Impact of polyunsaturated fatty acid consumption prior to ischemic stroke.

OBJECTIVE: The Japanese have higher levels of n-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in their diets. These facts may contribute to the lower rates of atherosclerosis in Japanese. The purposes of this study were to assess the PUFA levels in patients with subtypes of acute ischemic stroke and to assess the relationship between severity and PUFA levels. MATERIAL AND METHODS: We studied 75 patients with lacunar infarction (LI; n = 25), atherothrombotic infarction (AT; n = 32), and cardiogenic embolism (CE; n = 18). The patients underwent blood examinations in a fasting state next morning of hospitalization, including examination of low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglyceride (TG), blood glucose, hemoglobin A1c (HbA1c), uric acid, and fatty acid fractions of EPA, DHA, dihomo-γ-linolenic acid (DGLA), and arachidonic acid (AA). We used the modified Rankin Scale (mRS) to assess clinical severity at discharge. RESULTS: There was no significant difference in the EPA/AA and DHA/AA ratio among the three stroke subgroups, although the DGLA/AA ratio was significantly higher in patients with LI than in patients with CE. Considering the confounding factors, the mRS was negatively correlated with EPA/AA and positively correlated with age, DHA/AA, and blood glucose. CONCLUSIONS: High EPA/AA ratio was associated with good outcome in ischemic stroke. Our paper suggests that prestroke dietary habits affect the severity in patients with ischemic stroke.

Preoperative embolization of meningiomas with N-butyl cyanoacrylate.

Meningiomas are often embolized before their surgical resection to reduce blood loss during surgery. Polyvinyl alcohol (PVA) particles have been the most frequently used material for embolization of meningiomas. We have used n-butyl cyanoacrylate (NBCA) as the first-choice material since 2001. Thirty-one meningiomas were embolized with NBCA. We report the result of embolization of meningiomas with NBCA in comparison with PVA particles.

Striatal Distribution of Dopamine Transporters and Dopamine D2 Receptors at Different Stages of Parkinson's Disease. A CFT and RAC PET Study.

We investigated the alteration of dopaminergic system in striata of Parkinson's disease (PD) at different stages using positron emission tomography (PET), [(11)C]2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane (CFT) for dopamine transporter (DAT), and [(11)C]raclopride (RAC) for dopamine D2 receptor (D2R). We studied eight elderly healthy volunteers (Group A), 13 drug naïve patients with PD (Group B), and seven advanced PD patients with mild dyskinesia (Group D). Six patients in Group B were re-examined after antiparkinsonian therapy (Group C). Regions of interest were drawn on the cerebellar hemisphere, head of the caudate nucleus (CN), and anterior (AP) and posterior putamen (PP) in the PET images. We calculated uptake ratio index (URI), asymmetry index (AI) and presynapse-to-postsynapse ratio (PPR) to evaluate dopaminergic function. DAT was smaller in the three PD groups than the Group A. URI of RAC in the PP was significantly larger in Group B than in Groups A and C. AI of CFT in the putamen was larger in the PD groups than in normal subjects, and AI of RAC in the PP was the largest in the Group B. PPRs in the AP and PP were smaller in the three PD groups than in Group A. DAT decreased with disease progression in patients with PD. Binding of RAC was largest in the putamen of drug-naïve PD patients, but the enhanced binding could not be detected in the therapeutic patients with PD because of weak D2R affinity of RAC.

Anterior vertebral artery decompression with an ultrasonic bone curette to treat bow hunter's syndrome.

We report a patient with bow hunter's syndrome who was treated by anterior decompression of the vertebral artery (VA) using an ultrasonic bone curette (SONOPET). This 60-year-old man reported almost losing consciousness upon head rotation. Although the right VA appeared normal at the natural head position, upon left head rotation it became completely occluded at the transverse foramen of C2. We performed anterior decompression of the right VA at the axis using a high-speed drill and SONOPET. For anterior decompression of the VA in a deep and narrow operative field, we recommend use of SONOPET, which permits safe, easy bone dissection.

