Abstract:We investigated oxidative damage to the c-fos gene and to its transcription in the brain of Long-Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant ( p Ͻ 0.001) increase in the immunoreactivity to 8-hydroxy-2Ј-guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0 -30 min of reperfusion in all 27 animals treated with 15-90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (60 mg/ kg, i.p.), abolished the majority but not all of the oh8G/ oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c-fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c-fos gene, was increased 13-fold ( p Ͻ 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR-induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain. Key Words: Stroke-DNA repair-Oxidative stress-Gene expression-Neuroregeneration-Apoptosis. J. Neurochem. 73, 1164Neurochem. 73, -1174Neurochem. 73, (1999.An elevated sensitivity to oxidative stress in certain populations of brain cells may be a key mechanism underlying several neurological disorders (Liu et al., 1989;Jenner et al., 1992;Neve, 1996;Parshad et al., 1996;Kisby et al., 1997). Numerous studies have correlated an increase in calcium influx, glutamate, and reactive oxygen species (ROS) with neuronal death. ROS levels are known to be elevated after brain injury of the ischemia-reperfusion type (Hall and Braughler, 1989;Rosenthal et al., 1992;Poiries, 1994;Yoshida et al., 1994;Zhang et al., 1994;O'Neill et al., 1996;Smith et al., 1996) and following administration of excitotoxic drugs (Schulz et al., 1995;Ayata et al., 1997;Back et al., 1998). ROS are known to damage mitochondrial DNA (Driggers et al., 1993;Mecocci et al., 1993;Yakes and Van Houten, 1997;Murakami et al., 1998), nuclear genes (Liu et al., 1996;Taffe et al., 1996;Chen et al., 1997), and RNA (Liu et al., 1996;Nunomura et al., 1999). Damage to nucleic acids may lead to abnormal gene expression and premature neuronal death (Wolozin et al., 1996;Citron et al., 1997;Lamb, 1997;Iadecola et al., 1999).Activation of gene transcription in the ischemic brain following oxidative stress appears to signify a need for gene products to repair the injury. For example, transcription of one of the immediate early genes, the c-fos gene, is activated after head injury Yang et al., 1994). The product of the c-fos transcript then forms activator protein-1, which in turn activates various cellular functions, including the production of growth factors and DNA repair en...

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 +/- 3 (mean +/- SD per mm(2)). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 microg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of a human UDP-galactose:␤-N-acetylglucosamine ␤-1,3-galactosyltransferase, designated ␤3Gal-T1, revealed no ESTs with identical sequences but a large number with similarity. Three different sets of overlapping ESTs with sequence similarities to ␤3Gal-T1 were compiled, and complete coding regions of these genes were obtained. Expression of two of these genes in the Baculo virus system showed that one represented a UDP-galactose:␤-N-acetylglucosamine ␤-1,3-galactosyltransferase (␤3Gal-T2) with similar kinetic properties as ␤3Gal-T1. Another gene represented a UDP-galactose:␤-N-acetyl-galactosamine ␤-1,3-galactosyltransferase (␤3Gal-T4) involved in G M1 /G D1 ganglioside synthesis, and this gene was highly similar to a recently reported rat G D1 synthase (Miyazaki, H., Fukumoto, S., Okada, M., Hasegawa, T., and Furukawa, K. (1997) J. Biol. Chem. 272, 24794-24799). Northern analysis of mRNA from human organs with the four homologous cDNA revealed different expression patterns. ␤3Gal-T1 mRNA was expressed in brain, ␤3Gal-T2 was expressed in brain and heart, and ␤3Gal-T3 and -T4 were more widely expressed. The coding regions for each of the four genes were contained in single exons. ␤3Gal-T2, -T3, and -T4 were localized to 1q31, 3q25, and 6p21.3, respectively, by EST mapping. The results demonstrate the existence of a family of homologous ␤3-galactosyltransferase genes.

Alpha3-fucosyltransferases (alpha3-FucTs) catalyze the final step in the synthesis of a range of important glycoconjugates that function in cell adhesion and lymphocyte recirculation. Six members of this family of enzymes have been cloned from the human genome, and their expression pattern has been shown to be highly regulated. Each enzyme has a unique acceptor substrate binding pattern, and each generates a unique range of fucosylated products. Results from a range of studies have provided information on amino acids in the FucT sequence that contribute to the differential acceptor specificity for the FucTs, and to the binding of the nucleotide sugar donor GDP-fucose. These results, in conjunction with results obtained from the analysis of the disulfide bond pattern, have provided useful clues about the spatial distribution of amino acids that influence or directly contribute to substrate binding. This information is reviewed here, and a molecular fold prediction is presented which has been constructed based on the available information and current modeling methodology.

