Cloning of a Novel RNA Binding Polypeptide (RA301) Induced by Hypoxia/Reoxygenation*

Astrocytes have a critical role in the neuronal re- sponse to ischemia, as their production of neurotrophic mediators can favorably impact on the extreme sensitiv- ity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed two ribonucleoprotein domains and serine/argi-nine-rich domains, suggestive of their function as RNA splicing factor. Northern analysis displayed striking induction only in cultured astrocytes within 15 min of reoxygenation and reached a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrated expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/ reoxygenated astrocytes. Induction of RA301 mRNA was mediated, in large part, by endogenously generated re- active oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase which blocks oxygen-free radical formation by astrocytes. Similarly, in- creased expression of RA301 in supporting a neurotrophic function of astrocytes was suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expres- sion is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues. Pardee (11) with minor modifications. In brief, reverse transcriptase products obtained from total RNA (about 25 ng in each reaction) were used as PCR templates in 30 (cid:109) l of reaction mixture containing arbitrary primer (1 (cid:109) M ), dNTP (200 (cid:109) M of each), and 2 units of Taq polymerase (AmpliTaq, Takara Shuzo). The PCR was performed for 39 cycles with a setting of 95 °C for 30 s, 40 °C for 1 min, and 72 °C for 1 min (5 min for last cycle) by using the same arbitrary 12-mer oligonucleotide as both upstream and downstream primers. In one set of experiments, PCR products obtained from hypoxic and hypoxia/reoxy-genated astrocytes were screened using 60 different primers. After separation in 5% polyacrylamide gel electrophoresis, PCR products were stained with ethidium bromide. cDNA bands specifically amplified in hypoxia/reoxygenated astrocytes were cut out and eluted from the gels and reamplified by PCR using the same primers and conditions employed in differential display except for an increased concentration of dNTP mixture (40 (cid:109) M ). Cloning and Sequencing of cDNA Fragments— Reamplified cDNA fragments were cloned into the pT7Blue T-vector (Novagen). Plasmid DNA sequencing of cloned fragments was carried out using the Taq dye primer cycle sequencing core kit Biosystems Tokyo, Ja- pan) with either the M13 forward sequencing primer or reverse primer. The cDNA sequences were analyzed and compared for homology with those available in EMBL and GenBank TM DNA data bases, and NBRF protein data base. In the present study, one of the cDNA fragments (tentatively named RA301) was subjected to further analysis. By screening a rat cDNA library, a cDNA (2 kilobase pairs) that encom- passes the entire open reading frame of RA301 was isolated and se-quenced in both directions. Blot blot

Tissue injury consequent on ischemia results from two distinct types of mechanisms: first, adaptive processes to meet the challenges of oxygen deprivation and reduction in blood flow are marshalled, and second, the response to restoration of oxygenated blood flow, a central feature of which is the generation of oxygen-free radicals by a range of cells, is triggered. Characteristics of the cellular response to oxygen deprivation include redirection of energy metabolism, increased glucose utilization, and expression of oxygen-regulated proteins (1)(2)(3). During reoxygenation, distinct mechanisms are triggered, such as generation of oxygen-free radicals, and expression of heat shock proteins (HSP) 1 and glucose-regulated proteins, such as GRP78 (4). One common denominator of cellular mechanisms activated by hypoxia alone or hypoxia followed by reoxygenation is redirection of cellular biosynthetic processes leading to synthesis of new proteins and, thus, changes in the cellular phenotype.
Astrocytes, the most abundant cell type in the central nervous system, are relatively resistant to the environmental stress imposed by hypoxia/reoxygenation, maintaining their capacity to proliferate and to generate neurotrophic mediators (5,6). This contrasts with the relative inability of neurons to maintain viability in response to such environmental perturbations. As we previously found that inhibition of protein synthesis during early reoxygenation prevented effective astrocyte adaptation to hypoxia/reoxygenation, resulting in eventual cell death (7), we hypothesized that early during reexposure of cells to ambient oxygen critical gene products were expressed.
In this study we report the cloning of a novel putative RNA binding protein (RA301) based on differential display, which is strongly induced in cultured astrocytes early during reoxygenation, as well as in ischemic rat brain. Furthermore, inhibition of RA301 expression with a specific antisense probe reduced about 3-fold of release of interleukin-6 (IL-6), a neuroprotective cytokine, by astrocytes subjected to hypoxia/reoxygenation. The identification of a putative RNA binding protein, with potential properties of a splicing factor based on analysis of the deduced amino acid sequence and whose expression is induced by hypoxia/reoxygenation, is likely to provide new insights into the cellular biosynthetic response to ischemia.

