Targeted deletion of alkylpurine-DNA-N-glycosylase in mice eliminates repair of 1,N6-ethenoadenine and hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine

It has previously been reported that 1,N6-ethenoadenine (ɛA), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (ɛC), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from APNG-null mutant mice and wild-type controls. The ability of these extracts to cleave defined oligomers containing a single modified base was determined. The results showed that both testes and liver cells of these knockout mice completely lacked activity toward oligonucleotides containing ɛA and Hx, but retained wild-type levels of activity for ɛC and 8-oxoG. These findings indicate that (i) the previously identified ɛA-DNA glycosylase and Hx-DNA glycosylase activities are functions of APNG; (ii) the two structurally closely related mutagenic adducts ɛA and ɛC are repaired by separate gene products; and (iii) APNG does not contribute detectably to the repair of 8-oxoG.

The increased level of β1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control

β1,4-Galactosyltransferase (β4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian β4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by ≈200 bp. In the mammary gland, coincident with the increased β4GalT-I enzyme level (≈50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a β4GalT-I transcript in which the 5′- untranslated region (UTR) has been truncated from ≈175 nt to ≈28 nt. The 5′-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5′-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5′-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (≈14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, β4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.

Localization of α1,3-fucosyltransferase VI in Weibel–Palade bodies of human endothelial cells

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked α1→3 to N-acetylglucosamine. A family of α1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription–PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with β4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel–Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel–Palade bodies suggests a function independent of Golgi-associated glycosylation.

Genomic organization of α1 and β1 subunits of the mammalian soluble guanylyl cyclase genes

The structures of the genes encoding the α1 and β1 subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the α1 (2.5 kb) and β1 (3.3 kb) subunits are presented in this report. The α1 sGC gene is approximately 26.4 kb and contains nine exons, whereas the β1 sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse α1 and β1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5′ untranscribed regions of α1 and β1 subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5′ untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

Transcytosis of α1-acidic glycoprotein in the continuous microvascular endothelium

By using perfusions and bolus administration, coupled with postembedding immunocytochemical procedures, we have identified the structures involved in the transport of derivatized orosomucoid (α1-acidic glycoprotein) across the continuous microvascular endothelium of the murine myocardium. Our findings indicate that: (i) monomeric orosomucoid binds to the luminal surface of the endothelium; (ii) it is restricted to caveolae during its transport across the endothelium; (iii) it is detected in the perivascular spaces at early time points (by 1 min) and in larger quantities at later time points (>5 min) from the beginning of its perfusion or its intravascular administration; (iv) no orosomucoid molecules are found in the intercellular junctions or at the abluminal exits of interendothelial spaces; and (v) the vesicular transport of orosomucoid is strongly inhibited by N-ethylmaleimide (>80%). Because, by size and shape, the orosomucoid qualifies as a preferential probe for the postulated small pore system, our results are discussed in relation to the pore theory of capillary permeability.

Two Arabidopsis Mutants That Overproduce Ethylene Are Affected in the Posttranscriptional Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase1

The Arabidopsis mutants eto1 (ethylene overproducer) and eto3 produce elevated levels of ethylene as etiolated seedlings. Ethylene production in these seedlings peaks at 60 to 96 h, and then declines back to almost wild-type levels. Ethylene overproduction in eto1 and eto3 is limited mainly to etiolated seedlings; light-grown seedlings and various adult tissues produce close to wild-type amounts of ethylene. Several compounds that induce ethylene biosynthesis in wild-type, etiolated seedlings through distinct 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) isoforms were found to act synergistically with eto1 and eto3, as did the ethylene-insensitive mutation etr1 (ethylene resistant), which blocks feedback inhibition of biosynthesis. ACS activity, the rate-limiting step of ethylene biosynthesis, was highly elevated in both eto1 and eto3 mutant seedlings, even though RNA gel-blot analysis demonstrated that the steady-state level of ACS mRNA was not increased, including that of a novel Arabidopsis ACS gene that was identified. Measurements of the conversion of ACC to ethylene by intact seedlings indicated that the mutations did not affect conjugation of ACC or the activity of ACC oxidase, the final step of ethylene biosynthesis. Taken together, these data suggest that the eto1 and eto3 mutations elevate ethylene biosynthesis by affecting the posttranscriptional regulation of ACS.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions.

Differential Expression and Internal Feedback Regulation of 1-Aminocyclopropane-1-Carboxylate Synthase, 1-Aminocyclopropane-1-Carboxylate Oxidase, and Ethylene Receptor Genes in Tomato Fruit during Development and Ripening1

We investigated the feedback regulation of ethylene biosynthesis in tomato (Lycopersicon esculentum) fruit with respect to the transition from system 1 to system 2 ethylene production. The abundance of LE-ACS2, LE-ACS4, and NR mRNAs increased in the ripening fruit concomitant with a burst in ethylene production. These increases in mRNAs with ripening were prevented to a large extent by treatment with 1-methylcyclopropene (MCP), an ethylene action inhibitor. Transcripts for the LE-ACS6 gene, which accumulated in preclimacteric fruit but not in untreated ripening fruit, did accumulate in ripening fruit treated with MCP. Treatment of young fruit with propylene prevented the accumulation of transcripts for this gene. LE-ACS1A, LE-ACS3, and TAE1 genes were expressed constitutively in the fruit throughout development and ripening irrespective of whether the fruit was treated with MCP or propylene. The transcripts for LE-ACO1 and LE-ACO4 genes already existed in preclimacteric fruit and increased greatly when ripening commenced. These increases in LE-ACO mRNA with ripening were also prevented by treatment with MCP. The results suggest that in tomato fruit the preclimacteric system 1 ethylene is possibly mediated via constitutively expressed LE-ACS1A and LE-ACS3 and negatively feedback-regulated LE-ACS6 genes with preexisting LE-ACO1 and LE-ACO4 mRNAs. At the onset of the climacteric stage, it shifts to system 2 ethylene, with a large accumulation of LE-ACS2, LE-ACS4, LE-ACO1, and LE-ACO4 mRNAs as a result of a positive feedback regulation. This transition from system 1 to system 2 ethylene production might be related to the accumulated level of NR mRNA.

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor

Several G-protein coupled receptors, such as the β1-adrenergic receptor (β1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein–protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the β1-AR either as a glutathione S-transferase fusion protein in biochemical “pull-down” assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the β1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the β1-AR but not to that of the β2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of β1-ARs in HEK293 cells while having no effect on β2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in β1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193–7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein’s surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen–deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein’s interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

Cloning and expression of rat 25-hydroxyvitamin D3-1α-hydroxylase cDNA

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1α-hydroxylase (P4501α), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501α cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1α-hydroxylase activity. The sequence analysis showed that P4501α was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501α mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1α,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501α mRNA was greatly increased in the renal proximal convoluted tubules.

Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n = 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.