Background. Substantial hydroxyl radical (.OH)‐induced base lesions, recently found in the DNA of invasive ductal carcinoma of the female breast, are likely to be intimately related to oncogenesis. However, virtually no information was available regarding relationships between the different base lesions in the normal and cancerous breast. Such information is essential in understanding initial stages in the development of breast cancer and the potential of the base lesions as early predictors of cancer risk. Methods. The OH‐induced DNA base lesions in normal reduction mammoplasty tissue (RMT) were compared with those from invasive ductal carcinoma (IDC) and nearby microscopically normal tissue (MNT). Comparisons were then undertaken on relationships between the base lesion profiles in the normal and cancerous breast using 22 statistical models. Results. DNA from the RMT was characterized by a high ratio of ring‐opening products (e.g., 4,6‐diamino‐5‐formamidopyrimidine) to hydroxy‐adducts of adenine and guanine. A dramatic shift in this relationship in favor of carcinogenic hydroxy‐adducts (e.g., 8‐hydroxyguanine) was found in the cancerous breast. Statistical models with a high sensitivity (91%) and specificity (97%) provided a consistent means of classifying tissues (e.g., 96% correct). Conclusions. The dramatic shift in the DNA base lesion relationships in oncogenesis is attributed to alterations in the redox potential of the breast favoring oxidative conditions and cancer formation. These findings suggest that base lesion profiles are potential sentinels for cancer risk assessment. Further, intervention in controlling the tissue redox potential may provide benefit in delaying or preventing early oncogenic changes and the ultimate manifestation of cancer.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of the human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferase, designated ␤4Gal-T1, revealed a large number of ESTs with identical as well as similar sequences. ESTs with sequences similar to that of ␤4Gal-T1 could be grouped into at least two non-identical sequence sets. Analysis of the predicted amino acid sequence of the novel ESTs with ␤4Gal-T1 revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of ␤4Gal-T1. The likelihood that the identified ESTs represented novel galactosyltransferase genes was tested by cloning and sequencing of the full coding region of two distinct genes, followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represented genuine UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferases, thus designated ␤4Gal-T2 and ␤4Gal-T3. Genomic cloning of the genes revealed that they have identical genomic organizations compared with ␤4Gal-T1. The two novel genes were located on 1p32-33 and 1q23. The results demonstrate the existence of a family of homologous galactosyltransferases with related functions. The existence of multiple ␤4-galactosyltransferases with the same or overlapping functions may be relevant for interpretation of biological functions previously assigned to ␤4Gal-T1.During the last decade, more than 40 mammalian glycosyltransferases have been cloned and characterized (1, 2). The initial strategy for cloning glycosyltransferases was cumbersome purification of labile enzyme proteins followed by screening of cDNA libraries with antibodies or DNA probes based on amino acid sequence information (3-11). The introduction of transfection cloning by Lowe and co-workers (12) resulted in a marked increase in the cloning of novel glycosyltransferase genes (13-17). A third successful approach has taken advantage of conserved sequences in glycosyltransferases that share donor and/or acceptor substrates. Thus, searches for novel members of homologous glycosyltransferase gene families utilizing conserved sequence motifs for RT-PCR 1 cloning with degenerate primers have resulted in the identification and cloning of a number of novel genes (18,19).One part of the human genome project is the establishment of a data base of expressed sequence tags (ESTs), which currently has over 700,000 unique sequences. ESTs represent short 5Ј-and 3Ј-sequences (200 -500 bp) of cDNA clones from a large variety of human and animal organs (20). The EST data base is now estimated to contain sequence information from more than half the human genes; it therefore provides a unique source for identifying novel members of homologous gene families (21). The EST data base has recently been successfully utilized in searches for novel glycosyltransferase genes of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, where several novel members of this homologous gene family have been iso...