MATERIALS AND METHODS
Cell Culture and Conditions for Hypoxia/Reoxygenation-Rat primary astrocytes were obtained from neonatal rats by a modification of a previously described method (8). In brief, cerebral hemispheres were harvested from neonatal Sprague-Dawley rats within 24 h of birth, and brain tissues were digested at 37°C by Dispase II (3 mg/ml; Boehringer Mannheim). The mixture was plated in 175-cm 2 culture flasks (two brains/flask), and cells were grown in minimal essential medium supplemented with fetal calf serum (10%; CellGrow). After 10 days, culture flasks were incubated for 48 h with cytosine arabinofuranoside (10 g/ml; Wako Chemicals, Osaka, Japan) to prevent fibroblast overgrowth and agitated on a shaking platform (Bioshaker, BR-30L, Taitek, Tokyo, Japan) to separate astrocytes from remaining microglia and oligodendroglia. The adherent cell population was then identified by morphologic and immunohistochemical criteria (detection of glial fibril-* This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas 04268103 from the Ministry of Education, Science and Culture, Japan, by United States Public Health Service Grants HL42507 and HL50629, and PERC. We also thank Nanki Ikueikai and Chiba Geigy Science Foundation for supporting this study. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Dept. of Anatomy and Neuroscience, Osaka University Medical School, 2-2 Yamada-oka, Suita City 565, Japan. Tel.: 81-6-879-3221; Fax: 81-6-879-3229. 1 The abbreviations used are: HSP, heat shock protein; AGPC, acid guanidinium/phenol/chloroform; DPI, diphenyl iodonium; HSP72, 72-kDa heat shock protein; IL, interleukin; PCR, polymerase chain reaction; Tra-2, transformer-2. lary acidic protein). Cells were then replated at a density of 5 ϫ 10 4 cells/cm 2 in the above medium. When cultures achieved confluence, they were exposed to hypoxia using an incubator attached to an hypoxia chamber which maintained a humidified atmosphere with low oxygen tension (Coy Laboratory Products, Ann Arbor, MI) as described previously (9). Where indicated, after exposure to hypoxia, cultures were returned to the ambient atmosphere (reoxygenation), at which time the conditioned medium was rapidly exchanged with fresh medium. Oxygen tension in the medium was monitored using a blood gas analyzer (ABL-2, Radiometer, Sweden). Cell viability was assessed by several methods including morphological criteria, trypan blue exclusion, and lactate dehydrogenase release.
Preparation of Total RNA and cDNA-Total RNA was extracted and purified from astrocytes (about 5 ϫ 10 8 cells) exposed to hypoxia for 24 h and hypoxia/reoxygenation (24 h of hypoxia followed by 1 h of reoxygenation) by the acid guanidinium/phenol/chloroform (AGPC) method (10) using a kit from Clontech (Palo Alto, CA). For hypoxic samples, an RNA extraction procedure was performed inside the hypoxic chamber after all reagents were equilibrated in the hypoxic atmosphere. Then purified RNA (about 3 g) was reverse transcribed in a buffer containing Moloney murine leukemia virus reverse transcriptase (300 units; Life Technologies, Inc.), oligo(dT) primer (2.5 M, T 11 GT), and dNTP mixture (20 M of each) for 60 min at 37°C. Then cDNAs synthesized from RNAs were subjected to a differential display method (see below).
Differential Display-The polymerase chain reaction (PCR), recovery, and reamplification of cDNAs obtained from astrocytes exposed to hypoxia and hypoxia/reoxygenation were performed by the method described by Liang and Pardee (11) with minor modifications. In brief, reverse transcriptase products obtained from total RNA (about 25 ng in each reaction) were used as PCR templates in 30 l of reaction mixture containing arbitrary primer (1 M), dNTP (200 M of each), and 2 units of Taq polymerase (AmpliTaq, Takara Shuzo). The PCR was performed for 39 cycles with a setting of 95°C for 30 s, 40°C for 1 min, and 72°C for 1 min (5 min for last cycle) by using the same arbitrary 12-mer oligonucleotide as both upstream and downstream primers. In one set of experiments, PCR products obtained from hypoxic and hypoxia/reoxygenated astrocytes were screened using 60 different primers. After separation in 5% polyacrylamide gel electrophoresis, PCR products were stained with ethidium bromide. cDNA bands specifically amplified in hypoxia/reoxygenated astrocytes were cut out and eluted from the gels and reamplified by PCR using the same primers and conditions employed in differential display except for an increased concentration of dNTP mixture (40 M).
