules in the spleen was noted.Morpholine was a weak positive mutagen in L5178 mouse lymphoma assay, in BALB/3T3 malignant cell transformation and fibroblast transformation assays, and in sister chromatid exchange assays, but was negative in the Ames test with and without metabolic activation. At nontoxic doses Morpholine did not increase the rate of DNA repair in rat hepatocytes. Results of other mutagenic assays varied according to the system used.Nitrosation of Morpholine produces N-nitrosomorpholine, which has been mutagenic in a variety of test systems. Simultaneous exposure of laboratory animals to Morpholine and nitrites has caused a number of different cancers. A carcinogenic response was produced in rats in a long-term feeding study of Morpholine in which nitrites were present in the diet.In humans, Morpholine is absorbed and is considered to be a skin and eye irritant, as well as a skin sensitizer. A formulation containing 1% Morpholine indicated that the ingredient was neither an irritant nor sensitizer.Morpholine is not considered to be an animal carcinogen. It reacts easily with nitrosating agents, resulting in the formation of N-nitrosomorpholine. Under conditions of use, it is highly unlikely that Morpholine is totally free of carcinogenic nitrosoamines. Without quantitative data regarding the formation of N-nitrosomorpholine under conditions of use, it cannot be concluded that Morpholine is safe for use in cosmetic products.

Leukotriene (LT) receptor antagonists. Heterocycle-linked tetrazoles and carboxylic acids. LY203647

LY171883, 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl] ethanone, is an orally active antagonist of LTD4- and LTE4-induced responses in a variety of test systems. We prepared a new series of LT antagonists based on a proposed model of LY171883 binding to the LTE4 receptor in which the n-propyl and tetrazole moieties of LY171883 occupy those parts of the receptor to which the C1-C5 chain and the cysteinyl carboxyl of LTE4 bind, respectively. The new compounds have an acidic function corresponding to the glycine carboxyl of LTD4 linked through a heterocyclic group which is proposed to bind to the LTD4 receptor where the cysteinyl glycine amide bond of LTD4 binds. LY203647, 1-[2-hydroxy-3-propyl-4-[4-[2-[4-(1H-tetrazol-5-yl)butyl]-2H- tetrazol-5-yl]butoxy]phenyl] ethanone, showed good LTD4 antagonist activity with a suitable pharmacologic and toxicologic profile and has been chosen for clinical evaluation.

Genetic effects of ethanol

Alcoholics have a higher frequency of chromosomal aberrations and sister-chromatid exchanges (SCEs) in their peripheral lymphocytes. In human and mammalian cells in vitro, ethanol generally does not induce genetic damage, but it induces SCEs in the presence of an exogenous metabolic system. In human lymphocytes in vitro, ethanol induces SCEs in the presence of alcohol dehydrogenase. In animals in vivo, ethanol induces a variety of genetic effects, including SCEs, micronuclei, dominant lethal mutations and aneuploidy in mouse eggs. There is some indication that ethanol may lead to genetic damage in sperm. In bacteria, ethanol is at best marginally active. Ethanol leads to anomalous chromosome segregation in Aspergillus, to mutations in yeast, to chromosomal aberrations and SCEs in plant root tips and to disturbances of meiosis and micronuclei in tetrads in Zea and Tradescantia respectively. The first metabolite of ethanol, acetaldehyde is mutagenic in a variety of test systems. The mutagenic activity of acetaldehyde in bacteria is questionable, but there is no doubt of its mutagenic activity in a variety of eukaryotic test systems in vitro as well as in vivo.

Mutagenicity of extracts of brown and black masheri, pyrolysed products of tobacco using short-term tests

Black and brown varieties of masheri, which are pyrolysed tobacco products were analysed for their mutagenic potentials using a battery of test systems. Both materials were found to be mutagenic in Salmonella typhimurium strain TA98 with metabolic activation and also in V79 Chinese Hamster cells producing 8-azaguanine resistant mutations. Both varieties were found to induce statistically significant increases in micronuclei formation as compared to those produced in the solvent controls. Both varieties induced structural chromosomal aberrations in bone marrow cells of mice. Our data suggest that masheri is a potent mutagen in a variety of test systems and is likely to have a mutagenic potential vis à vis humans.

