The radioracemization of amino acids by ionizing radiation: Geochemical and cosmochemical implications

A number of optically active amino acids, both in the solid state and as sodium or hydrochloride salts in aqueous solution, have been exposed to ionizing radiation from a 3000 Ci⁶⁰Co -ray source to see if radioracemization might accompany their well-known radiolysis. -Ray doses causing 55–68% radiolysis of solid amino acids typically engendered 2–5% racemization, while aqueous solutions of the sodium salts of amino acids which underwent 53–66% radiolysis showed 5–11% racemization. Amino acid hydrochloride salts in aqueous solution, on the other hand, showed little or no radioracemization accompanying their radiolysis. Both radiolysis, and radioracemization were roughly proportional to -ray dose in the range studied (1–36×10⁶ rads). Mechanisms for the radioracemization of amino acids in the solid state and as aqueous sodium salts are discussed, and the absence of radioracemization for aqueous hydrochloride salts is rationalized. Isovaline, a non-protein amino acid which has been isolated from the Murchison meteorite, contains no -hydrogen atom and is therefore incapable of racemizationvia the chemical mechanisms by which ordinary amino acids racemize. Nevertheless, isovaline suffers radioracemization in the solid state to an extent comparable to that shown by ordinary amino acids, as do its sodium and hydrochloride salts in the solid state. The sodium salt of isovaline in aqueous solution, however, fails to racemize during its radiolysis. Several implicaitons of the newly described phenomenon of radiomization are pointed out for the fields of geochemistry and cosmochemistry.A portion of this research has been described previously at the 144th National Meeting of the American Association for the Advancement of Science, Washington D.C., Feb. 12–17, 1978, at the Fourth College Park Colloquium on Chemical Evolution, University of Maryland, College Park, Maryland, Oct. 18–20, 1978, and at the Carnegie Institution of Washington Conference: Advances in the Biogeochemistry of Amino Acids, Airlie House, Warrenton, Virginia, Oct. 29—Nov. 1, 1978.     

Uncoupling and isotope effects in γ-butyrobetaine hydroxylation

Replacement of unlabeled -butyrobetaine with -[2,3,4-²H₆]butyrobetaine has a profound effect on the stoichiometry between decarboxylation of 2-oxoglutarate and hydroxylation in the reaction catalyzed by human -butyrobetaine hydroxylase. The ratios between decarboxylation and hydroxylation are 1.16 with Unlabeled and 7.48 with deuterated -butyrobetaine as substrate. From these ratios an internal isotope effect of 41 has been calculated. DV in the overall reaction measured as 2- oxoglutarate decarboxylation is 2.5 and D_V/K is 1.0. For -butyrobetaine hydroxylase fromPseudomonas sp. AK 1, 2-oxoglutarate decarboxylation exceeds hydroxylation with 10% when deuterated -butyrobetaine is used. No excess was found with unlabeled substrate and no internal isotope effect could be calculated. D_V for the bacterial enzyme is 6.     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-γ

We investigated the influence of the combined use of sizofiran, a-1,3-glucan and a recombinant interferon- (rIFN-) upon biological activities of peritoneal macrophages (M). The number of peritoneal M and the production of cytokines (interleukin-1, interferon- and tumor necrosis factor) was increased by the combined treatment. Fully activated peritoneal M based on the increased number of elongated pseudopods were observed by electromicroscope. Sizofiran seems to assure a sufficient supply of M to kill tumor cells in the peritoneal cavity and co-administered rIFN- seems to directly stimulate the accumulated M in addition to its direct cytotoxicity against tumor cells. This combination therapy may be a step to the prevention of the recurrence of gynecological malignancies including ovarian cancer, after a negative second-look laparotomy.Abbreviations rIFN- recombinant interferon- - IL-1 interleukin-1 - TNF tumor necrosis factor - SLL second look laparotomy     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

Comparative study of the γ-butyrolactone and pantolactone contents in cells and musts during vinification by three Saccharomyces cerevisiae races

Summary Changes in the -butyrolactone and pantolactone contents in yeast cells and musts during fermentation and subsequent flor veil formation of Sherry wines were studied. Saccharomyces cerevisiae race cerevisiae, S. cerevisiae race bayanus and S. cerevisiae race capensis were used. During the alcoholic fermentation (first 31 days), -butyrolactone contents in musts and yeast cells were similar for the three yeast races tested. In this period, pantolactone was excreted to the must by bayanus and capensis races, and it was not detected in cerevisiae race cells. During flor veil formation (31 to 134 days), bayanus and capensis races yield higher -butyrolactone and pantolactone contents than cerevisiae race in the wines. In the final wines, pantolactone contents were always lower than those of -butyrolactone.     