Traumatic cerebral aneurysm associated with a contralateral traumatic carotid cavernous fistula. A case report.

SUMMARY: This report documents the management of a traumatic carotid aneurysm (TCA) with a traumatic carotid-cavernous fistula (T-CCF) of the contralateral internal carotid artery (ICA) following a closed head injury. A 38-year-old man presented with severe traumatic subarachnoid hemorrhage and pneumocephalus due to a severe head injury. Four months after the accident, the patient presented with clinical symptoms of exophthalmos and retroorbital bruit. Cerebral angiography showed a TCA of the IC-PC region, which coexisted with a contralateral T-CCF. Both lesions were successfully managed with an endovascular treatment using coils to isolate a fistula from the ICA, and direct surgical trapping of the intracranial ICA to eliminate a TCA. Post-operative angiography revealed a good cross-flow through the anterior communicating artery from the contralateral ICA, which was completely obliterated by the T-CCF. No additional surgical or endovascular procedure for traumatic vascular injuries was required. The patient remained asymptomatic during the clinical follow-up period of 24 months. The goal of traumatic carotid injuries is the selective elimination of the vascular pathologic injury with asymptomatic state, using direct surgery and/or an endovascular treatment.

Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.

OBJECTIVE: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. DESIGN: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. METHODS: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. RESULTS: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. CONCLUSIONS: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

[A case of iatrogenic carotid artery dissection treated with radial artery graft].

A case of iatrogenic intracranial artery dissection is reported. A 52-year-old female developed severe headache and nausea. Brain CT showed diffuse subarachnoid hemorrhage. On admission, carotid angiography revealed an aneurysm in the right middle cerebral artery and the intact right internal carotid artery. The aneurysm was clipped successfully. Carotid angiography on day 7 revealed dissection in the right internal carotid artery. Repeated angiograms at 10 and 31 days showed progression of the carotid artery dissection. Findings of ECD-SPECT on day 31 (Balloon occlusion test) suggested low perfusion of the right internal carotid artery territory. The patient underwent surgical reconstruction of the right internal carotid artery using a radial artery. She presented with right abducens nerve palsy three days after the radial artery graft. The patency of the radial artery graft was proved by the post-operative angiography. Internal carotid artery dissection may occur spontaneously or as a result of trauma. An iatrogenic dissection is an uncommon complication of cerebral angiography. There are no evidence-based guidelines for the treatment although anticoagulation therapy is most commonly used. The present case emphasizes the usefulness of radial artery graft for traumatic carotid artery dissection.

Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese black bear, Ursus thibetanus japonicus, during pregnancy.

The Japanese black bear, Ursus thibetanus japonicus, is a seasonal breeder and shows delayed implantation for several months during pregnancy. The objective of this study was to clarify the steroidogenic capability of the corpus luteum and placenta during pregnancy, including both delayed implantation and fetal development, by immunolocalization of steroidogenic enzymes in these organs of the Japanese black bear. Ovaries and placentae from 15 wild Japanese black bears, which had been killed legally by hunters and were thought to be pregnant, were used in an immunocytochemical study to localize the cholesterol side chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) by the avidin-biotin-peroxidase complex method using polyclonal antisera raised in mammals against P450scc, 3betaHSD, P450c17 and P450arom. P450scc and 3betaHSD were localized in all luteal cells throughout pregnancy. P450c17 was present in a few luteal cells, especially in the outer area of the corpus luteum throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. Cells positively immunostained for P450c17 were significantly smaller than negatively immunostained cells (P < 0.01). P450arom was present sporadically in a few luteal cells throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. The size of cells positively immunostained for P450arom was not significantly different from that of negatively immunostained cells. The whole placenta was negatively immunostained for P450scc, 3betaHSD and P450c17, but P450arom was present in the syncytiotrophoblasts and endothelial cells of maternal blood vessels. These results indicate that, in the Japanese black bear, corpora lutea are a source of progesterone which may play an important role in the maintenance of delayed implantation and fetal development during pregnancy. Corpora lutea have a minimum capability to synthesize androgen in small luteal cells and oestrogen in normal-sized luteal cells during pregnancy, and placentae have the ability to synthesize oestrogen during late pregnancy.