Cloning and Sequencing of cDNA Fragments-Reamplified cDNA fragments were cloned into the pT7Blue T-vector (Novagen). Plasmid DNA sequencing of cloned fragments was carried out using the Taq dye primer cycle sequencing core kit (Applied Biosystems Inc., Tokyo, Japan) with either the M13 forward sequencing primer or reverse primer. The cDNA sequences were analyzed and compared for homology with those available in EMBL and GenBank TM DNA data bases, and NBRF protein data base. In the present study, one of the cDNA fragments (tentatively named RA301) was subjected to further analysis. By screening a rat cDNA library, a cDNA (2 kilobase pairs) that encompasses the entire open reading frame of RA301 was isolated and sequenced in both directions.
Northern Blot Analysis-To confirm the induction of RA301 transcript by reoxygenation, Northern blot analysis was performed using a cDNA probe synthesized from an RA301 fragment as described previously (9). In brief, about 5 g of total RNA extracted from hypoxic or hypoxia/reoxigenated astrocytes by the AGPC method were subjected to agarose (1%), formaldehyde gel electrophoresis and transferred overnight to Immobilon N membrane (Millipore, Bedford, MA). RNA was then fixed to the membrane by exposure to UV irradiation prior to hybridization with cDNA probes. The membrane was prehybridized for 3 h at 65°C in hybridization buffer (6 ϫ SSC (NaCl, 0.9 M, sodium citrate, 90 mM, final pH 7.0), 5 ϫ Denhardt's solution (Ficoll, 0.5%; polyvinylpyrrolidone, 0.5%; bovine serum albumin, 0.5%), 0.5% SDS, and 100 g/ml heat-denatured salmon sperm DNA). The membrane was probed with 32 P-labeled cDNA fragment of RA301 by the random hexamer procedure (12). After hybridization, filters were washed twice with 2 ϫ SSC, 0.5% SDS, and 0.1 ϫ SSC, 0.5% SDS for 30 min at 65°C, exposed to x-ray film (Kodak X-OMAT, Eastman Kodak Co.), and subjected to autoradiography. Induction of RA301 mRNA was evaluated by comparison with ␤-actin mRNA. To assess induction of RA301 in astrocytes by hypoxia/reoygenation, total RNA was obtained from astrocytes exposed to hypoxia/reoxygenation at the indicated time, and Northern blotting was performed as above. In some experiments, either cycloheximide (10 g/ml) or diphenyl iodonium (DPI) (50 M (all reagents were from Sigma) was added to the culture 15 min prior to reoxygenation, and total RNA was extracted 30 min after reoxygenation. Reactive oxygen intermediates derived from astrocytes were measured by lucigenin-based chemiluminescence as described previously (6). To assess the effect of hypoxia/reoxygenation on other mammalian RNA binding factors or related proteins, RNAs obtained from astrocytes in normoxia, hypoxia (32 h), and hypoxia/reoxygenation (32 h/1 h) were also analyzed by Northern blotting using radiolabeled cDNA probes including U1-70K (13), U2AF 65 (14), and U2AF 35 (15).
Effect of RA301 Antisense Oligonucleotides on the Expression of IL-6 in Astrocytes by Hypoxia/Reoxygenation-IL-6 activity released by cultured astrocytes exposed to hypoxia/reoxygenation was studied as described elsewhere (6). In brief, cultured astrocytes were exposed to normoxia or hypoxia for 32 h followed by reoxygenation for 2, 4, 8, 16, and 32 h. Conditioned medium was assayed for IL-6 using the MH-60 assay. To assess the role of RA301 in the induction of IL-6 after reoxygenation, three antisense 20-mer phosphorothioate oligonucleotides corresponding to three different structures around the initiation codon (named YS-59, -60, and -61) were synthesized and added to the culture medium (10 M for each oligonucleotide) 30 min before reoxygenation (16). IL-6 activity in the culture supernatant was measured 8 h after reoxygenation. Sense oligonucleotide (3Ј-CGA CAG CGA GTA CTG AGG CC-5Ј) complementary to the antisense structure (YS-60; 5Ј-GCT GTC GCT CAT GAC TCC GG-3Ј) which most effectively inhibited IL-6 release was also added as a control for specificity (each oligonucleotide was synthesized by Yuki Gosei Kogyo Co., Ltd.).