Pharmacology of bevantolol hydrochloride

Bevantolol is a cardioselective, β-adrenoreceptor antagonist, devoid of intrinsic β sympathomimetic activity and with weak membrane-stabilizing and local anesthetic properties. The 3,4-dimethoxyphenylethylamino moiety, substituted on the side chain amine function, confers cardioselectivity, which has been confirmed by a number of experiments. In vitro, bevantolol demonstrated greater antagonism of atrial than tracheal responses to isoproterenol. In vivo, bevantolol preferentially inhibited isoproterenol-induced tachycardias in conscious and anesthetized dogs, compared with the nonselective agent propranolol. Conversely, its effect on blood pressure after isoproterenol was minimal compared with propranolol, reflecting its muted effect on β 2 peripheral receptors. A functional difference between bevantolol and propranolol was demonstrated in histamine-challenged guinea pigs. Bevantolol had little effect on the antiasthmatic effect of isoproterenol, whereas propranolol blocked it totally. Bevantolol's lack of intrinsic sympathomimetic activity was demonstrated in normal and reserpinized dogs, where it was devoid of intrinsic sympathomimetic activity at doses up to 10 mg/kg. Similarly, intravenous doses of 10 mg/kg had to be administered before direct myocardial depression occurred in the reserpinized animals. Metabolite 3, which is excreted in trace amounts in human urine, demonstrates intrinsic sympathomimetic activity when administered in pharmacologic doses to dogs; however, any clinical relevance remains to be established. Several laboratories have demonstrated that bevantolol interacts at α-adrenergic sites. These data require further investigation. The dose-related antihypertensive effect of bevantolol has been demonstrated in spontaneously hypertensive and 2 kidney, 1 clip renal hypertensive rats. Animal experiments also suggest that bevantolol may be useful in angina: It caused a favorable redistribution of blood flow in dogs in which the left circumflex artery had been stenosed. The redistributed flow improved contractile function in the ischemic myocardium. The pharmacologic profile of bevantolol shows it to be an interesting βadrenoreceptor antagonist in a variety of test systems.

Synthesis and antitumor activity of cyclophosphamide analogues. 4. Preparation, kinetic studies, and anticancer screening of "phenylketophosphamide" and similar compounds related to the cyclophosphamide metabolite aldophosphamide.

Phenyl ketone phosphorodiamidates [C6H5C(O)CH2CH2OP(O)NHR1NR2R3] were synthesized in conjunction with an ongoing investigation into the effects of substituents on the dynamical solution chemistry of the metabolites of cyclophosphamide (1a). In contrast to aldophosphamide (3a), which readily interconverts with its cyclic isomer 4-hydroxycyclophosphamide (2a), phenylketophosphamide (14a: R1 = H, R2 = R3 = CH2CH2Cl) exhibited an apparent "resistance" toward an intramolecular addition reaction such that 4-hydroxy-4-phenylcyclophosphamide (13a) could not be detected either spectroscopically (31P or 13C NMR) or chemically (NaCN trapping experiment). Control studies that compared the relative reactivities of 14a and methylketophosphamide [20: CH3C(O)CH2CH2OP(O)NH2N-(CH2CH2Cl)2] revealed that the factors that modulate the ring closure/opening reactions were not peculiar to the phenyl group; however, differences between phenyl and methyl profoundly influenced the rates of fragmentation of 14a and 20. 31P NMR spectroscopy was used to determine the rates at which each compound generated a cytotoxic alkylating agent. Under a standard set of reaction conditions [1 M lutidine buffer with added Me2SO (8:2), pH 7.4, 37 degrees C], the half-lives of 2a/3a, 14a, phenylketoifosfamide (14b: R1 = R2 = CH2CH2Cl, R3 = H), phenylketotrofosfamide (14c: R1 = R2 = R3 = CH2CH2Cl), and 20 were 72, 66, 63, 56, and 173 min, respectively. Analogues 14a and 14b exhibited good anticancer activity against a variety of test systems.