Mechanisms of adrenal damage induced by 7,12-dimethylbenz (alpha) anthrancene in female Sprague--Dawley rats.

The mechanisms of adrenal damage induced by 7,12-dimethylbenz (alpha) anthrancene (DMBA) in 50-day-old female Sprague--Dawley rats were investigated. A single dose of DMBA, either fed (30 mg) per os or injected (6 mg) in a caudal vein, caused inner cortical cell death (cells of the zonae fasciculata and reticularis) by an apoptotic mechanism. Apoptotic cells were identified by cell morphology, and terminal dUTP nick end labeling (TUNEL)-positive cells were seen at 12 hrs post-DMBA, reached a maximum at 36 h, and were accompanied by blood congestion followed by massive hemorrhage leading to post-apoptotic necrosis at 48 and 72 h. The apoptotic cascade involved the up-regulation of Bax, the down-regulation of Bcl-2, and the activation of caspase-3. At 72 h, regeneration as evidenced by the appearance of 5-bromo-2'-deoxyuridine-positive cells began to occur in the damaged inner cortical zones, with the cells proliferating toward the medulla thereafter. Regenerative cells expressed cytochrome P450 11 beta hydroxylase. The damage was repaired but calcification appeared at 2 weeks post-DMBA, leaving bow-shaped lesions in some adrenals.

The rate-determining step in P450 C21-catalyzing reactions in a membrane-reconstituted system.

Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17alpha-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 degrees C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17alpha-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 +/- 0.7 s(-)1, and that for 17alpha-hydroxyprogesterone was 1.8 +/- 0.5 s(-)1 at 37 degrees C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17alpha-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics.

Regulation of the successive reaction catalyzed by rat neuronal nitric oxide synthase.

The rat neuronal nitric oxide synthase (nNOS) catalyzes two monooxygenase reactions successively from L-arginine (L-Arg) to L-citrulline (L-Cit) via N(omega)-hydroxy-L-arginine (OH-Arg) without most of OH-Arg leaving the substrate-binding site. In the steady-state reaction conditions, the amount of OH-Arg produced is about 1/30-1/50 that of L-Cit. We found in this study using nNOS purified from an Escherichia coli expression system that the ratio of the amount of OH-Arg to L-Cit (OH-Arg/L-Cit) increased to about 1 at low concentration of NADPH. In one cycle of the nNOS reaction, the decrease in NADPH concentration was found to reduce the rates of two monooxygenase reactions but had little effect on the rate constant of OH-Arg dissociation from the enzyme. The addition of NADP(+), the competitive inhibitor for NADPH, caused the decrease in the rates of monooxygenase reactions in a single cycle of the reaction and the increase in the ratio of OH-Arg/L-Cit in the steady state. At low CaM concentrations, the ratio of OH-Arg/L-Cit was about the same as that at high CaM. In a single cycle of the nNOS reaction, the rate of monooxygenation was not altered by the CaM concentration but the amount of metabolized L-Arg decreased with the decrease in CaM concentration, showing that the amount of active nNOS was regulated by complex formation between nNOS and CaM. It becomes clear that there are two regulatory mechanisms for the successive reaction of nNOS. One controls the rates of monooxygenations and the other controls the amount of active species of nNOS.

Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast.

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase. To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.

Ependymal cyst producing alpha-fetoprotein. Case report.

This 17-year-old man was admitted to the hospital due to progressive headache and diplopia. Neuroradiological studies revealed a cystic mass in the pineal region without a parenchymal lesion. In addition, serum alpha-fetoprotein (AFP) levels were elevated. A cyst-to-third-ventricle and cistern fenestration was performed, but the cyst enlarged 3 months after the first operation. In the second operation, subtotal resection of the cyst was performed. The AFP level in the cyst fluid was very high preoperatively but was decreased postoperatively. The patient was discharged with no neurological deficit. Pathological examination of resected tissue showed a single layer of cuboidal cells that resembled an ependymal structure. The cells were immunoreactive for AFP immunostain, which indicated AFP production from these cells.

