Assay of cytotoxicity and mutagenicity of alkylating agents by using Neurospora spheroplasts

A system relying on the use of Neurospora crassa spheroplasts has been developed for the assay of cytotoxicity and mutagenicity of chemical compounds. Mutagenicity was assayed by using reversion of alleles in the am gene selected to recognize certain specified transitions and also undefined point mutations. Cytotoxicity was quantified by measuring a 'cytotoxicity parameter', m, which appears in the exponential function that fits the survival/dose curve for each compound (under standard incubation conditions). Of the compounds tested, nitrogen mustard (Cl(CH2)2 NMe(CH2)2Cl) was cytotoxic and non-mutagenic, and ethyl nitrosourea was highly mutagenic but not cytotoxic. Of the remaining compounds tested, methyl nitrosourea, butadiene diepoxide, and cis platin (cis diammonia platinum II chloride) all showed comparable mutagenicity per survivor, although the values of m covered a wide range. Differences were found between the different compounds in the effects of the uvs-2 allele on survival and on the preponderance of G to A transitions.     

Prolonged lactational infertility in adolescent rhesus monkeys

The present study examined the effects of first pregnancy and nursing behavior on postpartum infertility in seasonally breeding rhesus monkeys to assess whether prolonged lactational infertility observed in adolescent mothers is due to a particular pattern of nursing or to decrements in body growth rates. After a successful first pregnancy, a significant percentage of the lactating adolescent mothers (57.1%; n = 8) failed to exhibit an ovulation with normal luteal phase during the subsequent breeding season. In contrast, the remaining lactating adolescents (42.9%, n = 6) and all of the adult mothers (100%, n = 6) exhibited ovulations with a normal luteal phase. Age alone was not the critical variable, since all nonlactating adolescents exhibited ovulations with normal luteal phase parameters in the subsequent breeding season. The luteal phase abnormalities exhibited by the subset of lactating adolescent females were characterized by an inadequate luteal phase (ILP) and by significantly lower serum levels of progesterone, estradiol, and bioactive luteinizing hormone. The occurrence of these ILP ovulations was associated with more frequent nursing bouts prior to ovulation and during the subsequent luteal phase. In contrast, nursing patterns for adult females who had ovulations with normal luteal phases were more similar to those of the infertile lactating adolescents exhibiting significantly longer and more frequent nursing bouts, suggesting that fully adult females may be less sensitive to the inhibitory aspects of a suckling stimulus. Differences in luteal phase function among lactating adolescents were not related to differential rates of ponderal or skeletal growth. A still-developing neuroendocrine system may thus render a significant proportion of adolescent females more sensitive to suckling-induced suppression of gonadotropin secretion.     

Uterine asynchrony: a cause of embryonic loss

During early gestation, hormonal events associated with corpora lutea formation and embryonic synthesis of proteins, prostaglandins, and steroids result in synthesis and release of endometrial secretory products into the uterine lumen. The embryo, inherently and in response to secretory products of the uterus, develops and grows. However, considerable embryonic mortality occurs when uterine secretions become altered in such a manner that they are asynchronous to the developing embryo. Factors that advance or retard development of the uterus and embryo have been utilized to document utero-embryonic asynchrony, and it has been observed that the uterus will not "wait" for embryos to become synchronous. However, the reverse is possible: embryonic development can be accelerated or decelerated. Furthermore within the uterus, localized areas might also exist that favor development of some embryos at the expense of others. This review will consider causes of utero-embryonic asynchrony and offer models of embryonic loss associated with an asynchronous environment in cattle, sheep, and swine.     

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.     

Diaphragm metabolism during supramaximal phrenic nerve stimulation

The metabolic changes accompanying diaphragm fatigue caused by supramaximal stimulation of the phrenic nerves are incompletely described. In particular, we wished to determine whether the occurrence of anaerobic metabolism correlated with fatigue as defined by decline in force generation. In 10 anesthetized mechanically ventilated mongrel dogs we measured arterial pressure, transdiaphragmatic pressure (Pdi), phrenic arterial flow (Qdi-Doppler flow probe), arterial and phrenic venous blood gases, and lactate levels. From these we derived indexes of diaphragm O2 consumption (VO2) and lactate production. Bilateral phrenic nerve pacing was carried out (50 Hz, duty cycle 0.4, 24 contractions/min) for two 15-min pacing periods separated by a 45-min rest period. Over each pacing period Pdi decreased from approximately 16 to approximately 10 cmH2O (P less than 0.01, no significant difference between periods). Initially, during pacing, Qdi and VO2 each increased fivefold over prepacing base line. Qdi remained elevated at this level whereas VO2 decreased over the pacing period by approximately 25%. Hence, the change in VO2 over the pacing period was due primarily to changes in O2 extraction. During the first pacing period lactate production was observed early and declined throughout the pacing period. No lactate production was observed during the second pacing period, although Pdi, VO2, and Qdi responses were the same for both pacing periods. Phrenic venous PO2 remained greater than 30 Torr throughout both pacing periods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial characterization of T cell components related to defined VH (VT) markers

Certain antigen-binding surface molecules and factors of T cells possess serological determinants related to immunoglobulin (Ig)-heavy-chain-variable regions (VH). We obtained sufficient quantities (greater than 100 micrograms) of homogenous VH-related T-cell molecules (VTM) for biochemical studies from normal murine thymocytes and by growing large quantities of monoclonal T-cell leukemia lines expressing the determinants. A solid phase immune adsorbent prepared from the IgG fraction of rabbit anti-IgT serum was used to isolate VTM from formic acid-solubilized T cells. The VTM from murine thymocytes and T-cell lines had Mr of 65,000-68,000. The VTM from distinct cell lines differ by isoelectric focusing and resolution of tryptic peptides indicating clonal restriction. VTM lack conventional light- or heavy-chain-constant region determinants but cross-react with antisera directed against defined VHa allotypes and JH peptides. The detection of a cross-reaction with a synthetic JH peptide is consistent with recently published data identifying JH-related sequences in putative T-cell receptor genes. The amino acid compositions of the VTM were distinct from those of mammalian Ig, major histocompatibility complex (MHC) antigens, and viral glycoproteins, but significant similarities occur with Ig V regions or heavy chains of primitive vertebrates. The results indicate that the VH-bearing T-cell products are not classical Ig, but bear limited VH-cross-reactive determinants.     

