Recovery from sublethal and potentially lethal damage in an X-ray-sensitive CHO cell

It has been suggested that DNA strand breaks are the molecular lesions responsible for radiation-induced lethality and that their repair is the basis for the recovery of irradiated cells from sublethal and potentially lethal damage. EM9 is a Chinese hamster ovary cell line that is hypersensitive to killing by X rays and has been reported to have a defect in the rate of rejoining of DNA single-strand breaks. To establish the importance of DNA strand-break repair in cellular recovery from sublethal and potentially lethal X-ray damage, those two parameters, recovery from sublethal and potentially lethal damage, were studied in EM9 cells as well as in EM9's parental repair-proficient strain, AA8. As previously reported, EM9 is the more radiosensitive cell line, having a D0 of 0.98 Gy compared to a D0 of 1.56 Gy for AA8 cells. DNA alkaline elution studies suggest that EM9 cells repair DNA single-strand breaks at a slower rate than AA8 cells. Neutral elution analysis suggests that EM9 cells also repair DNA double-strand breaks more slowly than AA8 cells. All of these data are consistent with the hypothesis that DNA strand-break ligation is defective in EM9 cells and that this defect accounts for increased radiosensitivity. The kinetics and magnitude of recovery from sublethal and potentially lethal damage, however, were similar for both EM9 and AA8 cells. Six-hour recovery ratios for sublethal damage repair were found to be 2.47 for AA8 cells and 1.31 for EM9 cells. Twenty-four-hour recovery ratios for potentially lethal damage repair were 3.2 for AA8 and 3.3 for EM9 cells. Both measurements were made at approximately equitoxic doses. Thus, the defect in EM9 cells that confers radiosensitivity and affects DNA strand-break rejoining does not affect sublethal damage repair or potentially lethal damage repair.     

Apparent identity of cerebral tyrosylsulfotransferase activities using either a cholecystokinin derivative or an acidic amino acid polymer as substrate

The tyrosylsulfotransferase activities of rat cerebral fractions transferring [35S]sulfate groups from 3'-phosphoadenosine 5'-[35S]phosphosulfate to either Boc-cholecystokinin-8 (in non-sulfated form) or the acidic amino acid polymer (Glu, Ala, Tyr)n (6:3:1) were compared. They appear similar regarding subcellular distribution (both being enriched in the microsomal fraction) and inhibition by an excess of the acidic amino acid polymer, NaCl or 2,6-dichloro 4-nitrophenol. These results obtained with artificial substrates suggest that identical (or closely similar) tyrosylsulfotransferases are responsible for sulfation of tyrosine residues of several secretory proteins from various tissues.     

Breeding system ofDaphniopsis ephemeralis: adaptations to a transient environment

Populations of the vernal cladoceranDaphniopsis ephemeralis are found in woodland ponds throughout southern Ontario. The species reproduces by cyclic parthenogenesis, and genotype frequencies at allozyme loci are ordinarily in good agreement with Hardy-Weinberg expectations. Occasional heterozygote deficiencies are apparently the consequence of admixture of ephippial hatchlings produced in temporally separated bouts of sexual reproduction. Considerable heterogeneity in genotypic frequencies exists among local populations in southwestern Ontario, indicating that gene flow among populations is restricted. Inbreeding coefficients suggest that populations receive an average of 0.3 migrants per generation. The completion of a sexual life cycle is made possible despite the brief persistence of populations by the emergence of males from ephippial eggs and by the production of equal numbers of male and female progeny in the first parthenogenetic brood.     

Characterization of Erwinia chrysanthemi extracellular proteases: cloning and expression of the protease genes in Escherichia coli.