Kinetic studies on the successive reaction of neuronal nitric oxide synthase from L-arginine to nitric oxide and L-citrulline.

Rat neuronal nitric oxide synthase (nNOS) was heterologously expressed in Escherichia coliand purified. The conversion of L-arginine to N(omega)-hydroxy-L-arginine and further to L-citrulline in one cycle of the reaction of the purified nNOS was measured with the reaction rapid quenching method using (3)H-L-arginine as the substrate. It was found that most of the produced (3)H-N(omega)-hydroxy-L-arginine was successively hydroxylated to (3)H-L-citrulline without leaving the enzyme. From the analysis of time courses, the rate constants for each reaction step, and also for the dissociation of the intermediate, were estimated at various temperature in which the rates for the first and the second reactions were not much different each other but the rate for the dissociation of (3)H-N(omega)-hydroxy-L-arginine from the enzyme was significantly slow. Under the steady-state reaction condition, almost all of the nNOS was estimated to be active from the amount of burst formation of L-citrulline in the pre-steady state. The rate constant for the dissociation of the product L-citrulline from nNOS was calculated from the combination of results of the rapid quenching experiments and the metabolism of L-arginine in the presence of an excess amount of substrate, which was the smallest among all the rate constants in one cycle of the nNOS reaction. The activation energies for all the reaction steps were determined from the temperature dependence of the rate constants, which revealed that the rate-determining step of the nNOS reaction in the steady state was the dissociation of the product L-citrulline from the enzyme.

EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.

Pregnenolone, pregnenolone sulfate, and cytochrome P450 side-chain cleavage enzyme in the amphibian brain and their seasonal changes.

To clarify whether the amphibian brain synthesizes de novo neurosteroids, we examined pregnenolone, pregnenolone sulfate ester, and cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc), an enzyme converting cholesterol to pregnenolone, using amphibians. Pregnenolone and its sulfate ester in the brain, gonad, and plasma of Xenopus laevis were measured by a specific pregnenolone RIA. The concentrations of these two steroids in the female brain were significantly larger than those in the ovary and plasma. A similar tendency was evident in the male. In both sexes, pregnenolone and its sulfate ester were concentrated more highly in the cerebellum than in the telencephalon, diencephalon, or midbrain. An immunoreactive protein band of electrophoretic mobility in the proximity of bovine adrenal P450scc was detected in the Xenopus brain as well as the testis by Western blot analysis. Immunohistochemical analysis indicated that Purkinje cells in the Xenopus cerebellum were specifically immunostained with the P450scc antibody. P450scc-like immunoreactive cells were further found in several telencephalic and diencephalic regions, such as the pallium mediale and nucleus preopticus, in the Xenopus brain. A similar localization of P450scc-like immunoreactive cells was evident in Rana nigromaculata, a seasonally breeding amphibian. In the present study, seasonal changes in pregnenolone and its sulfate ester were further examined as a possible physiological change using R. nigromaculata. In both sexes, pregnenolone concentrations in the brain were almost constant during the seasonally breeding cycle. In contrast, the pregnenolone sulfate concentration in the brain was significantly lower in the hibernating quiescent phase and higher in the breeding and postbreeding active phases, independent of the plasma steroid level. These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.

Extracavernous arteriovenous fistulae. Report of five cases.

SUMMARY: We report five cases of arteriovenous fistulae (AVFs) of the carotid system. Two were traumatic non penetrating injuries and involved the subarachnoid, extracavernous part of the intracranial internal carotid artery; two were spontaneous and involved the internal carotid artery in its extracranial portion; one was a spontaneous AVF of the ascending pharyngeal artery. All the symptoms due to these AVFs were not related to the location of the fistula, but to the congestive venous drainage. The revealing symptoms regressed and/or improved after transarterial detachable balloon embolisation that led to complete occlusion of the AVFs.