Characterization of bacterial mutagenicity mediated by 13-hydroxy- ent-kaurenoic acid (steviol) and several structurally-related derivatives and evaluation of potential to induce glutathione S-transferase in mice

Stevioside is a sweet-tasting diterpene glycoside that is derived from Stevia rebaudiana (Bertoni) Bertoni (Compositae). It is used commercially in Japan and other parts of the world as a sucrose substitute. Whereas stevioside demonstrates no mutagenic activity in a variety of test systems, the aglycone, steviol (13-hydroxy- ent-kaurenoic acid), is mutagenic toward Salmonella typhimurium strain TM677 in the presence of a metabolic activating system derived from the liver of Aroclor 1254-pretreated rats. The required activating component is localized in the microsomal fraction of rat liver, suggestive of a cytochrome P-450-mediated reaction. Partially purified epoxide hydrolase does not inhibit steviol-induced mutagenicity, indicating that an active metabolite is not an epoxide that serves as a substrate for this enzyme preparation. The 13-hydroxy group of steviol is required for the expression of mutagenicity since ent-kaurenoic acid is nonmutagenic, and acetylation of steviol at this position negates mutagenicity. Similarly, diterpenes bearing a strong structural resemblance to steviol, cafestol and kahweol, were found to demonstrate no mutagenic activity toward Salmonella typhimurium TM677, as were their respective acetates and palmitic acid esters. Conversely, 19- O-β- d-glucopyranosyl steviol, a potential hydrolysis product of stevioside, is mutagenic and bactericidal in the presence of a metabolic activating system. Additionally, in contrast to the nonmutagenic diterpenes cafestol and kahweol that are effective as inducers of glutathione S-transferase activity, evaluation by administration to mice proved steviol, isosteviol and various steviol glycosides to be inactive in this process. Thus, structural differences among these naturally occurring and semi-synthetic diterpenes appear to impart major differences in biological activity that may relate to human health upon dietary ingestion.

Assessment of the impact of the emission of certain organochlorine compounds on the aquatic environment: Part II: Allylchloride, 1,3- and 2,3-dichloropropene

On the basis of the available scientific information the impact of pollution of the aquatic environment by the chloropropenes: allyl chloride, and 1,3- and 2,3-dichloropropene has been evaluated. These chloropropenes have low persistence in the aquatic environment and their bioaccumulation is negligible. The available data indicate moderate aquatic toxicity, but further data obtained under well-controlled experimental conditions are needed to substantiate this conclusion. The acute mammalian toxicity of the chloropropenes is moderate. These compounds possess mutagenic activity in a variety of test systems. Weak carcinogenic effects have been observed in experimental animals.

On the genotoxicity of the pesticides Endodan and Kilacar in 6 different test systems

Two pesticides, the fungicide Endodan (ethylene thiuram monosulphide) and the insecticide-acaricide Kilacar (bis(parachlorophenyl)cyclopropyl methanol), produced or used in the neighbouring countries of Bulgaria and Greece were investigated in a coordinated research programme for their genotoxic effects in a variety of test systems. This included the Ames test, Aspergillus nidulans for mitotic segregation, in vitro human lymphocyte cell cultures for SCE and chromosomal aberrations, in vivo bone marrow cells in hamsters and rats and the dominant lethal test in rats. The genotoxicity of Endodan was found to range from negative to slightly positive in different test systems. At concentrations of 7.5 and 12.0 μg/plate together with S9 mix it induced base-pair substitutions in the TA100 strain of Salmonella typhimurium at a rather low level. At a dose of 93 mg/kg b.w. it also caused chromosomal aberrations in acutely treated hamster bone marrow cells. A significant increase of SCE was also found in human lymphocyte cultures at a concentration of 20.0 μg/ml. Endodan was found to be negative in A. nidulans for somatic segregation, lymphocyte cultures for chromosomal aberrations and mitotic activity and in rats for dominant lethals and chromosomal aberrations. Kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at concentrations of 2.5 and 5.0 μg/plate together with S9 mix. At concentrations of 1.0, 1.5 and 2 μg/ml Kilacar increased the number of mitotic segregants in A. nidulans by 160%, 220% and 156% respectively over the control. In Syrian hamster bone marrow cells after acute administration at concentrations of 0, 40, 80 and 160 mg/kg, the MI was 5.50, 4.30, 3.10 and 1.30 respectively, and an increase in chromosomal aberrations of about 300% over the control was observed with a concentration of 80 mg/kg. In human lymphocytes no significant changes were observed in either MI or SCE. In the dominant lethal test after chronic treatment of male rats at doses of 5.1, 10.2 and 102.0 mg/kg b.w. no significant mutagenic effect was found although a decrease was shown in the percentage of females with implants mated with treated males in the first week.