Erwinia chrysanthemi, a phytopathogenic enterobacterium, secretes three antigenically and structurally distinct proteases, A, B, and C and produces a protease inhibitor, a low-molecular-weight, heat-stable protein which remains mostly intracellular and which binds specifically to the A, B, and C proteases. The structural genes for proteases A, B, and C and for the inhibitor are clustered on a ca. 40-kilobase DNA fragment present in cosmid pEW4. Escherichia coli strains harboring pEW4 secrete the three proteases into the medium during the exponential phase of growth, without intracellular accumulation and in the absence of detectable cell lysis. An 8.5-kilobase EcoRI fragment derived from the cosmid encodes proteases B and C and the inhibitor as well as functions involved in the synthesis or secretion (or both) of the proteases. The inhibitor is not required for protease synthesis or secretion.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Co-purification and characterization of ATP-sulfurylase and adenosine-5'-phosphosulfate kinase from rat chondrosarcoma

The two sulfate-activating enzymes, ATP-sulfurylase (EC 2.7.7.4) and adenosine-5'-phosphosulfate kinase (adenylylsulfate kinase, EC 2.7.1.25), were each purified about 2000-fold from crude rat chondrosarcoma homogenate. Throughout a purification protocol which included Sephacryl S-300 gel filtration, DEAE-Sephadex ion exchange, hydroxylapatite, and ATP-agarose affinity chromatography, these two activities consistently co-purified. ATP-sulfurylase and adenosine-5'-phosphosulfate kinase each showed a pH optima of 7.0-7.4 and a bimodal temperature optima of 46 and 52-54 degrees C. Both activities preferred Mg2+ as their divalent cation source over Mn2+, Co2+, or Zn2+. The apparent Km values determined for adenosine 5'-phosphosulfate in both assays was 1-5 microM; the Km for pyrophosphate in the sulfurylase reaction was 40 microM and for ATP in the kinase reaction was 5 mM. Gel electrophoresis indicated major bands at Mr = 160,000 in nondenaturing systems and 35,000-37,000 and 60,000 under dissociative conditions, whereas gel filtration of the most highly purified fractions yielded a coincident peak in the molecular weight range 260,000.     

Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins

Summary We have analyzed and compared the amino acid sequences of the type 4 fimbrial subunits fromPseudomonas aeruginosa, Moraxella bovis, M. nonliquefaciens, Bacteroides nodosus, Neisseria gonorrhoeae, andN. meningitidis. We propose a consensus sequence for the highly conserved aminoterminal regions of these proteins. In the variable regions, a domain corresponding to an epitope common toN. gonorrhoeae andN. meningitidis fimbriae is conserved, both in sequence and in environment, in fimbrial subunits fromB. nodosus. The subunits fromM. bovis andP. aeruginosa do not show any homologies to this sequence. In all of the subunits, the carboxy-terminal half of the molecule consists of a series of fairly hydrophobic domains. The last three domains, two of which include the cysteines of the disulfide bridge inN. gonorrhoeae, P. aeruginosa, andM. bovis, are more or less conserved in sequence in all of the proteins including that ofB. nodosus. We propose that these conserved hydrophobic regions, which have the potential to form a series of beta-sheets, form a structural framework around which more variable hydrophilic sequences determining immunological profile are arranged. The evolutionary relationships of the contemporary proteins and the distribution of type 4 fimbriae are also discussed.     

Nucleic acid-like structures III. Oligomerization of 3′-deoxyadenosine 2′,5′-diphosphoimidazolide

Summary The activated dimonophosphate of 3-deoxyadenosine (cordycepin) undergoes oligomerization to produce a new family of pyrophosphate-linked oligomers in which the average repeating unit involves a nine-atom structural group. The presence of a poly(U) template increase the relative yields of higher oligomers, although the template-free reaction is itself extremely efficient.For the previous paper in this series see Schwartz et al. (1987)     

An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels

Partially deglycosylated chondroitin sulfate proteoglycan (CSPG) or peptide fragments obtained from CSPG are not readily detectable in gels by staining with Alcian blue 8GX or ammoniacal silver using the technique of Oakley et al. (B. Oakley, D. Kirsh, and N. Morris (1980) Anal. Biochem. 105, 361). Sequencial staining with both reagents allows visualization of intact CSPG or peptides derived from proteoglycans in polyacrylamide gels at protein concentrations as low as 2 ng/mm2, or glucuronic acid and galactosamine concentrations of 1 ng/mm2 or less. This method is significantly more sensitive and has broader applicability than that described by H. Min and M. Cowman (1986) Anal. Biochem. 155, 275) for staining glycosaminoglycan fragments in polyacrylamide gels.