Shake Flask Biodegradation of 14 Commercial Phthalate Esters

An acclimated shake flask CO₂ evolution test was used to study the biodegradability of 14 commercial phthalate esters that are commonly used as plasticizers. Both CO₂ evolution (ultimate biodegradation) and loss of parent phthalate esters (primary biodegradation) were measured. With only a few exceptions, primary biodegradation was 90% or higher, and ultimate biodegradation was in excess of 55% of theoretical results in 28 days. The results showed that all of the commercial phthalate esters were susceptible to biodegradation by mixed populations of microorganisms from natural sources. The results also provide considerable insight into the utility and reproducibility of a standard biodegradation test that is being recommended for widespread screening of chemicals.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

The pericellular boundary layer modulates acetylcholine receptor stability in cultured myotubes

Degradation of acetylcholine receptors in cultured chicken myotubes was measured by release into the medium of radioactivity from 125I-labeled alpha-bungarotoxin. Disturbance of the pericellular boundary layer by stirring of the culture medium shortened the half-life of receptor in the membrane from 24 to 12 h. The effect could not be explained by dissociation of toxin-receptor complexes or by conditioning of the bulk phase of the medium. The rates of synthesis and degradation of total cell protein and the degradation of lactoperoxidase-iodinated surface protein were not affected by medium stirring. The loss of glucosamine-labeled material from the cells was enhanced by stirring, however, and this resulted entirely from the increased shedding of high molecular weight glycosubstances from the cells. Cells in stirred cultures contained lower levels of surface coat material stainable with colloidal thorium. These results indicate that glycosubstances of the pericellular matrix protect ACh receptors from degradation.     

Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function

A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk^?) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 × 10⁶ D) was used as a carrier, the presence of either 20 mM NH₄Cl, 1 μM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-P_i) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 × 10⁶ D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH₄Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-P_i coprecipitate, whereas NH₄Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.     

Cell division cycle in mammalian cells : VIII. Mapping of G1 into six segments using temperature-sensitive cell cycle mutants

The G1 blocks in three temperature-sensitive (ts) Syrian hamster cell-cycle mutants have been mapped in relation to other G1 landmarks. Two mutants reported here, ts-559 and ts-694, show defective progression only in G1. When shifted from the permissive temperature of 33 degrees C to the non-permissive temperature of 39 degrees C, G1 cells of these two mutants show no further cell cycle progression, while cells in S, G2 and mitosis progress through the cell cycle but become blocked after entering G1. The two mutants complement each other, and also complement the previously reported mutant ts-550C with blocks in both G1 and G2 of the cell cycle. The locations of the G1 blocks in both ts-559 and ts-694 are before the hydroxyurea arrest point. The G1 ts point in ts-694 is prior to the isoleucine deprivation and serum starvation points, while the G1 block in ts-559 is after the serum starvation point but before the isoleucine block. Other G1 block points which have been reported are in mutants of different species and isolated in different laboratories, causing difficulties for relative positioning of the blocks in G1. The mutants for mapping in this study have been isolated from the same cell line. The G1 ts arrest points of ts-559 and ts-694, and that found in ts-550C, together with nutritional deprivations and metabolic inhibitors, provide seven reference points which divide G1 into six segments, each of which is bracketed by two adjacent points: mitosis, ts-694 block, serum starvation arrest point, ts-559 block, isoleucine deprivation arrest point, ts-550C block, hydroxyurea or excess-thymidine arrest segment.     

Discrimination by male ring-tailed Lemurs (Lemur catta) between the scent marks of male and those of female conspecifics

Olfaction plays an important role in the social communication of all prosimians. (The experiment reported in this paper forms part of an intensive chemobehavioral study of olfaction in Lemur catta (ring-tailed lemur) being carried out in this laboratory.) Five male Lemur cattawere tested on their behavioral responses to paired scent stimuli. Responses measured were (1) total investigation time, (2) arm-marking, (3) ABO/BO rubbing, and (4) flehmen. Males showed a strong discrimination between the scent stimuli,giving higher levels of response to female scent on measures 1, 3, and 4. This response suggests an olfactory-related preference by males for female scent under controlled conditions. This preference may be a consequence of the females’ dominance over males and the brevity of estrus in L. catta,both of which would favor such choice behavior.     

Transfer RNA genes of Euglena gracilis chloroplast DNA

Summary Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2–6 genes. None of the known genes contains introns, nor codes for the 3-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.     

A followup study of the academic attainment and classroom behavior of children with elevated dentine lead levels

To investigate the impact of low-level lead exposure on children's behavior within a natural setting, we assessed the school performance of a sample of 141 elementary school children classified according to dentine lead level. Twenty-two children had “elevated” levels (≥20 parts per million), 71 had “midrange” levels (10.0–19.9 parts per million), and 48 had “low” levels (<10 parts per million). Four types of outcome data were collected: (1) scores on a standardized group intelligence test, (2) teachers' ratings, (3) incidence of academic failure (assignment to remedial aid, grade retention), and (4) observations of classroom behavior. In general, higher dentine lead levels were associated with less favorable school performance, with most covariance-adjustedp-values in the range of 0.05–0.15. The incidence of grade retention was the outcome most strongly related to lead level. The pattern of results suggests a consistent, though weak relationship between children's dentine lead levels and elementary school performance.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.