Production of Anti-RA301 Peptide Antibody and Western Blotting-To obtain antibody reactive with RA301 antigen, the peptide with code, amino acid residues 167-183 (see Fig. 1A), extra cysteine residue was introduced at the N terminus for conjugation) was synthesized and were conjugated with bovine serum albumin using a kit obtained from Sigma. Rabbits were immunized by conventional methods and antiserum was obtained from three rabbits immunized with 100 g of synthetic peptides followed by the evaluation of titer of the serum by enzyme-linked immunosorbent assay. After astrocytes (about 1 ϫ 10 6 cells) were exposed to hypoxia/reoxygenation, cells were harvested at the indicated times, washed three times with phosphate-buffered saline, pelleted, and lysed in buffer containing 2% SDS. Then about 10 g of protein were applied to SDS-polyacrylamide gel electrophoresis (10% gel), and RA301 antigen was visualized by the method described by Towbin et al. (17) using the antiserum. Where indicated, either antisense oligonucleotides (YS-59, -60, and -61) (10 M), sense oligonucleotide against YS-60 (10 M), or cycloheximide (10 g/ml) was added to the culture 30 min prior to reoxygenation. To evaluate the effect of RA301 sense and antisense oligonucleotides on expression of other stress protein, the same samples were also subjected to immunoblotting using anti-72 kDa HSP (HSP72) monoclonal antibody (Amersham Corp., Sweden). Protein content was determined using the Bio-Rad microprotein assay kit.
Expression of RA301 in Ischemic Brain-Expression of RA301 antigen was studied by temporal (2 h) unilateral occlusion of the middle cerebral artery in male Sprague-Dawley rats (250 g) as described by Tamura and Graham (18). Blood gasses, pH, and mean arterial blood pressure were monitored throughout the operation. Experiments were completed only if these physiological variables remained within normal limits. The normal limits for mean arterial blood pressure were set at 90 -130 mm Hg, those for PCO 2 at 30 -50 mm Hg, for PO 2 at 100 -130 mm Hg, and for arterial blood pH at 7.25-7.45. At the indicated time points, rats were sacrificed, and the brains were separated and quickly frozen at Ϫ80°C. Brain tissues were then homogenized with a polytron (setting 5, 20 strokes) in 2 ml of phosphate-buffered saline (pH 7.4) containing 2% SDS. Following centrifugation (10,000 ϫ g) for 10 min at 4°C, tissue homogenates were filtered twice through 0.22-m cellulose filters (Sumilon, Osaka, Japan), and about 10 g of protein were subjected to Western blotting, as above. For in situ hybridization, serial coronal sections (5 m in thickness) were obtained from the frozen brains with a cryostat, pretreated, and hybridized with either antisense or sense cRNA probes according to the method described previously (19). cRNA probes were in vitro transcribed from a template (RA301 cDNA fragment; 480 base pairs) subcloned into pBluescript plasmid (Stratagene, La Jolla, CA) under the presence of 35 S-UTP (NEG-039H, DuPont NEN). After hybridization, sections were washed in high stringency conditions, dried, and exposed to x-ray film. Two days later, the film was developed, and brain images were examined. Then the sections were covered with photographic emulsion (NTB-2, Kodak). After a 2-week exposure, the slides were developed, counterstained, and examined under a microscope with dark-field illumination.
Statistical Analysis-Where indicated, statistical analysis was performed by Student's non-paired t test for the multiple comparison, following analysis of variance.

Viability of Astrocytes Exposed to Hypoxia/Reoxygenation-
Oxygen tension in the medium fell to 8 torr within 3-5 h after cultures were transferred to the hypoxia chamber. Cell viability was maintained throughout hypoxia, and following replace-ment of cultures back to normoxia (reoxygenation), based on lack of lactate dehydrogenase release into culture supernatants, continued trypan blue exclusion, adherence to the culture substrate, and unchanged morphologic features (data not shown).