Relative agonist potency as a means of differentiating alpha-adrenoceptors and alpha-adrenergic mechanisms.

Relative potencies (ED50), affinities (assessed as dissociation constants) and efficacies (i.e. ability to activate the receptor) of agonists are useful in subclassifying and differentiating alpha-adrenoceptors. The postsynaptic alpha-adrenoceptor of rat aorta is of the alpha 1-subtype, but may differ from the alpha 1-adrenoceptor of rabbit and guinea pig aortae, based on comparison of relative potencies of selected agonists. By evaluating the relative potency of agonists, qualitative and quantitative species differences between alpha 1-adrenoceptors in rat and guinea pig are observed in a variety of test systems in vivo and in vitro. By comparing the relative potencies of aromatic hydroxyl-substituted phenethylamines and imidazolines at alpha 1-adrenoceptors in guinea pig aorta, differences in the ability of these two classes of alpha-adrenoceptor agonists to bind to, and subsequently activate, alpha 1-adrenoceptors have been observed. Evaluating the relative potencies of agonists, when used in conjunction with other techniques, provides a valuable method for classifying alpha-adrenoceptors and for studying alpha-adrenergic mechanisms.

Induction of cytolytic activity by anti-T3 monoclonal antibody. Activation of alloimmune memory cells and natural killer cells from normal and immunodeficient individuals.

The ability of the monoclonal antibody directed at the T3 antigen (anti-T3) to induce cytolytic activity was investigated since several agents that can activate T cells induce the acquisition of cytolytic activity in a variety of test systems. Pretreatment of human alloimmune memory cells, generated in primary long-term mixed lymphocyte cultures, with anti-T3 resulted in the induction of statistically significant specific secondary cytolytic activity and natural killer (NK) cell-like activity. No such augmentation or induction of cytolytic activity was found with anti-T3 pretreatment when syngeneic cells or inappropriate allogeneic cells (HLA-A, B antigens different from the original priming stimulus) were used as target cells and pretreatment of memory cells with anti-T4 or anti-T8 did not induce cytolytic activity to allogeneic or syngeneic target cells. Differential effects were observed when anti-T3 was added to the cytotoxicity assay in which anti-T3 pretreated alloimmune memory cells were effectors. The addition of anti-T3 to the assay prior to the introduction of target cells resulted in 39 +/- 8% inhibition of specific secondary cytolytic activity and only 5 +/- 8% inhibition of NK cell activity. NK cell activity mediated by large granular lymphocyte-enriched fraction of peripheral blood mononuclear cells (PBM) obtained from normal individuals was significantly augmented by anti-T3 when NK-sensitive cell lines MOLT-4 or K-562 were used as target cells. This augmentation in NK cell activity was not associated with nonspecific cytotoxicity to syngeneic or allogeneic PBM, and anti-T3 failed to activate the LGL fraction depleted of T cells. The monoclonal antibodies, anti-T4 or anti-T8, did not increase NK cell activity. NK cell activity mediated by PBM from eight immunodeficient individuals (four with acquired immunodeficiency syndrome and four with renal allografts) was also significantly augmented by anti-T3 pretreatment. Our findings, in addition to providing a rationale for the frequent occurrence of re-rejection episodes in renal graft recipients treated with anti-T3, suggest that anti-T3 might be utilized to enhance the cytotoxic armamentarium of immunodeficient patients.

Inhibition of platelet thromboxane A 2 synthase activity by sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinymethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro , as well as in rhesus monkey platelets ex vivo . A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI 2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 at 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation,. Similar effects were obtained by oral administration of 1–5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxgenase inhibitors and equal to PGI 2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cylooxygenase inhibitors suggesting that its efficacy dependened in part of endogenous PGI 2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of patho[hysiological states.