Molecular switch of F0F1-ATP synthase,G-protein,and other ATP-driven enzymes

Exchange-out of amide tritium from labeled -subunit of ₃₃ complex of F₀F₁-ATP synthase was not accelerated by ATP, suggesting that hemagglutinin-type transition of coiled-coil structure did not occur in -subunit. Local topology of nucleotide binding site and switch II region of G-protein resemble those of F₁- subunit and other proteins which catalyze ATP-triggered reactions. Probably, binding of nucleotide to F₀F₁-ATP synthase induces conformational change of the switch II-like region with transforming subunit structure from open to closed form and this transformation results in loss of hydrogen bonds with the subunit, thus enabling the subunit to move.     

The context of T-cell receptor gamma chain genes among wild mouse species

We have examined the context of mouse T-cell receptor gamma (Tcr ) chain variable (V ) and constant (C) genes among a panel of geographically isolated species of mice. Our Southern hybridization survey with C reveals that essentially three C genes are found among mouse species extending phylogenetically from inbred mice through the feral species Mus pahari. However, a V DNA probe detects three to nine V restriction fragment bands among the same group of mice. These results suggest that certain feral mice such as M. pahari, M. platythrix, and M. shortridgei have amplified numbers of V genes. Studies of individual mice from these particular species indicate the highly amplified V content is not the result of a catastrophic gene duplication or deletion event. We conclude that certain species of mice maintain increased content of V presumably for increased diversity in a Tcell response.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose ofγ-rays

The effect was studied of a low dose of-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.     

Tumor sensitivity to IFN-γ is required for successful antigen-specific immunotherapy of a transplantable mouse tumor model for HPV-transformed tumors

Purpose: Many human tumors lose responsiveness to IFN-, providing a possible mechanism for the tumor to avoid immune recognition and destruction. Here we investigate the importance of tumor responsiveness to IFN- in the successful immunotherapy of TC1 tumors that were immortalized with human papillomavirus proteins E6 and E7. Methods: To investigate the role of IFN- in vivo, we constructed a variant of TC1, TC1.mugR, that is unresponsive to IFN- due to overexpression of a dominant negative IFN- receptor. Results: Using recombinant Listeria monocytogenes that express HPV-16 E7 (Lm-LLO-E7) to stimulate an antitumor response, we demonstrate that sensitivity to IFN- is required for therapeutic efficacy in that Lm-LLO-E7 induces regression of TC1 tumors but not TC1.mugR. In addition, we show that tumor sensitivity to IFN- is not required for inhibition of tumor angiogenesis by Lm-LLO-E7 or for trafficking of CD4⁺ and CD8⁺ T cells to the tumor. However, it is required for penetration of lymphocytes into the tumor mass in vivo. Conclusions: Our findings identify a role for IFN- in immunity to TC1 tumors and show that loss of tumor responsiveness to IFN- poses a challenge to antigen-based immunotherapy.Mary E. Dominiecki and Gregory L. Beatty contributed equally to this work.     

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtCAL1 and AtCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtCA and AtCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtCAs. All plant species analyzed contain both AtCA and AtCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtCAL sequences show a deviation of functionally important active site residues with respect to CAs but could form active interfaces in the interaction with AtCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.     

Introgression of the Haynaldia villosa genome into γ-ray-induced asymmetric somatic hybrids of wheat

To study the effect of -ray treatment on donor and derived somatic hybrids, we carried out -ray donor treatment experiments with a wide range of -ray dosages and asymmetric somatic hybridization between protoplasts of wheat (Triticum aestivum L. Jinan 177) and protoplasts of Haynaldia villosa Schur. treated with different dosages of -rays (40, 60 and 80 Gy, respectively). We first screened the putative hybrids by isozyme analysis, followed by characterization of nuclear and organellar genome composition of the hybrids. Genomic in situ hybridization on mitotic metaphases demonstrated that the donor chromosome elimination in the hybrids increased with increased -ray dosage. Intergenomic chromosome recombination/translocations were observed in the hybrids from different dosages of -rays. PCR amplification of 5S rDNA spacer sequences showed that only some of the regenerated hybrid clones inherited donor 5S rDNA sequences, suggesting that the donor DNA was also eliminated randomly. Restriction fragment length polymorphism analysis using mitochondrion (mt) and chloroplast (cp) gene-specific probes showed that the hybrid calli contained mt genomes of both parents and the cp genome of only one of the parents. Recombinations between parental mt as well as cp genes were found in the hybrid clones. Furthermore, development of the hybrid clones was dependent on the -ray dosage used for the donor treatment. Regenerated plants were only obtained from fusion combinations of low (40 Gy) and intermediate (60 Gy) dose irradiation. The possible role and significance of -rays on the introgression of small segments of donor chromosomes to the receptor is discussed.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