A novel putative member of the human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferase family, designated ␤4Gal-T4, was identified by BLAST analysis of expressed sequence tags. The sequence of ␤4Gal-T4 encoded a type II membrane protein with significant sequence similarity to other ␤1,4-galactosyltransferases. Expression of the full coding sequence and a secreted form of ␤4Gal-T4 in insect cells showed that the gene product had ␤1,4-galactosyltransferase activity. Analysis of the substrate specificity of the secreted form revealed that the enzyme catalyzed glycosylation of glycolipids with terminal ␤-GlcNAc; however, in contrast to ␤4Gal-T1, -T2, and -T3, this enzyme did not transfer galactose to asialo-agalacto-fetuin, asialo-agalactotransferrin, or ovalbumin. The catalytic activity of ␤4Gal-T4 with monosaccharide acceptor substrates, Nacetylglucosamine as well as glucose, was markedly activated in the presence of ␣-lactalbumin. The genomic organization of the coding region of ␤4Gal-T4 was contained in six exons. All intron/exon boundaries were similarly positioned in ␤4Gal-T1, -T2, and -T3. ␤4Gal-T4 represents a new member of the ␤4-galactosyltransferase family. Its kinetic parameters suggest unique functions in the synthesis of neolactoseries glycosphingolipids.A family of human UDP-galactose:␤-N-acetylglucosamine ␤1,4-galactosyltransferases (␤4Gal-Ts) 1 was recently identified (1-3). Four genes within this family encode ␤4-galactosyltransferases, which form the Gal␤1-4GlcNAc linkage (2, 4 -6). The kinetic parameters and expression patterns of these enzymes differ and they are predicted to show some degree of overlap in biological function (2, 3, 6). Two ␤4-galactosyltransferases, ␤4Gal-T1 and -T2, can function as lactose synthases in the presence of ␣-lactalbumin (2, 3, 7), whereas ␤4Gal-T3 and ␤4Gal-T5 2 are largely insensitive to ␣-lactalbumin modulation (2, 6, 8). ␤4Gal-T1, -T2, and -T3 catalyze transfer of galactose to lactoseries glycosphingolipids, but ␤4Gal-T3 only efficiently catalyzes synthesis of the first N-acetyllactosamine unit in lactoseries glycolipids (2). In contrast, ␤4Gal-T5 was reported to be inactive with a glycolipid substrate (Lc 3 Cer) 2 as well as with the glycoprotein acceptor, asialo-agalacto-transferrin (6). A rat lactosylceramide synthase was recently purified and cloned by Nomura et al. (9), and it appears to represent the ortholog of the human member of the gene ␤4-galactosyltransferase family designated ␤4Gal-T6 (10). Thus, the formation of Gal␤1-4Glc(NAc) linkages in different glycoconjugates and their varying oligosaccharide structures may be catalyzed by different ␤4-galactosyltransferases.Analysis of ESTs suggested the existence of additional members of the human ␤4Gal-T gene family (1-3), and recently, Lo et al. (10) compared the full coding sequences of six homologous human genes and suggested a nomenclature based on sequence similarity: ␤4Gal-T1 (5, 11, 12), ␤4Gal-T2 (2), ␤4Gal-T3 (2), ␤4Gal-T4, ␤4Gal-T5 (6), and ␤4Gal-T6 (9). So far, all genes except one, ...

Background. The authors previously have shown by gas chromatography‐mass spectrometry that the hydroxyl radical (.OH) induces alterations in the DNA base structure of the female breast, which are premalignant markers of breast cancer. Fourier transform‐infrared (FT‐IR) spectroscopy also has a high potential for revealing a broad array of structural changes in DNA that may provide important new insight into breast cancer etiology and prediction. Methods. DNA from normal reduction mammoplasty tissue, invasive ductal carcinoma, and nearby microscopically normal tissue was analyzed by FT‐IR spectroscopy. Statistical models based on DNA spectral properties were developed and compared with a statistical model previously used with base modifications. Results. Substantial differences were found in the spectral properties of DNA from women with normal and cancerous breast tissue, indicating an ability to discriminate cancerous tissue from noncancerous tissue with a sensitivity and specificity of 83%. Most importantly, the normal population was divided into subgroups in which a nonrandom progression was identified and a cancer‐like DNA phenotype that was highly correlated (r ⩾ 0.90) with that of the patients with cancer was exhibited in 59% of the women. The spectral data, which also were highly correlated with the base‐model data, were used to establish a model for predicting the probability of breast cancer. Consistent with the high cancer reoccurrence rate in the ipsilateral breast, 8 of 10 of the microscopically normal tissue specimens remaining after tumor excision were classified as cancerous using this model. Conclusions. Progressive structural changes in the DNA of the normal female breast, leading to a premalignant cancer‐like phenotype in a high proportion of women, are the basis for a new paradigm for understanding the etiology of breast cancer and predicting its occurrence at early stages of oncogenesis. The results also suggest therapeutic strategies for potentially reversing the extent of DNA damage, which may be useful in disease prevention and treatment. Cancer 1995;75:503‐17.