Pulmonary toxicity of inhaled coal liquid aerosols (boiling range 230–450°C)

The biological activity of materials produced in the direct liquefaction of coal is being assessed by a variety of test systems. In this study, the pulmonary toxicity of process solvent (PS) from the solvent refined coal-I (SRC-I) process was determined by histamine aerosol challenge tests and pulmonary function and morphologic evaluations. Guinea pigs inhaled aerosols of PS (boiling range, 230 to 450°C) for 6 hr/day, 5 days/week, for up to 12 days in three different experiments. In the first experiment, 8-week-old animals inhaled 0 (controls), 0.15, or 0.60 mg/liter PS aerosols with a mass median aerodynamic diameter (MMAD) of 1.3 μm. Exposure to 0.15 mg/liter PS for 12 days resulted in depressed weight gain and marked hypersensitivity to inhaled histamine compared with sham-exposed control animals. Four of five animals exposed to 0.6 mg/liter PS died of respiratory failure during exposure. During the second experiment, 14-week-old animals inhaled 0 (controls) or 0.19 mg/liter PS (MMAD, 1.3 μm) for 1, 3, or 12 days. Hypersensitivity to aerosolized histamine occurred only after 12 days exposure to PS aerosols. At that time, morphologic lung evaluations showed mild to moderate pneumonitis and accumulation of exudate in bronchioles of PS-exposed animals. In the third experiment, pulmonary function evaluations were conducted on 4-week-old animals exposed to 0 (controls) or 0.19 mg/liter PS for 8 days. Functional changes measured in these animals (compared to controls) included increased gas trapping at low lung volumes, decreased quasi-static compliance, and decreased diffusion capacity of the lung for carbon monoxide. These studies showed that measurable changes in lung function were produced in guinea pigs after 8 to 12 days exposure to 0.15 or 0.19 mg/liter PS and that exposure to PS affected weight gain only in younger animals (4 and 8 weeks old) but not in 14-week-old animals.

Report of the Federal Panel on Formaldehyde.

The Federal Panel on Formaldehyde concluded that definitive experiments exist which demonstrate the mutagenicity and carcinogenicity of formaldehyde under laboratory conditions. Formaldehyde induces both gene mutations and chromosomal aberrations in a variety of test systems. Inhalation of formaldehyde causes cancer of the nose in rats. The concentrations of formaldehyde in inhaled air that caused nasal cancer in Fisher 344 rats are within the same order of magnitude as those to which humans may be exposed. The data presently available do not permit a direct assessment of the carcinogenicity of formaldehyde to man. Epidemiologic studies on exposed human populations are in progress and may further clarify the situation. Other experimental and human studies on toxic effects such as teratogenicity and reproductive disorders are as yet inadequate for a health risk assessment. The CIIT 24 month study on animal carcinogenicity has not yet been completely evaluated. Additional data are expected on the effects of prolonged exposure to lower doses of formaldehyde and on the possible carcinogenicity of formaldehyde in the mouse. The panel recommends that, for a comprehensive health risk assessment, further experiments be conducted on the effects of other modes of exposure (ingestion and skin penetration), the effects in humans, and on the pharmacokinetics of formaldehyde in man and animals and the possible role for formaldehyde in reproductive and chronic respiratory disorders. It is the conclusion of the panel that formaldehyde should be presumed to pose a carcinogenic risk to humans.

Interaction between activated nordihydroguaiaretic acid and deoxyribonucleic acid

Nordihydroguaiaretic acid (NDGA) is a natural product of the lignan family that has been shown to possess antimicrobial and antineoplastic properties in a variety of test systems. NDGA was observed by u.v.-visible spectroscopy to be unstable in an aqueous environment; however, by these same techniques NDGA was shown to be stable in the presence of mercaptoethanol. It is suggested that an activated NDGA is an intermediate in the O 2-mediated oxidation of NDGA and that the activated NDGA forms a stable complex when reacted with duplex DNA. This DNA-activated NDGA complex was detected and studied by both fluorescence spectroscopy and CsCl density gradient techniques. The addition of DNA quenched the fluorescence of activated NDGA in a concentration-dependent fashion. Furthermore, the exposure of activated NDGA lowered the buoyant density of DNA, also, in a concentration-dependent manner. Since mononucleotides did not quench the fluorescence of activated NDGA, and heat-denatured DNA was less effective than fully duplex DNA in its ability to interact with activated NDGA, the duplex structure of DNA was determined to be important in the complex formation. Whereas activated NDGA bound to both poly dG · poly dC and poly (dA · dT), there appeared, by one method of analysis, to be a preference for poly dG · poly dC. Activated NDGA-DNA complex was stable to dialysis, but dissociated in the presence of Sarkosyl. No changes in the viscosity or melting temperature of DNA was induced by the addition of activated NDGA. These data suggest a mechanism in which the activated NDGA was bound to the more apolar regions of duplex DNA that are located in either the major and/or minor grooves.

