Abundant alkali-sensitive sites in DNA of human and mouse sperm

The DNA of human and mouse sperm cells was analyzed by single-cell microgel electrophoresis, by agarose gel electrophoresis, and by alkaline elution--three techniques that can detect single-strand DNA breaks and/or labile sites. Under these conditions a surprisingly large number of single-strand DNA breaks, approximately 10(6) to 10(7) per genome, were detected in human and mouse sperm but not in human lymphocytes or in mouse bone marrow cells. These breaks were also present in chicken erythrocyte DNA, which is also highly condensed. These breaks were not observed under neutral pH conditions nor under denaturing conditions not involving alkali, suggesting that these sites are alkali-sensitive and do not represent preexisting single-strand breaks. The high frequency of such sites in sperm from healthy mouse and human donors suggests that they represent a functional characteristic of condensed chromatin rather than DNA damage.     

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells

Effects on [Ca2+]i levels of endothelin-l (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2-loaded neuroblastoma NG108-15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.     

Proportion of beta-D-glucuronidase-negative Escherichia coli in human fecal samples.

Convenient assays and reports that almost all clinical isolates of Escherichia coli produce beta-D-glucuronidase (GUR) have led to great interest in the use of the enzyme for the rapid detection of the bacterium in water, food, and environmental samples. In these materials, E. coli serves as an indicator of possible fecal contamination. Therefore, it was crucial to examine the proportion of GUR-negative E. coli in human fecal samples. The bacterium was isolated from 35 samples, and a mean of 34% and a median of 15% were found to be GUR negative in lauryl sulfate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide. E. coli from three samples were temperature dependent for GUR production: very weakly positive at 37 degrees C but strongly positive at 44.5 degrees C. These results remind us of differences between fecal and clinical E. coli populations, of diversity in GUR regulation and expression in natural populations of E. coli, and of the need for caution in using GUR for the detection of fecal E. coli.     

Mechanisms of excitation-contraction coupling and calcium liberation in striated muscles of vertebrates

In vertebrate skeletal muscle, the main part of excitation-contraction coupling occurs at the level of the triad, where membranes of T-system and of junctional SR are facing each other. From place to place, the junctional gap is bridged by "feet" structures which include the SR Ca2+ channel. Half of them are closely apposed to tubular intramembranous structures assumed to be DHP-sensitive voltage-sensors which are similar to tubular Ca2+ channels and act by controlling Ca2+ release from SR. During a twitch, the release of Ca2+ activator from SR is controlled both by voltage-sensors via the feet structures and by a tubular Na+ current via a Na+-induced Ca2+ release mechanism. During long-duration mechanical responses, additional mechanisms are involved: a Ca2+-induced Ca2+ release which can be activated by ICa; the release of Ca2+ from membrane, controlled by the operation of a Na+/Ca2+ exchanger and/or new arrangements of surface membrane charges. An IP3-mediated Ca2+ release could be involved too. All these mechanisms can be regulated by intracellular biochemical or ionic processes.     

Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogues

The structure of the GTP-binding site of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and include 8-azido-GTP, P3-(4-azidoanilino)-P1-5'-GTP (AA-GTP), 5'-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)-GTP (ANPAP-GTP), the 2',3'-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5'-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2'- and 3'-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T alpha subunit of transducin and was localized on the 21-kDa tryptic fragment of T alpha. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T alpha and T beta gamma subunits of transducin. The labeling on T alpha was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T alpha and T beta gamma. This observation implies that the guanine ring of the bound GTP on T alpha could be in close proximity with T beta gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of abasic sites in DNA duplexes: deoxyadenosine stacks into the helix opposite acyclic lesions

Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated APP 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C).d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated APE 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the APP 9-mer and APE 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H2O and D2O solution between -5 and 5 degrees C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and E14) and stacks between the flanking G4.C15 and G6.C13 Watson-Crick base pairs in both the APP 9-mer and APE 9-mer duplexes. The helix is right-handed at and adjacent to the abasic site, and all glycosidic torsion angles are anti in both 9-mer duplexes. Proton NMR parameters for the APP 9-mer and APE 9-mer duplexes are similar to those reported previously for the APF 9-mer duplex (F = furan) in which a cyclic analogue of deoxyribose was embedded in an otherwise identical DNA sequence [Kalnik, M. W., Chang, C. N., Grollman, A. P., & Patel, D. J. (1988) Biochemistry 27, 924-931]. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the APP 9-mer and APE 9-mer duplexes (5 degrees C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles. These same torsion angles are also distorted in the APF 9-mer but assume a different conformation than those in the APP 9-mer and APE 9-mer duplexes.     

Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxin binding chain

Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.     

Interactions between Pesticide Genes: Model and Experiment

In response to years of intense selection pressure by organophosphate insecticides, several different insecticide resistance mechanisms have evolved in natural populations of the mosquito Culex pipiens. We examined interactions between two of the most important mechanisms using a four-compartment model of insecticide pharmacokinetics. The joint effect of different mechanisms of resistance can be expressed in terms of epistasis at the physiological level in this model. The type of epistasis predicted by the model depends on the particular physiological mechanisms of resistance involved. Resistance due to a reduced penetration of the insecticide combines multiplicatively with other resistance factors, but resistance due to detoxicative processes and to insensitivity of the target site combines additively. How the pattern of epistasis at the physiological level is translated into fitness epistasis in natural populations of this mosquito depends on the intensity and pattern of insecticide selection in the field.     

The combined effects of chlorine and ammonia on litter breakdown in outdoor experimental streams

The response of Potamogeton crispus L. breakdown to controlled doses of different levels of chlorine and chlorine + ammonia was investigated over two years in outdoor experimental streams. In 1985, downstream riffles of 2 streams were dosed (observed in-stream concentrations) at ca. 10 μg/L Total Residual Chlorine (TRC), one stream at 64 μg/L TRC and one stream at 230 μg/L TRC. Two control streams were not dosed and the upstream riffles of each stream served as within stream controls. In 1986, the downstream riffle of one stream was dosed at 70 μg/L TRC and a second stream was dosed at 200 μg/L TRC. Four streams were also dosed with 2.5 mg/L NH₃-N: one stream with no chlorine, one stream with ca. 10 μg/L TRC, one with 56 μg/L TRC, and one with 150 μg/L TRC. A seventh stream was dosed for 2 h at 2000 μg/L TRC and 2.5 mg/L ammonia and then allowed to recover (recovery stream). Each year, litter decomposition (degree day k values) was measured during two 35 day trials (Jun–Jul and Aug–Sep). In 1985, when streams were dosed with chlorine alone, decomposition was significantly reduced with the high (230 μg/L TRC) chlorine dose. Downstream decomposition was 27% (Jun–Jul) and 59% (Aug–Sep) of the upstream (control) rate. No other chlorine effects were found during this period. In Jun–Jul 1986, there was significantly lower decomposition in the downstream dosed sites of the 200 μg/L TRC alone stream, the 146 μg/L TRC + ammonia stream and the recovery stream; downstream decay rates were (respectively) 56%, 42% and 64% of the upstream control sites. No other up-down pairs were different in July 1986. In Aug–Sep, all three streams with chlorine + ammonia (6, 56 and 146 μg/L TRC + 2,5 mg/L ammonia) and the 70 μg/L TRC alone stream had significantly lower decomposition rates in the downstream dosed sites. For these streams, downstream decay rates ranged from 46% (high chlorine + ammonia) to 73% (low chlorine + ammonia) of the upstream control rates. No other up-down pairs were different during this trial. Up and downstream sites of the stream dosed with 2.5 mg/L ammonia alone were nearly identical for both trials (< 3% difference). These results indicate that TRC at less than 250 μg/L can significantly reduce litter decomposition and strongly suggest that addition of ammonia to chlorinated water can increase the toxic effect of chlorine. currently at the Department of Fisheries and Wildlife currently at the Department of Fisheries and Wildlife     

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency

Summary Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.Abbreviations PD Pseudo arylsulfatase-A Deficiency - ARA Arylsulfatase-A