Isolation of the cDNA for RA301 by Differential Display-Using the primer 5Ј-CCT TTC CGA CGT-3Ј for differential display, a cDNA, termed RA301, was amplified from the cDNAs prepared from pooled RNA of hypoxia/reoxygenated astrocytes. The deduced amino acid sequence, based on nucleotide sequencing of the RA301 cDNA (accession no. D49708, in DDBJ, GenBank TM , EMBL data bases), revealed several motifs specifically associated with RNA binding factors, including two serine/arginine-rich domains, an RNA recognition motif, and a glycine octamer domain (Fig. 1A). The total number of amino acids expected is 288, and the predicted molecular mass is about 36 kDa. The amino acid sequence of RA301 has greatest homology (38.2%) to transformer-2 (Tra-2), a Drosophila splicing factor (Fig. 1B). The glycine octamer structure (Fig. 1A) is homologous to the glycine-rich domain of several RNA splicing factors, such as SC35 (20) and SF2/ASF (21) (Fig. 1C). Northern analysis of RNA from hypoxia/reoxygenated astrocytes using the full-length cDNA of RA301 as the probe demonstrated a band corresponding to 2 kilobase pairs, suggesting that the cDNA of 2 kilobase pairs was likely to be full-length ( Fig. 2A).
Induction of RA301 mRNA in Astrocytes Exposed to Hypoxia/Reoxygenation-Compared with normoxic cultured astrocytes, where no band was detectable, Northern analysis showed a dense band after cultures were exposed to hypoxia, followed by reoxygenation ( Fig. 2A). In contrast, hypoxia alone did not up-regulate RA301 mRNA. Maximal expression of the mRNA was observed 30 -60 min following replacement of hypoxic astrocytes into normoxia (Fig. 2B), and the increase in levels of RA301 mRNA was evident as early as 15 min after reoxygenation (Fig. 2C). By 4 h after exposing hypoxic astrocytes to normoxia, levels of RA301 mRNA had declined to close to the pretreatment base line.
Addition of cycloheximide (10 g/ml) to hypoxic astrocytes simultaneously with their placement in normoxia did not block induction of RA301 mRNA (Fig. 2C), whereas this concentration of cycloheximide blocked incorporation of [ 3 H]leucine into material precipitable in trichloroacetic acid by Ͼ90%. In contrast, the neutrophil-type NADPH oxidase inhibitor, DPI, when present at the start of reoxygenation, completely blocked the appearance of RA301 mRNA (Fig. 2C). Under these conditions, DPI prevents generation of oxygen-free radicals by reoxy-genated astrocytes based on chemiluminescence using lucigenin as the substrate and the cytochrome c reduction assay (6). Since DPI can also inhibit the nitric oxide pathway, experiments were performed in which L-arginine-free medium was employed, or the competitive inhibitor of nitric oxide synthase L-NMMA was present. However, neither of these agents had a similar effect to DPI on RA301 mRNA (data not shown). These data suggested that oxygen-free radicals formed early during the period of reoxygenation were likely to trigger increased expression of RA301 mRNA, rather than induction of a polypeptide requiring de novo protein biosynthesis.
Induction of RA301 in astrocytes exposed to hypoxia followed by reoxygenation was not part of a generalized event reflecting up-regulation of multiple RNA binding factors and related proteins. By Northern analysis, U1-70K, U2AF 65 , and U2AF 35 did not display increased expression at the mRNA level, compared with the results previously obtained with RA301 (Fig. 3).
Expression of RA301 in Astrocytes Exposed to Hypoxia/ Reoxygenation: Effect of Antisense/Sense Oligonucleotides-Rabbit antiserum was raised against the synthetic peptide corresponding to residues 167-183 in RA301 immunoblotted a band corresponding to a molecular mass of about 36 kDa (Fig.  4A). This band was not observed in lysates of normoxic and hypoxic astrocytes, but was readily detectable in extracts from hypoxia/reoxygenated cells (Fig. 4A). Immunoreactive RA301 was observed within 2 h of reoxygenation, being maximal by 4 h (Fig. 4A). To modulate expression of RA301, antisense oligonucleotides were employed. Antisense oligonucleotide (YS-60) blocked induction of RA301 antigen in astrocytes exposed to hypoxia/reoxygenation, although sense oligonucleotide at the same concentration was without effect (Fig. 4B). Further evidence of the specificity of YS-60 for inhibiting expression of RA301 was its lack of effect on induction of HSP72 which also occurs in astrocytes exposed to hypoxia/reoxygenation (Fig.  4C). Consistent with a central role for de novo biosynthesis in the production of RA301 antigen, cycloheximide (10 g/ml) inhibited its expression (Fig. 4B).