γ-Tubulin is a component of the Spitzenkörper and centrosomes in hyphal-tip cells ofAllomyces macrogynus

Summary A monoclonal antibody was used to localize -tubulin in hyphal tip cells of the chytridiomycete fungusAllomyces macrogynus, and its distribution determined with standard epifluorescence and laser scanning confocal microscopy. The results demonstrate that -tubulin is a component of the Spitzenkörper and centrosomes. Immunoblot analysis of total soluble protein extracts separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified a single 56 kDa -tubulin-related polypeptide. Localization of -tubulin to the Spitzenkörper ofA. macrogynus provides evidence that the Spitzenkörper in this fungus functions as a microtubule-organizing center.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPB spindle pole body - YpSs yeast extract-inorganic phosphate-soluble starch     

Forskolin Modulates Acetylcholine Receptor Gating by Interacting with the Small Extracellular Loop Between the M2 and M3 Transmembrane Domains

1. Forskolin acts as an allosteric modulator of muscle-type nicotinic acetylcholine receptors. Receptors from mouse muscle and Torpedo electroplax demonstrate differential sensitivity to inhibition by forskolin. Previous work from this laboratory suggested that the subunit is responsible for this differential sensitivity.2. We have used a series of mouse/Torpedo species-chimeric subunits to further define the site of forskolin interaction with the subunit. Analysis of the patterns of forskolin inhibition of receptors containing mouse/Torpedo chimeric subunits along with the mouse , , and subunits suggests that forskolin interacts with the small extracellular domain that links the M2 and M3 transmembrane domains (the M2–M3 linker).3. We suggest that the M2–M3 linker domain plays an important role in the transduction of ligand binding to the conformational changes that result in channel opening.     

γ-Linolenic acid production from Spirulina platensis

Various Spirulina strains were assayed for their productivity of -linolenic acid (Lnn). Spirulina platensis ARM-346 was found to accumulate large amounts of Lnn. Urea as a nitrogen source was found to be most effective giving a yield of 13.5 mg Lnn/g dry cell mass. With increase in temperature the Lnn content was found to increase along with biomass. The optimum temperature for maximum Lnn and biomass production was found to be 35°C. The Lnn content was highest at 0.06% (w/v) NaCl and 0.07% (w/v) K₂HPO₄. Cells cultivated in the orange region of the electromagnetic spectrum as energy source showed a high content of Lnn, and there was less biomass compared to cells grown in red light. When the culture was left in the dark for various times, after 144 h it contained about 26% more Lnn than in conventionally cultivated cells.     

Restriction fragment length polymorphisms associated with immunoglobulin heavy chain gamma genes in Tunisians

Summary DNA polymorphisms in the human immunoglobulin gamma () region have been studied in random Arabo-Berber Tunisians and in a large Tunisian Berber kindred. Haplotypes have then been designated, based on variation in the BamHI restriction fragments containing the C1, C2, C4, and C genes. Two new haplotypes, in addition to the four previously described, have been observed. These new haplotypes, designated H5 and H6, were confirmed by family studies. The H5 haplotype was associated with black African Gm haplotypes · (Gm^{1,17;..;5,6,11} and Gm^1,17;..;5,11) (Gm^{a,z;..;blc3bo} and Gm^a,z;..;blbo) and probably represents a common haplotype in the black population. The haplotype H6 may be derived from H5. One of 39 random Tunisians was homozygous for a multigene deletion. DNA polymorphisms of the C genes, in conjuction with Gm markers, provide highly variable genetic markers important for the characterization of human populations.     

Carotenoids and Fatty Acids in Red Yeasts Sporobolomyces roseus and Rhodotorula glutinis

Rhodotorula glutinis and Sporobolomyces roseus, grown under different aeration regimes, showed differential responses in their carotenoid content. At higher aeration, the concentration of total carotenoids increased relative to the biomass and total fatty acids in R. glutinis, but the composition of carotenoids (torulene > -carotene > -carotene > torularhodin) remained unaltered. In contrast, S. roseus responded to enhanced aeration by a shift from the predominant -carotene to torulene and torularhodin, indicating a biosynthetic switch at the -carotene branch point of carotenoid biosynthesis. The overall levels of total carotenoids in highly aerated flasks were 0.55 mol-percent and 0.50 mol-percent relative to the total fatty acids in R. glutinis and S. roseus (respectively), and 206 and 412 g g^–1 dry weight (respectively).