Calcium ion as a second messenger for o-nitrophenylsulfenyl-adrenocorticotropin (NPS-ACTH) and ACTH in bovine adrenal steroidogenesis.

o-Nitrophenyl sulfenyl-modified ACTH (NPS-ACTH) stimulated steroidogenesis acutely in bovine fasciculata-reticularis cells without increase in cellular cAMP synthesis. Application of NPS-ACTH to the cultured cells induced Ca2+ signals in individual cells as detected by video-enhanced microscopic fluorescence measurements. The percentage of Ca2+ signaling cells corresponded well with the increase of steroidogenesis induced by NPS-ACTH below 1 nM. Treatment of the cells with nicardipine, a Ca2+ channel blocker, suppressed the Ca2+ signals except for the transient increase just after the addition of NPS-ACTH and also blocked completely the stimulative effect on the steroidogenesis of NPS-ACTH below 1 nM. At a dosage of NPS-ACTH higher than 10 nM, the stimulative effect of steroidogenesis was partly suppressed by nicardipine and also by AA-861, a lipoxygenase inhibitor. The action of NPS-ACTH might be mediated by both Ca2+ and lipoxygenase metabolite(s) of arachidonic acid as dual second messengers. The effect of ACTH in pM range on the steroidogenesis was suppressed completely by the treatment with nicardipine and AA-861 at the same time, indicating that the action was mediated by both Ca2+ and the lipoxygenase metabolite(s) but not by cAMP. cAMP plays a significant role as a second messenger for ACTH action only at ACTH concentrations greater than 10 pM.

Kinetic studies on bovine cytochrome p45011 beta catalyzing successive reactions from deoxycorticosterone to aldosterone.

The reactions for the synthesis of aldosterone from deoxycorticosterone were investigated kinetically in the membrane-reconstituted system with bovine cytochrome P45011 beta at 37 degrees C. Reaction rapid-quenching experiments for the metabolism of deoxycorticosterone by P45011 beta showed that aldosterone was produced via corticosterone, not via 18-hydroxydeoxycorticosterone. The kinetic analysis revealed that aldosterone was formed successively from fractions of intermediate metabolites which did not dissociate from P45011 beta. The rate of each reaction step in the successive reactions was estimated from the combination of results of the rapid-quenching experiments and the metabolism of deoxycorticosterone in the presence of an excess amount of substrate, in which the dissociation of final product, aldosterone, from the enzyme was the slowest step in the synthesis from deoxycorticosterone. Under steady-state reaction conditions, the interaction of P45011 beta with P450scc stimulates the production of corticosterone from deoxycorticosterone by about 10-fold but inhibits further reactions from corticosterone. The rapid-quenching experiments showed, however, that the rate constant for the 11 beta-hydroxylation of deoxycorticosterone for corticosterone production in the presence of P450scc was almost the same as that without P450scc. The interaction of P45011 beta with P450scc in the reaction system for deoxycorticosterone metabolism was found to slow the rate of the subsequent 18-hydroxylation of the produced corticosterone and to accelerate the dissociation of the corticosterone from P45011 beta, which stimulated the corticosterone production and inhibited the further reaction for aldosterone synthesis in the steady-state reaction.

Existence and localization of a protein in zebra finch brain with similar structural features as the large subunit of the splicing factor U2AF.

From brains of the adult male zebra finch, we purified a peptide that has a homologous sequence of the large subunit of human U2AF. U2AF is a non-small-nuclear ribonucleoprotein (snRNP) splicing factor required for pre-spliceosome assembly. U2AF consists of a large and a small subunit, whereas only a large subunit is required for in vitro splicing. To show that U2AF large-subunit-like protein exists in the brain of zebra finches, we conducted immunoblot analysis on brain extract, using antiserum against an isolated peptide with primary structures similar to the U2AF large subunit (ppU2AFls). The immunoblot analysis showed that a protein with a molecular weight of about 60 kd reacted with the anti-ppU2AFls-antibody to a putative peptide of the U2AF large subunit. To examine the localization of this protein in the brain, we also conducted immunohistochemical analysis with the anti-peptide. An intense immunoreaction was restricted within the cellular nucleus throughout the brain, suggesting that this protein may contribute to the pre-spliceosome assembly in almost all of the regions of the brain.