Cytotoxic T lymphocytes specific for I region determinants do not require interactions with H-2K or D gene products

Gene products coded for by the major hisocompatibility complex (MHC) can serve as target antigens for cytotoxic T lymphocytes (CTL) (1). A variety of test systems are available which have yielded information consistently reinforcing the importance of this complex of genes in the generation and effector phases of the cytotoxic immune response. Originally, it was shown that allogeneically-induced CTL had specificity primarily for the products of the K and D loci of the mouse H-2 complex (2). More recently this has also been found to be the case for xenogeneic immunizations (3,4). Additional examples of T cell-mediated lysis have been reported involving viral-infected or chemically- modified syngeneic stimulating and target cells in which homology at H-2K or H-2D was required between the responding and target cells for appreciable lysis to occur (5-7). Moreover, CTL specific for minor histocompatability antigens are able to lyse only target cells bearing these membrane antigens and sharing a common H-2K or H2-D gene product with the effector (8,9). Two hypotheses have been proposed to explain the requirement for H-2 identity between effector and targets in these systems. CTL may recognize new antigenic determinants created by the interaction of the modifier with syngeneic K and D gene products. Alternately, a dual recognition system my exist, requiring an antigen-specific receptor as well as a second receptor with specificity for homologous H-2K or H-2D determinants (5). Neither model can be excluded at this time. The I region also contains genes coding for histocompatibility loci since animals differing at the I-A or I-C regions of the H-2 complex reject skin grafts (10-12), though less rapidly than mice differing at the H-2K or H-2D regions, Also CTL can be generated to I region determinants but less efficiently than CTL specific for H-2K or H-2D gene products (12-14). The question can therefore be raised, whether the I region minor histocompatibility loci function independently from the H-2K or H-2D loci or whether I region-specific cytolysis requires the participation of H-2K or H-2D gene products of the target cell. This communication illustrates the generation of CTL showing specificity for I region determinants in primary mixed lymphocyte cultures. Further, we demonstrate by genetic analysis and byt eh use of speficit alloantisera that CTL directed to Ia determinants (a) do not see these antigens as modifications of H-2K or H-2D gene products but as independent gene products coded for by the I region, and (b) they do not require interaction with target cells bearing the same H-2K or H-2D gene product as the effect CTL.

Microcomputer-Controlled Precision Pneumatic Pressure Generator

An increasing variety of avionics require precision pneumatic pressure stimuli during organization and intermediate maintenance. The accuracy and long-term stability requirements approach the best mercurial standards. Pressure stimuli must be coordinated and varied at precise rates that are beyond the frequency response of mercury columns and many electromechanical sensors. Test systems are frequently required to simultaneously monitor and evaluate unit under test (UUT) response and generate pneumatic stimuli. In addition, the environment is frequently hostile, maintainability is paramount, performance must always be known and warmup time must be nil. Comprehensive trade studies, tests, and analyses were performed. This initial work indicated a new pressure generator should be a "smart" instrument and should use a pressure sensor not a density sensor. Current field and production experience indicates performance objectives have been exceeded and this new type of precision pressure generator can be readily incorporated in a variety of test systems or be used "stand-alone". This paper discusses the design implementation, features, and characteristics of this microcomputer controlled precision pneumatic pressure generator-a new "smart" test instrument.

Quantitative analysis of a direct writing method for dye dilution curves.

A method for continuous inscription of dye dilution curves was critically analyzed by comparing calculated flow rates with measured flows in a variety of test systems. In the same model system, flows calculated from injection of dye into water tended to be less than measured values, while rates of flow estimated from dye injection into blood were usually greater than known values. All measured flows were determined by stopwatch and calibrated cylinder. Right heart bypass in the dog permitted rigid analysis of the technique under relatively physiological conditions. With accurately measured rates of flow delivered into the pulmonary artery, the mean error of estimated values was less than +5%. Submitted on May 21, 1956