Expression of RA301 mRNA and Antigen in Ischemic Rat Brain-A rat brain ischemia model was employed to assess expression of RA301 in vivo. In situ hybridization showed increased levels of mRNA in the ischemic hemisphere, versus lower levels in nonischemic hemisphere (Fig. 5, A and B). RA301 mRNA was maximally expressed about 12 h after 2-h unilateral occlusion of the middle cerebral artery and remained elevated up to 24 h, returning to base-line levels by about 3 days (data not shown). RA301 mRNA appeared diffusely induced in the ischemic hemisphere (Fig. 5, A and B). Detailed analysis with microautoradiography (Fig. 5, D and E) suggested that astrocytes expressed RA301 mRNA in addition to neurons of the ischemic cerebral cortex. Controls with the sense probe demonstrated no specific staining pattern in ischemic hemisphere (data not shown). Immunoblotting of homoge-nates derived from ischemic hemisphere demonstrated an approximately 36-kDa band not observed with extracts from nonischemic control brain (Fig. 5C).
Effect of RA301 Expression on Increased Release of IL-6 Activity by Astrocytes Exposed to Hypoxia/Reoxygenation-IL-6 is a neurotrophic cytokine which, in previous studies, has been shown to be synthesized and released in increased amounts by astrocytes subjected to hypoxia/reoxygenation (6). To explore a possible role for RA301 in IL-6 production by astrocytes, antisense oligonucleotide was employed. YS-60 blocked by Ͼ65% elaboration of IL-6 activity into the conditioned medium of astrocytes exposed to hypoxia/reoxygenation (Fig. 6A), under conditions where expression of RA301 was largely blocked (Fig.  4B). Two other antisense oligonucleotides, YS-59 and YS-61, did not effectively modulate expression of RA301 antigen (data not shown) and had no effect on astrocyte release of IL-6 activity (Fig. 6B). None of these oligonucleotides affected the activity of exogenous IL-6 added to the MH-60 bioassay.

DISCUSSION
Exposure of cells to a period of oxygen deprivation followed by reoxygenation imposes a major metabolic stress. The period of hypoxia, in which there is a shift to anaerobic glycolysis, is associated with events such as up-regulation of the non-insulin-dependent glucose transporter (2), activation of NF-IL-6, and transcription of target genes, including IL-6 and tumor necrosis factor-␣ (22), which we have hypothesized primes the cells for the subsequent phase of reoxygenation. It is also likely that other events, such as activation of AP-1, which is stimulated by antioxidants, will occur during hypoxia, further modifying biosynthetic mechanisms (23,24). In contrast, during reoxygenation, generation of oxygen-free radicals occurs. This is especially striking in vivo when leukocytes are attracted to reperfused tissues and activated, and their formation of reactive oxygen intermediates is induced. Previous studies have drawn attention to a role for oxygen-free radicals in triggering production of polypeptide mediators relevant to the biology of ischemia; reactive oxygen intermediates appear to initiate expression of IL-1 and IL-8 in mononuclear phagocytes (25,26)  and to induce synthesis and elaboration of IL-6 in astrocytes (6). One mechanism through which such reactive oxygen species impact on the biosynthetic apparatus is through activation of the transcription factor NF-kB (27), thereby accelerating the rate of transcription of particular mRNAs. Another means for coordinately regulating expression of protein products is through processing of multiple RNA species.
Our findings demonstrate that a novel 36-kDa polypeptide, RA301, is induced by oxygen-free radicals produced endogenously by cultured astrocytes exposed to hypoxia followed by reoxygenation. Based on the deduced amino acid sequence, domains characteristic of RNA binding/splicing proteins are present, including two serine/arginine-rich motifs, an RNA recognition motif, and a glycine-rich motif. RA301 bears closest homology to Tra-2, an RNA splicing factor important in sex determination in Drosophila (28). Thus, RNA splicing factors, such a Tra-2, can have a fundamental role in altering cellular phenotype by maturation of nascent RNAs. Further studies will be required to prove that RA301 also has properties of an RNA splicing factor.
The presence of RA301 in ischemic regions of rat brain further emphasizes its potential expression in loci where it is likely to impact on ultimate expression of proteins in cellular elements subject to ischemia. The importance of such de novo protein products in the cellular adaptation to hypoxia/reoxygenation is illustrated by the induction of cell death which invariably follows addition of cycloheximide to reoxygenated astrocytes following 8 h of reoxygenation (7). These studies provide a first step in characterizing a gene product of potential importance in the cellular response to ischemia, RA301, and raise multiple questions concerning its potential impact on redirecting essential biosynthetic events in astrocytes, and, potentially, other cells exposed to hypoxia/reoxygenation. The concomitant attenuation of the production of IL-6 by astrocytes exposed to hypoxia/reoxygenation when RA301 expression was blocked by antisense oligonucleotide suggests that RA301 may affect synthesis of critical gene products.