Conformations of complexes derived from the interactions of two stereoisomeric bay-region 5-methylchrysene diol epoxides with DNA

The reaction mechanisms of two isomeric bay-region diol epoxides of 5-methylchrysene (trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II) with double-stranded DNA in aqueous solutions were studied utilizing kinetic flow dichroism and fluorescence techniques. As in the case of the previously studied benzo(a)pyrene-7,8-diol-9,10-oxide isomers (BaPDE), both DE-I and DE-II rapidly form intercalation-type complexes (association constants K = 2700 and 1500 M-1 respectively in a neutral 5mM phosphate solution). The physically bound diol epoxide molecules react on time scales of minutes to form predominantly tetraols; a greater fraction (6 +/- 1%) of DE-I than of DE-II (2-3%) molecules react with the DNA to form covalent products. The DE-II isomer is characterized by a greater reactivity than DE-I, and the rates of reaction are markedly accelerated in the presence of DNA in both cases. The linear dichroism spectra of the covalent adducts reveal that the conformations of both types of adducts are similar, with the long axes of the phenanthrenyl chromophores tilted, on the average, at angles of 38-52 degrees with respect to the average orientations of the transition moments (at 260 nm) of the DNA bases. The conformations of the covalently bound DE-I and DE-II molecules resemble those observed in the case of the highly tumorigenic (+) enantiomer of anti-BaPDE.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of indomethacin on the activation and effector function of suppressor cells from tumor-bearing mice

Summary The spleens of mice with large M-1 fibrosarcomas contain two populations of suppressor cells with the properties of macrophages and T cells. In this study, we tested the effect of indomethacin on suppressor cell activation and effector function. Neither the activation nor the effector function of the suppressor macrophages was inhibited by indomethacin, and the activity of suppressor macrophages correlated with the tumor size. In contrast, the treatment of tumor-bearing mice with indomethacin from the day of injection of tumor cells completely blocked the in vivo activation of suppressor T cells. Indomethacin did not, however, depress suppressor T cell activity if mice were treated only during the third week of tumor growth. The effector function of the suppressor T cells, as assessed in mixing assays, was partially blocked by indomethacin, while selective suppression by low-molecular-weight factors was completely blocked if indomethacin was present in the cultures. Furthermore, the in vitro activation of suppressor cells by soluble factors secreted by tumor-bearer spleen cells was completely blocked by indomethacin, and this inhibition was reversed by prostaglandin E₁. These data are consistent with the hypothesis that prostaglandins are involved in the activation, but not the effector function, of tumor-activated suppressor T cells.     

Linkage of a polymorphic marker for the type III collagen gene (COL3A1) to atypical autosomal dominant Ehlers-Danlos syndrome type IV in a large Belgian pedigree

Summary We have examined a large family in which eleven members have a form of autosomal dominant Ehlers-Danlos syndrome type IV. Analysis of fibroblast cultures from affected individuals showed a partial deficiency of type III collagen production. The protein produced was, however, normal in all aspects examined. Using a restriction site polymorphism associated with the structural gene for human type III collagen (COL3A1), we have found tight linkage between the low frequency polymorphic allele and the clinical expression of the disease (lod=3.86 at =0), identifying the type III collagen gene as the disease locus.     

Bacteriostatic and bactericidal modes of action of bis(tributyltin)oxide on Legionella pneumophila.

The modes of action of bis(tributyltin)oxide (TBTO) doses between 1 X 10(4) and 6 X 10(7) molecules per cell on a single environmental isolate of Legionella pneumophila were studied by monitoring the following parameters: (i) growth, (ii) cell viability, (iii) 14C-amino acid incorporation, (iv) 14CO2 production from 14C-amino acids, (v) [3H]uridine incorporation, (vi) [3H]thymidine incorporation, (vii) oxygen consumption, (viii) cellular ATP levels, and (ix) adenylate energy charge. The amount of TBTO associated with the cells in these laboratory cultures was also compared with that remaining in the suspending medium. Most of the TBTO (68 to 88%) was found to be associated with the cells. This result explained why the cellular responses which were measured did not correlate with the TBTO concentration, but rather with the dose of TBTO to which the cells were exposed. At the lower TBTO doses tested (10(4) to 10(7) molecules per cell) a log-normal relationship was observed between the reduction in growth rate and the TBTO concentration. At intermediate TBTO doses (ca. 10(7) molecules per cell) growth stasis occurred, with nearly 100% of the cells in these cultures remaining viable for at least 5 h after treatment. The cellular function which seemed to be primarily affected at these levels of TBTO was the energy conversion mechanism, since the decline in the rates of CO2 production, oxygen consumption, and macromolecular synthesis was preceded by an immediate (within 1 min) drop in the intracellular levels of ATP and the adenylate energy charge. At the higher TBTO doses greater than 10(7) molecules per cell) an initial, precipitous, drop in the number of viable cells was observed, which was followed by a further exponential reduction of viable cells in the treated culture. This dramatic increase in bactericidal activity with a slight increase in the TBTO dose indicated that the modes of bacteriostatic and bactericidal action of TBTO were different.