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.

Lowering blood homocysteine with folic acid based supplements: meta-analysis of randomised trials

Objective: To determine the size of reduction in homocysteine concentrations produced by dietary supplementation with folic acid and with vitamins B-12 or B-6.

Fluid resuscitation with colloid or crystalloid solutions in critically ill patients: a systematic review of randomised trials

Objective: To determine the effect on mortality of resuscitation with colloid solutions compared with resuscitation with crystalloids.

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.